[Whole exome sequencing and analysis of a Chinese family with familial pulmonary sarcoidosis].

Objective: To analyze the clinical features and the results of the whole exome sequencing (WES) of a Chinese family containing both pulmonary sarcoidosis patients and healthy members, and to find potent genes and variants that may be involved in the pathogenesis of sarcoidosis. Methods: Three patients with pulmonary sarcoidosis and 1 healthy member was included from a Chinese Han family in the north of China diagnosed in November 2016, which characterized as 2 consecutive generations including 2 males and 1 female, aged from 23 to 69 years old. The proband is Ⅱ-6. Pulmonary sarcoidosis was diagnosed by clinical features, imaging and pathological findings, and clinical data such as family history were collected. Whole blood samples were taken and WES (Illumina NovaSeq S2) was performed. The pathogenicity analysis and gene annotation analysis were performed by ExAC, SIFT, Polyphenv2, Metascape databases. Results: It was found that 27 genes were highly pathogenic in the database filtering result. After gene annotation analysis, we found that ZC3H12A gene can negatively regulate the differentiation of Th17 cells, which may be involved in the onset of pulmonary sarcoidosis. Sanger sequencing confirmed the c.1361 A>G variant in 3 sarcoidosis patients but normal in healthy member. Conclusions: In patients with familial pulmonary sarcoidosis, the genetic background could regulate immune response which is one of the pathogenic mechanisms of sarcoidosis. The whole exome test and gene ontology analysis showed that Ⅱ-2, Ⅱ-6 and Ⅲ-1 pulmonary sarcoidosis patients in this family were all shared the same variant on ZC3H12A gene, which played a pivotal role in differentiation of Th17 cells and is a potent pathogenesis gene in this Chinese pulmonary sarcoidosis family.

[The role of histone deacetylases in the pathogenesis of idiopathic pulmonary fibrosis and cryptogenic organizing pneumonia].

Objective: To explore the role of histone deacetylases(HDAC) in the pathogenesis of idiopathic pulmonary fibrosis(IPF) and cryptogenic organizing pneumonia(COP). Methods: Fifteen IPF patients [14 males and 1female, age 40-73 years, mean age (59±8) years] and 15 COP patients [5 males and 10 females, age 41-71 years, mean age (59±8) years] from Peking Union Medical College Hospital were recruited from March 2018 to October 2018. Fifteen healthy donors[4 males and 11females, age 43-70 years, mean age (58±6) years] were enrolled as controls. Peripheral blood mononuclear cells (PBMC) were isolated by density gradient centrifugation. The nuclear and cytoplasmic proteins were extracted by Nuclear Extraction Kit. HDAC activity was measured by fluorimetric method. The relations between HDAC activity and clinical parameters were analyzed with SPSS. Results: The HDAC activity of cytoplasmic protein and nuclear protein from patients with IPF were (724±216) nmol/L and (2 309±708) nmol/L, which were higher than that of health controls (409±105) nmol/L and (1 572±611) nmol/L (P<0.01 for both). So as to the HDAC activity of cytoplasmic protein and nuclear protein from patients with COP which were (718±245) nmol/L and (3 310±1 005) nmol/L (P<0.01 for both).The HDAC activity of nuclear protein from COP patients was higher than that from IPF patients (Z=-2.840, P=0.005). The HDAC activity of nuclear protein was negatively correlated with FEV(1) and D(L)CO in IPF patients (r=-0.574, P=0.025; r=-0.583, P=0.029), and negatively correlated with FVC and TLC in COP patients(r=-0.846, P=0.016; r=-0.900, P=0.015). Conclusion: HDAC may be involved in the pathogenesis of COP and IPF.

[The role of B cell activating factor in the differential diagnosis of usual interstitial pneumonia].

Objective: To explore the differential diagnostic role of B cell-activating factor(BAFF) in idiopathic pulmonary fibrosis (IPF) and usual interstitial pneumonia (UIP) associated with autoimmune diseases (AIDs). Methods: Plasma levels of BAFF were measured by ELISA method in 23 patients with AIDs-UIP, 34 patients with IPF, and 21 healthy subjects as control. The correlation between plasma BAFF levels and other clinical results from patients was analyzed. Receiver operating characteristics (ROC) analysis for distinguishing AIDs-UIP from IPF patients was examined and the maximal area under curve (AUC) was found. Results: Plasma levels of BAFF were significantly elevated in AIDs-UIP patients and IPF patients compared to healthy subjects(P<0.001 and P=0.002, respectively). AIDs-UIP patients had higher level of BAFF than IPF patients(P=0.030). Plasma BAFF levels in AIDs-UIP patients were inversely correlated with pulmonary function results, including FVC%(r=-0.435, P=0.040)and TLC%(r=-0.449, P=0.041), as well as DLCO%(r=-0.491, P=0.024). When the cut off value of BAFF was set as 1.5 ng/ml to distinguish AIDs-UIP patients from IPF patients, the sensitivity and the specificity was 64.5% and 90.0%, respectively, and the area under ROC curve reached the maximum of 0.784(P=0.000, 95% CI: 66.3%-90.5%). Conclusions: Plasma BAFF levels were significantly higher and inversely correlated with pulmonary function, reflecting the severity of AIDs-UIP patients. Plasma BAFF levels may be a useful marker for distinguishing AIDs-UIP from IPF.

Resistin-like molecule-ß (RELM-ß) targets airways fibroblasts to effect remodelling in asthma: from mouse to man.

BACKGROUND: RELM-ß has been implicated in airways inflammation and remodelling in murine models. Its possible functions in human airways are largely unknown. The aim was to address the hypothesis that RELM-ß plays a role in extracellular matrix deposition in asthmatic airways. METHODS: The effects of RELM-ß gene deficiency were studied in a model of allergen exposure in mice sensitised and challenged with Aspergillus fumigatus (Af). RELM-ß expression was investigated in bronchial biopsies from asthmatic patients. Direct regulatory effects of RELM-ß on human lung fibroblasts were examined using primary cultures and the MRC5 cell line in vitro. RESULTS: Sensitisation and challenge of wild-type mice with Af-induced release of RELM-ß with a time course coincident with that of procollagen in the airways. Af-induced expression of mRNA encoding some, but not all ECM in the lung parenchyma was attenuated in RELM-ß-/- mice. RELM-ß expression was significantly increased in the bronchial submucosa of human asthmatics compared with controls, and its expression correlated positively with that of fibronectin and α-smooth muscle actin. In addition to epithelial cells, macrophages, fibroblasts and vascular endothelial cells formed the majority of cells expressing RELM-ß in the submucosa. Exposure to RELM-ß increased TGF-ß1, TGF-ß2, collagen I, fibronectin, smooth muscle α-actin, laminin α1, and hyaluronan and proteoglycan link protein 1 (Hapl1) production as well as proliferation by human lung fibroblasts in vitro. These changes were associated with activation of ERK1/2 in MRC5 cells. CONCLUSION: The data are consistent with the hypothesis that elevated RELM-ß expression in asthmatic airways contributes to airways remodelling at least partly by increasing fibroblast proliferation and differentiation with resulting deposition of extracellular matrix proteins.

Effects of sodium butyrate on growth performance, haematological and immunological characteristics of weanling piglets.

The experiment was conducted to study the effects of sodium butyrate (SB) on growth, haematological and immunological characteristics in weanling pigs. A total of 100 male piglets (Duroc×Landrace×Yorkshire) with a body weight of 8.0 ± 0.2 kg weaned at the age of 28 days were randomly assigned to two treatments with five replicates and 10 pigs per replicate. Piglets received a basal diet (control group) or diets supplemented with 1000 mg/kg SB. The feeding trial lasted for 21 days. The results showed that dietary SB significantly decreased (p < 0.05) diarrhoea incidence of weaned piglets, but did not affect (p > 0.05) the average daily feed intake (ADFI), average daily gain (ADG) and feed to gain (F/G). Furthermore, piglets fed dietary SB had higher (p < 0.05) serum concentrations of glucose and triglycerides and lower (p < 0.05) serum concentrations of urea nitrogen, cortisol, D-lactic acid and diamine oxidase when compared with the control group. However, dietary SB did not affect concentrations of serum albumin, total protein, insulin and glucagon (p > 0.05). There were no significant (p > 0.05) treatment effects on serum IgA and IgM, whereas serum IgG concentration and IgA+ cell count in jejunum from pigs fed SB were significantly higher (p < 0.05) than in those given the basal diet. In conclusion, the present study indicated that dietary SB significantly decreased diarrhoea incidence of weaned piglets and increased the efficiency of nitrogen utilization. Also, dietary SB could regulate and enhance the immune function of piglets by increasing the serum IgG concentration and IgA+ cell count in jejunum. Our results suggest that SB may reduce some of the adverse effects of weaning stress and play an important role in maintaining the integrity of intestinal mucosa.

Molecular analysis of intestinal bacterial microbiota of broiler chickens fed diets containing fermented cottonseed meal.

The study was conducted to investigate the effects of dietary inclusion of fermented cottonseed meal (FCM) on the ileal and cecal bacterial microbiota of broiler chickens. A total of 300 newborn yellow-feathered broiler chickens were randomly divided into 2 treatments with 3 replicates each (50 birds per replicate): control and 80 g/kg of FCM group. The feeding trial lasted for 42 d. Ileal and cecal digesta samples were collected from 8 chicks per replicate at 21 and 42 d of age to determine the composition of bacterial microbiota using denaturing gradient gel electrophoresis, cloning, sequencing, and real-time quantitative PCR analysis. The results demonstrated that the microbial composition in the ileum and cecum were considerably affected by the diet. The similarity dendrogram of banding profiles showed a more rapid stabilization of intestinal bacterial microbiota in broilers fed diets supplemented with FCM, compared with that of the birds fed the control diet. No significant difference was observed in total number of bands and Shannon-Weaver index, indicating that FCM had no effects on bacterial diversity. However, enumeration of bacteria in the ileal and cecal contents by quantitative PCR showed an increased (P < 0.05) population of lactobacilli, as well as a decreased (P < 0.05) Escherichia coli number by the dietary inclusion of FCM. In summary, dietary inclusion of FCM did not affect the intestinal microbial diversity but shifted intestinal microbiota, with a more homogenous population and an increased colonization of lactobacilli. The results also support the concept that dietary FCM inclusion could promote the beneficial bacteria in the intestinal tract.

Interleukin-25 promotes basic fibroblast growth factor expression by human endothelial cells through interaction with IL-17RB, but not IL-17RA.

BACKGROUND: Unlike other IL-17 family members, the Th2-derived cytokine IL-25 (IL-17E) induces (promotes) Th2 responses. One or both of the two receptors for IL-25 (IL-17RA, IL-17RB) is expressed on inflammatory cells and tissue structural cells, suggesting that in addition to promoting Th2-type inflammation IL-25 may also act on structural cells at sites of Th2-type inflammation such as in the asthmatic bronchial mucosa to promote remodelling changes. OBJECTIVE: Our previous studies showed elevated expression of IL-25 and IL-17RB immunoreactivity in asthmatic airways with co-localization of the latter to endothelial cells. We therefore hypothesized that IL-25 acts on endothelial cells through this receptor to induce production of the key angiogenic and remodelling cytokine basic fibroblast growth factor (bFGF). METHODS: Polymerase chain reaction (PCR) immunocytochemistry/immunohistochemistry and ELISA were employed to detect expression of IL-17RB, IL-17RA and bFGF by human vascular endothelial cells (HUVEC) and immunoreactivity for IL-25 and bFGF in asthmatic bronchial biopsies. Receptor-blocking antibodies, PCR and an in vitro angiogenesis assay were used to investigate whether IL-25 acts on IL-17RB or IL-17RA to induce bFGF expression and angiogenesis. PCR was also employed to investigate the signalling pathways involved in IL-25-mediated bFGF expression. RESULTS: HUVEC constitutively expressed IL-17RB, IL-17RA and bFGF. Production of the latter was further increased by IL-25, but attenuated after blockade of the IL-17RB, but not the IL-17RA receptor. Neutralization of endogenous VEGF and bFGF completely abrogated IL-25-induced angiogenesis which was also inhibited by blocking IL-17RB, but not IL-17RA. The PI3K-specific inhibitor LY294002 also completely attenuated IL-25-induced bFGF expression. Immunoreactivity for IL-25 and bFGF was elevated in the asthmatic bronchial mucosa and the expression of each correlated with the other. CONCLUSIONS AND CLINICAL RELEVANCE: Our data support the hypothesis that IL-25 contributes to elevated bFGF in asthmatic airways by acting on the endothelial cell IL-17RB receptor through PI3K-signalling pathways. Targeting the pathways might benefit therapy of airways remodelling.

A2B5-positive oligodendrocyte precursor cell transplantation improves neurological deficits in rats following spinal cord contusion associated with changes in expression of factors involved in the Notch signaling pathway.

BACKGROUND: Oligodendrocyte precursor cells (OPCs) are myelinated glial cells of the central nervous system (CNS), able to regenerate oligodendrocytes and myelin. This study aimed to elucidate the effect of A2B5-positive (A2B5+) OPC transplantation in rats with spinal cord contusion (SCC) and to investigate changes in expression of various factors involved in the Notch signaling pathway after OPC transplantation. METHODS: OPCs were obtained from induced pluripotent stem cells (iPSCs) originating from mouse embryo fibroblasts (MEFs). After identification of iPSCs and iPSC-derived OPCs, A2B5+ OPCs were transplanted into the injured site of rats with SCC one week after SCC insult. Behavioral tests evaluated motor and sensory function 7 days after OPC transplantation. Real-time quantitative polymerase chain reaction (RT-qPCR) determined the expression of various cytokines related to the Notch signaling pathway after OPC transplantation. RESULTS: IPSC-derived OPCs were successfully generated from MEFs, as indicated by positive immunostaining of A2B5, PDGFα and NG2. Further differentiation of OPCs was identified by immunostaining of Olig2, Sox10, Nkx2.2, O4, MBP and GFAP. Importantly, myelin formation was significantly enhanced in the SCC+ OPC group and SCI-induced motor and sensory dysfunction was largely alleviated by A2B5+ OPC transplantation. Expression of factors involved in the Notch signaling pathway (Notch-1, Numb, SHARP1 and NEDD4) was significantly increased after OPC transplantation. CONCLUSIONS: A2B5+ OPC transplantation attenuates motor and sensory dysfunction in SCC rats by promoting myelin formation, which may be associated with change in expression of factors involved in the Notch signaling pathway.

Down-regulation of MIAT suppresses osteosarcoma progression by acting as a ceRNA for miR-141-3p to regulate SIX1-mediated PI3K/AKT pathway.

OBJECTIVE: Osteosarcoma (OS) is a frequent bone malignancy. Long non-coding RNA myocardial infarction associated transcript (MIAT) has been reported to be involved in the development of human cancers, including OS. However, the mechanism underlying MIAT in OS progression remains largely unclear. PATIENTS AND METHODS: The expression levels of MIAT and sineoculis homeobox homolog 1 (SIX1) in OS tissues and cells were detected by quantitative real-time polymerase chain reaction and Western blot. Cell viability, apoptosis, migration and invasion of OS cells were determined by MTT, flow cytometry and trans-well assays, respectively. The target interaction among MIAT, miR-141-3p and SIX1 was analyzed by bioinformatics analysis and luciferase reporter assay. Phosphatidylinositide 3-kinases (PI3K)/protein kinase B (AKT) pathway was evaluated by Western blot. RESULTS: MIAT and SIX1 expression levels were enhanced in OS tissues and cells. Knockdown of MIAT or SIX1 repressed cell viability, migration and invasion but promoted apoptosis in OS cells. Moreover, overexpression of SIX1 reversed the inhibitive role of MIAT silence in OS progression. Furthermore, MIAT could increase SIX1 expression by competitively sponging miR-141-3p. Besides, inhibition of MIAT blocked PI3K/AKT pathway by decreasing SIX1 in OS cells. CONCLUSIONS: MIAT silence suppresses OS progression through inactivating PI3K/AKT signaling by sponging miR-141-3p to regulate SIX1, indicating a novel target for the treatment of OS.

Type I and II collagen regulation of chondrogenic differentiation by mesenchymal progenitor cells.

Chondrogenic differentiation by mesenchymal progenitor cells (MPCs) is associated with cytokines such as transforming growth factor-beta 1 (TGF-beta1) and dexamethasone. Extracellular matrix (ECM) also regulates the differentiation by MPCs. To define whether ECM plays a functional role in regulation of the chondrogenic differentiation by MPCs, an in vitro model was used. That model exposed to dexamethasone, recombinant human TGF-beta1(rhTGF-beta1) and collagens. The results showed that MPCs incorporated with dexamethasone and rhTGF-beta1 increased proliferation and expression of glycosaminoglycan (GAG) after 14 days. Type II collagen enhanced the GAG synthesis, but did not increase alkaline phosphatase (ALP) activity. When adding dexamethasone and rhTGF-beta1 MPCs increased mRNA expression of Sox9. Incorporation with type II collagen, dexamethasone and rhTGF-beta1, MPCs induced mRNA expression of aggrecan and enhanced levels of type II collagen, and Sox9 mRNA. In contrast, incorporation with type I collagen, dexamethasone and rhTGF-beta1 MPCs reduced levels of aggrecan, and Sox9 mRNA, and showed no type II collagen mRNA. Altogether, these results indicate that type I and II collagen, in addition to the cytokine effect, may play a functional role in regulating of chondrogenic differentiation by MPCs.

Transdermal iontophoretic delivery of enoxacin from various liposome-encapsulated formulations.

The major purpose of this work was to study the effect of various liposome formulations on the iontophoretic transport of enoxacin through excised rat skin. The electrochemical stability of these liposomes was also evaluated. The encapsulation percentage of enoxacin was significantly enhanced after 6 h incubation in an electric field; whereas the fusion of liposomes was inhibited by application of electric current. The results of iontophoretic drug transport showed that the permeability of enoxacin released from liposomes was higher compared with that of free drug. The iontophoretic permeability of enoxacin released from liposomes increased with a decrease in the fatty acid chain length of the phospholipid, which may be due to the different phase transition temperatures of the phospholipids. Incorporation of charged phospholipid resulted in an alteration of the transdermal behavior of enoxacin: the iontophoretic permeation as well as the amount of enoxacin partitioned in skin was greatly reduced after incorporation of stearylamine in liposomes, which can be attributed to the competitive ion effect. The enoxacin released from stratum corneum-based liposomes showed the highest amount of enoxacin partitioned into skin depot. The results of employing cathodal iontophoresis on negative charged liposomes suggested that the liposomal vesicles or phospholipids may carry enoxacin into deeper skin strata via the follicular route.

Acrokeratosis paraneoplastica (Bazex syndrome) with adenocarcinoma of the colon: report of a case and review of the literature.

Acrokeratosis paraneoplastica is a rare disease and is uncommon even in patients with upper aerodigestive tract cancer. We report a 63-year-old man with a 1-month history of numerous pruritic lesions and vesicles on both feet. Although he had received local therapy, progressive dense scale formation involving both palms and both soles was found. Colonoscopy was performed because of hematochezia, and it revealed an early colon cancer. After the resection of the cancer, the skin lesions began to fall off dramatically. To the best of our knowledge, there is no report of acrokeratosis paraneoplastica associated with colon cancer in the literature. This is the first case report of acrokeratosis paraneoplastica associated with early colon cancer.

Exercise modality and selected coronary risk factors: a multivariate approach.

To evaluate group differences in coronary risk which could be attributed to the modality of habitual exercise, selected physiologic and lipid indices of coronary artery disease (CAD) were measured in 57 endurance trained (ET), strength trained (ST), or sedentary (SED) men (19 per group, aged 21 to 44 yr). Initial data reduction accomplished with principle component analysis identified three factors with eigenvalues greater than one. Orthogonal rotation of the preliminary solution demonstrated that low density lipoprotein cholesterol (LDL-C), percent body fat (%BF) and VO2max, and high density lipoprotein cholesterol (HDL-C) could be used to represent Factors 1, 2, and 3, respectively. The subsequent MANOVA using these variables proved significant. Post hoc analysis via simultaneous confidence intervals indicated that LDL-C group differences were not significant. Values for %BF and HCL-C in the ST group (14.0% and 1.17 mmol.l-1, respectively) were between but did not differ significantly from respective values in the ET (11.8% and 1.34 mmol.l-1) and SED (18.7% and 1.13 mmol.l-1) groups. However, %BF and HDL-C differences between the ET and SED groups were significant. The VO2max of the ET subjects (63.2 ml.kg-1.min-1) was significantly higher than that of either the ST or SED subjects (49.5 and 46.7 ml.kg-1.min-1, respectively). These results suggest that ET is the most effective modality of exercise for CAD risk reduction while benefits derived from ST are minimal.

Capsaicin and nonivamide as novel skin permeation enhancers for indomethacin.

The study was conducted in vitro to investigate the changes of indomethacin transdermal permeation pretreated by capsaicin and nonivamide, two compounds chemically similar to Azone. The combined effect of low frequency ultrasound (20 kHz) and enhancers on the indomethacin permeation was also evaluated. The experimental data demonstrated that capsaicin and nonivamide significantly enhanced the flux of indomethacin across nude mouse skin. Enhancement effects of both analogues were very similar and depended predominantly on the concentration tested. Histological examination coupled with visual scores indicated the safety of capsaicin and nonivamide on skin structure. Simultaneous application of ultrasound and enhancers significantly increased skin permeation of indomethacin compared with either ultrasound or enhancers alone. Better effect was obtained by the combination with capsaicin than nonivamide.

Transdermal iontophoretic delivery of diclofenac sodium from various polymer formulations: in vitro and in vivo studies.

The objective of this study was to evaluate the in vitro and in vivo transdermal iontophoresis of various diclofenac sodium polymer formulations. The excised rat skin, human skin as well as cellulose membrane were used to examine the in vitro drug permeation whereas the microdialysis technique was used to monitor the drug concentration in vivo. Polymer solutions based on polyvinylpyrrolidone (PVP) and hydroxypropyl methylcellulose (HPMC) binary system showed higher drug permeability than that of single polymer vehicle. The effect of formulations on drug permeation through cellulose membrane was quite different from those through rat skin and human skin, which can be explained by the different permeation pathways between them. It appeared to be a membrane-controlled mechanism but not the vehicle matrix-controlled mechanism for diclofenac hydrogels when using skin as the diffusion barrier. The recovery of diclofenac sodium in the in vivo microdialysis was approximately 80-90%, indicating this technique can be used in the intradermal drug monitoring. For all the polymer formulations tested, there was a good relationship between the in vitro and in vivo drug permeation. A synergistic effect on drug permeation was observed when transdermal iontophoresis combined with the pretreatment of cardamom oil as a permeation enhancer.

MRI and histology of collagen template disc implantation and regeneration in rabbit temporomandibular joint: preliminary report.

INTRODUCTION: We aimed to evaluate regeneration of injured temporomandibular joint (TMJ) discs following reconstituted collagen template implantation in rabbits using contrast-enhanced magnetic resonance imaging (MRI) and to correlate these findings with histology. METHODS: Twenty-four adult rabbits were divided into five groups: group A, partial discectomy without implantation (n = 6); group B, partial discectomy with collagen template implantation (n = 6); group C, partial discectomy with subdermal graft implantation (n = 6); group D, sham operation (n = 4); and group E, control (n = 2). All rabbits received baseline MRI scans before surgery and follow-up MRI studies at 3 months after surgery. All rabbits were sacrificed for histologic analysis after the follow-up MRI. RESULTS: In group A, follow-up MRI showed marked joint effusion in all six injured TMJs, which was accompanied by bony erosion at the tympanic fossa and mandibular condyle. In group B, MRI showed a homogenous low signal intensity in five of six discs, suggestive of regeneration. One disc showed higher signal intensity at its lateral portion than that of the original disc, indicating partial regeneration. MRI of group C depicted a low signal intensity, bandlike regenerative structure in four of the six discs. One disc with partial regeneration demonstrated relatively high signal intensity. The disc in the sixth animal of this group showed no evidence of regeneration. All of the MRI findings were in agreement with the histologic findings. CONCLUSION: TMJ discs can regenerate following implantation of a reconstituted collagen template in discectomied rabbits. Contrast-enhanced MRI can be used to monitor and determine the degree of disc regeneration.

Surgical and pathologic observations of epididymal tubules during microscopic epididymal sperm aspiration for intracytoplasmic sperm injection.

Microscopic epididymal sperm aspiration (MESA) for sperm retrieval and intracytoplasmic sperm injection (ICSI) is currently our routine treatment for selected male patients with obstructive azoospermia. In order to refine the surgical technique and obtain better quality sperm for our assisted reproductive technology program, we observed the epididymal tubules in 40 sessions of surgical exploration of the epididymis for sperm aspiration. Epididymal tubules with long-term obstruction could be divided into three groups on the basis of clinical observations and pathology findings: markedly dilated, mildly dilated, and nondilated. All of the markedly dilated epididymal tubules (grade III, n = 10) were azoospermic and ICSI could not be done. Epididymal sperm obtained from the mildly dilated tubules (grade II, n = 9) resulted in poorer fertilization (49%) and pregnancy (33%) rates than sperm obtained from nondilated epididymal tubules (grade I, n = 21, fertilization rate 72%, pregnancy rate 57%). These findings demonstrate that nondilated epididymal tubules are best for sperm retrieval and successful ICSI. We believe this observation will be a good surgical parameter for urologic surgeons performing MESA.