A global phylogenomic analysis of the shiitake genus Lentinula.

Lentinula is a broadly distributed group of fungi that contains the cultivated shiitake mushroom, L. edodes. We sequenced 24 genomes representing eight described species and several unnamed lineages of Lentinula from 15 countries on four continents. Lentinula comprises four major clades that arose in the Oligocene, three in the Americas and one in Asia-Australasia. To expand sampling of shiitake mushrooms, we assembled 60 genomes of L. edodes from China that were previously published as raw Illumina reads and added them to our dataset. Lentinula edodes sensu lato (s. lat.) contains three lineages that may warrant recognition as species, one including a single isolate from Nepal that is the sister group to the rest of L. edodes s. lat., a second with 20 cultivars and 12 wild isolates from China, Japan, Korea, and the Russian Far East, and a third with 28 wild isolates from China, Thailand, and Vietnam. Two additional lineages in China have arisen by hybridization among the second and third groups. Genes encoding cysteine sulfoxide lyase (lecsl) and γ-glutamyl transpeptidase (leggt), which are implicated in biosynthesis of the organosulfur flavor compound lenthionine, have diversified in Lentinula. Paralogs of both genes that are unique to Lentinula (lecsl 3 and leggt 5b) are coordinately up-regulated in fruiting bodies of L. edodes. The pangenome of L. edodes s. lat. contains 20,308 groups of orthologous genes, but only 6,438 orthogroups (32%) are shared among all strains, whereas 3,444 orthogroups (17%) are found only in wild populations, which should be targeted for conservation.

Rice apoplastic CBM1-interacting protein counters blast pathogen invasion by binding conserved carbohydrate binding module 1 motif of fungal proteins.

When infecting plants, fungal pathogens secrete cell wall-degrading enzymes (CWDEs) that break down cellulose and hemicellulose, the primary components of plant cell walls. Some fungal CWDEs contain a unique domain, named the carbohydrate binding module (CBM), that facilitates their access to polysaccharides. However, little is known about how plants counteract pathogen degradation of their cell walls. Here, we show that the rice cysteine-rich repeat secretion protein OsRMC binds to and inhibits xylanase MoCel10A of the blast fungus pathogen Magnaporthe oryzae, interfering with its access to the rice cell wall and degradation of rice xylan. We found binding of OsRMC to various CBM1-containing enzymes, suggesting that it has a general role in inhibiting the action of CBM1. OsRMC is localized to the apoplast, and its expression is strongly induced in leaves infected with M. oryzae. Remarkably, knockdown and overexpression of OsRMC reduced and enhanced rice defense against M. oryzae, respectively, demonstrating that inhibition of CBM1-containing fungal enzymes by OsRMC is crucial for rice defense. We also identified additional CBM-interacting proteins (CBMIPs) from Arabidopsis thaliana and Setaria italica, indicating that a wide range of plants counteract pathogens through this mechanism.

Mpn491, a secreted nuclease of Mycoplasma pneumoniae, plays a critical role in evading killing by neutrophil extracellular traps.

Neutrophils play an important role in antimicrobial defense as the first line of innate immune system. Recently, the release of neutrophil extracellular traps (NETs) has been identified as a killing mechanism of neutrophils against invading microbes. Mycoplasma pneumoniae, a causative agent of respiratory infection, has been shown to be resistant to in vitro killing by neutrophils, suggesting that the bacterium might circumvent bactericidal activity of NETs. In this study, we investigated whether M. pneumoniae possesses resistance mechanisms against the NETs-mediated killing of neutrophils and found that the bacterium degrades the NETs induced upon M. pneumoniae infection. The NETs-degrading ability of M. pneumoniae required the production of a secreted nuclease, Mpn491, capable of using Mg2+ as a cofactor for its hydrolytic activity. Moreover, the inactivation of the nuclease resulted in increased susceptibility of M. pneumoniae to the NETs-mediated killing of neutrophils. The results suggest that M. pneumoniae employs Mpn491 as a means for evading the killing mechanism of neutrophils.

Lentinula edodes Genome Survey and Postharvest Transcriptome Analysis.

Lentinula edodes is a popular, cultivated edible and medicinal mushroom. Lentinula edodes is susceptible to postharvest problems, such as gill browning, fruiting body softening, and lentinan degradation. We constructed a de novo assembly draft genome sequence and performed gene prediction for Lentinula edodesDe novo assembly was carried out using short reads from paired-end and mate-paired libraries and by using long reads by PacBio, resulting in a contig number of 1,951 and an N50 of 1 Mb. Furthermore, we predicted genes by Augustus using transcriptome sequencing (RNA-seq) data from the whole life cycle of Lentinula edodes, resulting in 12,959 predicted genes. This analysis revealed that Lentinula edodes lacks lignin peroxidase. To reveal genes involved in the loss of quality of Lentinula edodes postharvest fruiting bodies, transcriptome analysis was carried out using serial analysis of gene expression (SuperSAGE). This analysis revealed that many cell wall-related enzymes are upregulated after harvest, such as ß-1,3-1,6-glucan-degrading enzymes in glycoside hydrolase (GH) families GH5, GH16, GH30, GH55, and GH128, and thaumatin-like proteins. In addition, we found that several chitin-related genes are upregulated, such as putative chitinases in GH family 18, exochitinases in GH20, and a putative chitosanase in GH family 75. The results suggest that cell wall-degrading enzymes synergistically cooperate for rapid fruiting body autolysis. Many putative transcription factor genes were upregulated postharvest, such as genes containing high-mobility-group (HMG) domains and zinc finger domains. Several cell death-related proteins were also upregulated postharvest.IMPORTANCE Our data collectively suggest that there is a rapid fruiting body autolysis system in Lentinula edodes The genes for the loss of postharvest quality newly found in this research will be targets for the future breeding of strains that keep fresh longer than present strains. De novoLentinula edodes genome assembly data will be used for the construction of a complete Lentinula edodes chromosome map for future breeding.

The exp2 gene, which encodes a protein with two zinc finger domains, regulates cap expansion and autolysis in Coprinopsis cinerea.

Cap expansion in agaricoid mushroom species is an important event for sexual reproduction because meiosis occurs in basidia under the cap, and basidiospores can be released by opening the cap. However, molecular mechanisms underlying cap expansion in basidiomycetes remain poorly understood. We aimed to elucidate the molecular mechanisms of cap expansion in basidiomycetes by analyzing the unique cap-expansionless UV mutant #13 (exp2-1) in Coprinopsis cinerea. Linkage analysis and consequent genome sequence analysis revealed that the gene responsible for the mutant phenotypes encodes a putative transcription factor with two C2H2 zinc finger motifs. The mutant that was genome-edited to lack exp2 exhibited an expansionless phenotype. Some of the genes encoding cell wall degradation-related enzymes showed decreased expression during cap expansion and autolysis in the exp2 UV and genome-edited mutant. The exp2 gene is widely conserved in Agaricomycetes, suggesting that Exp2 homologs regulate fruiting body maturation in Agaricomycetes, especially cap expansion in Agaricoid-type mushroom-forming fungi. Therefore, exp2 homologs could be a target for mushroom breeding to maintain shape after harvest for some cultivating mushrooms, presenting a promising avenue for further research in breeding techniques.

The Coprinopsis cinerea septin Cc.Cdc3 is involved in stipe cell elongation.

We have identified and characterized a Coprinopsis cinerea mutant defective in stipe elongation during fruiting body development. In the wild-type, stipe cells elongate at the maturation stage of fruiting, resulting in very slender cells. In the mutant, the stipe cells fail to elongate, but become rather globular at the maturation stage. We found that the mutant phenotype is rescued by a gene encoding a homolog of Saccharomyces cerevisiae CDC3 septin, Cc.Cdc3. The C. cinerea genome includes 6 septin genes, 5 of which, including Cc.cdc3, are highly transcribed during stipe elongation in the wild type. In the mutant, the level of Cc.cdc3 transcription in the stipe cells remains the same as that in the mycelium, and the level of Cc.cdc10 transcription is approximately 100 times lower than that in the wild-type stipe cells. No increase in transcription of Cc.cdc3 in the mutant may be due to the fact that the Cc.cdc3 gene has a 4-base pair insertion in its promoter and/or that the promoter region is methylated in the mutant. Overexpressed EGFP-Cc.Cdc3 fusion protein rescues the stipe elongation in the transformants, localizes to the cell cortex and assembles into abundant thin filaments in the elongating stipe cells. In contrast, in vegetative hyphae, EGFP-Cc.Cdc3 is localized to the hyphal tips of the apical cells of hyphae. Cellular defects in the mutant, combined with the localization of EGFP-Cc.Cdc3, suggest that septin filaments in the cell cortex provide the localized rigidity to the plasma membrane and allow cells to elongate cylindrically.

Successful cultivation of black morel, Morchella sp. in Japan.

True morels (Morchella spp.) are economically important edible fungi cultivated mainly in China. Japan is one of the top importers of morels, but there are no reports on the distribution of major cultivated species. To investigate the possibility of black morel cultivation in Japan, phylogenetic analysis, mating-type analysis, and field cultivation tests were conducted using domestically collected strains. A total of 172 strains were isolated from the spores of wild ascomata collected from 15 locations. Mating-type analysis for 118 strains revealed 28 strains had only MAT1-1-1, 40 strains had only MAT1-2-1, and 48 strains had both MAT genes. Seven strains were inoculated in March 2020 at the field cultivation test site. Mycelial growth and conidial layer formation were observed within a month. Ascomata were observed in April 2021 for one of the tested strains. Phylogenetic analysis revealed that both the observed ascomata and fruited strains were Morchella sp. Mel-21, which is one of the cultivated species in China. Moreover, no antagonism was observed in the somatic incompatibility test between strains isolated from observed ascomata and spawn strain. These results suggest that the ascomata originated in the inoculated spawn, a finding that will contribute to commercial morel cultivation in Japan.

Quantification of Epstein-Barr virus DNA is helpful for evaluation of chronic active Epstein-Barr virus infection.

Chronic active Epstein-Barr virus infection (CAEBV) presents with chronic or recurrent infectious mononucleosis-like symptoms, such as low-grade fever, liver dysfunction, lymphadenopathy, and hepatosplenomegaly. Immunological methods are useful for the diagnosis of viral infections. However, CAEBV patients do not necessarily have high titers of Epstein-Barr virus (EBV)-specific antibodies. Hosts that are immunocompromised after hematopoietic stem cell transplantations sometimes suffer from systemic EBV-associated hemophagocytic lymphohistiocytosis (EBV-HLH) and EBV-positive lymphoma. Patients with EBV-associated diseases are often diagnosed by analyses of bone marrow. Cytomegalovirus (CMV) can cause serious pneumonia or retinitis in immunocompromised hosts. In order to noninvasively understand the clinical status of patients with EBV-associated diseases, we conducted real-time polymerase chain reaction (PCR) methods in their peripheral blood in order to quantify EBV and CMV DNA levels, which reflect viral activity. Here, we describe a 30-year-old Japanese female patient with CAEBV. The patient had repeated fever, fatigue, and liver dysfunction. The histopathological results of liver biopsies were positive for EBV-encoded RNA-1. Acute hepatitis was associated with the EBV infection. The whole-blood EBV DNA levels were high and above 1.0 × 107 copies/mL. After immunosuppressive and antiviral therapies, EBV DNA levels lowered. However, she had to receive bone marrow transplantation because of her EBV-HLH. As the number of lymphocytes increased in the post-transplantation period, EBV DNA levels gradually increased again. The simultaneous detection of CMV DNA was more sensitive than the CMV antigenemia test that is often used to diagnose CMV infections. Unfortunately, the patient died due to a fungal infection. Observing EBV DNA levels closely with real-time quantitative PCR methods is helpful for evaluating the changes in the clinical course.

Identification of upregulated genes in Tricholoma matsutake mycorrhiza.

Many plant roots associate with fungi to form mycorrhizae; tree roots especially associate with ectomycorrhizal fungi, such as Tricholoma species. Tricholoma matsutake is an economically important fungus in Asian countries and usually inhabits forests primarily composed of Pinus densiflora (Japanese red pine). In this study, to understand the mycorrhizal association between T. matsutake and P. densiflora, genes specifically expressed in mycorrhiza compared with those expressed in mycelia and fruiting bodies were identified by RNA-seq. This revealed that genes for chromatin, proteasomes, signal transduction, pheromones, cell surface receptors, cytoskeleton, RNA processing and transporters from T. matsutake were highly expressed in mycorrhiza. It also identified 35 mycorrhiza-induced small secreted proteins (MiSSPs) that were highly expressed in mycorrhiza. Meanwhile, genes for proteases, defence-related proteins, cell-wall degradation, signal transduction, pinene synthesis, plant hormones and transporters from P. densiflora were highly expressed in mycorrhiza. These genes may be involved in mycorrhizal formation and maintenance. A MiSSP, 1460819, was highly expressed in mycorrhiza, and this expression was maintained for 24 months. These results provide insight into the mycorrhizal association between T. matsutake and P. densiflora.

Endo-ß-1,3-glucanase GLU1, from the fruiting body of Lentinula edodes, belongs to a new glycoside hydrolase family.

The cell wall of the fruiting body of the mushroom Lentinula edodes is degraded after harvesting by enzymes such as ß-1,3-glucanase. In this study, a novel endo-type ß-1,3-glucanase, GLU1, was purified from L. edodes fruiting bodies after harvesting. The gene encoding it, glu1, was isolated by rapid amplification of cDNA ends (RACE)-PCR using primers designed from the N-terminal amino acid sequence of GLU1. The putative amino acid sequence of the mature protein contained 247 amino acid residues with a molecular mass of 26 kDa and a pI of 3.87, and recombinant GLU1 expressed in Pichia pastoris exhibited ß-1,3-glucanase activity. GLU1 catalyzed depolymerization of glucans composed of ß-1,3-linked main chains, and reaction product analysis by thin-layer chromatography (TLC) clearly indicated that the enzyme had an endolytic mode. However, the amino acid sequence of GLU1 showed no significant similarity to known glycoside hydrolases. GLU1 has similarity to several hypothetical proteins in fungi, and GLU1 and highly similar proteins should be classified as a novel glycoside hydrolase family (GH128).

An endo-ß-1,6-glucanase involved in Lentinula edodes fruiting body autolysis.

A ß-1,6-glucanase, LePus30A, was purified and cloned from fruiting bodies of the basidiomycete Lentinula edodes. ß-1,6-glucanases degrade ß-1,6-glucan polysaccharides, a unique and essential component of fungal cell walls. The complementary DNA of LePus30A includes an open reading frame of 1,575 bp encoding an 18 amino acid signal peptide and the 506 amino acid mature protein. Sequence analysis indicated that LePus30A is a member of glycoside hydrolase family 30, and highly similar genes are broadly conserved among basidiomycetes. The purified LePus30A catalyzed depolymerization of ß-1,6-glucan endolytically and was highly specific toward ß-1,6-glucan polysaccharide. It is known that the cell walls of fruiting bodies of basidiomycetes are autodegraded after harvesting by means of enzymatic hydrolysis. The transcript level of LePus30A gene (lepus30a) was significantly increased in fruiting bodies after harvesting. Moreover, LePus30A showed hydrolyzing activity against the cell wall components of L. edodes fruiting bodies. These results suggest that LePus30A is responsible for the degradation of the cell wall components during fruiting body autolysis after harvest.

Monitoring twenty-six chronic myeloid leukemia patients by BCR-ABL mRNA level in bone marrow:a single hospital experience.

Chronic myeloid leukemia (CML) is caused by the BCR-ABL oncogene. The Philadelphia chromosome (Ph) from a reciprocal translocation, t(9;22) (q34;q11) causes a fusion gene, BCR-ABL, that encodes a constitutively active tyrosine kinase. Treatment of CML by imatinib is effective to control the tyrosyl phosphorylation of the protein related to the cell signaling. BCR-ABL mRNA is overexpressed in the minimal residual disease (MRD), known as an early sign of relapse. Between December 2005 and June 2008, we measured BCR-ABL mRNA levels in the bone marrow (BM) from patients by quantitative real-time polymerase chain reaction (RQ-PCR) in Aomori Prefectural Central Hospital. Eighty-six samples from 26 patients were collected. Among the 26 CML patients, 11 patients (42%) were in the pretreatment group. Seven (64%) of the 11 patients achieved complete molecular response (CMR). In the post-treatment group consisting of the remaining 15 patients, 9 (60%) patients achieved CMR. The patients receiving imatinib at a dose over 300 mg per day required 13 (6-77) months [median (range)] to achieve CMR. On the other hand, the patients receiving a dose below 300 mg per day required 29.5 (11-84) months [median (range)]. When BCR-ABL mRNA was detected during the treatment course of patients with CMR, careful observation of BCR-ABL mRNA was useful for tracking the clinical course of patients. In conclusion, the BCR-ABL mRNA level was useful for monitoring the clinical course in 26 patients with CML.

[SURGEON QUESTIONNAIRE FOR ESTABLISHING A SURGEON EDUCATION SYSTEM FOR TRANSURETHRAL LASER RESECTION OF THE PROSTATE (HoLEP)].

(Introduction)HoLEP's role in the surgical management of benign prostatic hyperplasia (BPH) is steadily growing. In this study, a questionnaire containing questions about perioperative management was submitted to HoLEP surgeons to help establish standard surgical training procedures. (Methods)We sent a comprehensive 17 questionnaires on HoLEP procedures to 18 surgeons. The questionnaire asked, "Which method are you using, the 1-LOBE or 3-LOBE method?", "What educational methods are being used for surgeons?", "How long is the catheter insertion period after HoLEP?", and "What is the most difficult problem encountered in surgical HoLEP education and what aspect of training is the most emphasized?" (Results)Sixteen (88.9%) surgeons answered these questionnaires. Five surgeons reported using the one lobe method, five surgeons reported using the three lobe method, and four surgeons answered that it depends on the case. Regarding educational methods, the main answer was that it is important to evaluate pre-HoLEP imaging tests such as MRI and cystoscopy and to simulate surgery for education. Regarding the postoperative catheter insertion period, 1 day: 1 surgeon, 2 days: 9 surgeons, 3 days: 3 surgeons, 4 days or more: 1 surgeon. The most important thing reported for surgical education was to help beginners understand the characteristics of lasers, including direction, distance to prostate tissue, and adenoma removal. (Conclusions)The surgeons' responses clearly indicated some differences in practices between institutions. More detailed data from these results will provide a step towards designing standardized surgical and educational protocols for HoLEP.

The inhibitory effects of mushroom extracts on sucrose-dependent oral biofilm formation.

Mushrooms contain large quantities of alpha-glucans. Shiitake (Lentinula edodes), Japan's most popular edible mushroom, has been reported to contain about 6% (weight/dried weight) of alpha-(1,3)-glucan. This glucan is one of the major components of oral biofilm formed by the cariogenic bacteria Streptococcus mutans and Streptococcus sobrinus. We found that extracts from shiitake and other edible mushrooms could reduce preformed biofilms of S. mutans and S. sobrinus in the presence of dextranase. We also investigated the alpha-glucanase activities of shiitake mushroom extracts and their effects on biofilm formation. The extracts possessed alpha-glucanase activity and degraded water-insoluble glucans from mutans streptococci. The extracts strongly inhibited the sucrose-dependent formation of biofilms by S. mutans and S. sobrinus in the presence of dextranase. Our results suggest that some components of mushrooms, including alpha-glucanases, might inhibit the sucrose-induced formation of oral biofilms.

Characterization of the post-harvest changes in gene transcription in the gill of the Lentinula edodes fruiting body.

We compared the gene expression patterns of Lentinula edodes fresh fruiting bodies and fruiting bodies 3 days after harvest, by suppression subtractive hybridization, to characterize the physiologic changes that occur after harvest, such as gill browning and cell wall lysis of the fruiting body, which are responsible for the loss of food quality and value. We found increase of transcription levels of several enzyme encoding genes, such as, two phenol oxidases encoding genes (tyr tyrosinase, lcc4 laccase), and several cell wall degradation-related enzyme-encoding genes, such as mixed-linked glucanase (mlg1), chitinases (chi1, chi2), chitin deacetylase (chd1), and chitosanase (cho1), after harvesting. We isolated a putative transcription factor-encoding gene (L. edodes exp1) with high similarity to exp1 from Coprinopsis cinerea, which is involved in autolysis of the cap during spore diffusion. Transcription of L. edodes exp1 increased post-harvest, which suggests that its target genes are up-regulated after harvesting. These enzymes and the transcription factor may be involved in L. edodes fruiting body senescence.

The basidiomycetous mushroom Lentinula edodes white collar-2 homolog PHRB, a partner of putative blue-light photoreceptor PHRA, binds to a specific site in the promoter region of the L. edodes tyrosinase gene.

We isolated a cDNA homolog of Neurospora crassa wc-2 from the basidiomycetous mushroom Lentinula edodes and termed it phrB cDNA. The deduced PHRB (313 amino acid residues) contained a PAS domain and a zinc-finger motif. Random binding-site selection analysis of the PHRB produced in Escherichia coli revealed that it bound to a 7-bp sequence with the consensus sequence 5'GATA/TTG/T/AC3'. Electrophoretic mobility-shift assay showed that it also bound to the consensus sequence 5'GATATTC3' in the promoter region of the L. edodes tyrosinase gene (Le.tyr). In vitro GST-pulldown immunoblot analysis disclosed that PHRB interacts with a putative blue-light photoreceptor of L. edodes (PHRA), the homolog of N. crassa WC-1, through the PAS B- and/or PAS C domain of PHRA. The expression of phrB and Le.tyr genes in pre-primordial mycelia of L. edodes is induced by light exposure, suggesting that PHRB can regulate the expression of the Le.tyr gene in a light-dependent manner.

The tyrosinase-encoding gene of Lentinula edodes, Letyr, is abundantly expressed in the gills of the fruit-body during post-harvest preservation.

The gill browning of Lentinula edodes fruit-bodies during preservation is thought to be due to melanin biosynthesis catalyzed by tyrosinase. We isolated a genomic DNA sequence and cDNA encoding a putative tyrosinase from the white rot basidiomycete Lentinula edodes (shiitake mushroom). The gene, named Letyr, consists of a 1,854-bp open reading frame interrupted by eight introns, and encodes a putative protein of 618 amino acid residues with an estimated molecular mass of 68 kDa. Amino acid residues known to be involved in copper-binding domains were conserved in the deduced amino acid residues of LeTyr. Transcriptional and translational expression of Letyr in the gills of the fruit-body increased during preservation after harvest. This correlation between Letyr expression and fruit-body preservation suggests that tyrosinase gene expression contributes to gill browning.

WT1 mRNA level in peripheral blood is a sensitive biomarker for monitoring minimal residual disease in acute myeloid leukemia.

The Wilms' tumor gene 1 (WT1) encodes a transcription factor that is involved in normal cellular development and cell survival. WT1 mRNA is overexpressed in the minimal residual disease (MRD) of patients with hematopoietic malignancy patients, particularly acute myeloid leukemia (AML). MRD represents the condition with the low levels of leukemia cells in the bone marrow and is known as a sign of recurrence. In hematopoietic malignancies, definition of remission is based on the lack of MRD at submicroscopic level. Between December 2005 and June 2008, we started to measure WT1 mRNA levels in the peripheral blood (PB) from patients by quantitative real-time PCR in Aomori Prefectural Central Hospital. Three hundreds and eight samples from 95 patients were evaluated. The patients included AML (55 patients), acute lymphoblastic leukemia (11), myelodysplastic syndrome (20), malignant lymphoma (5), chronic myeloid leukemia (1), prostatic carcinoma (1), and leukopenia (2). Among the 55 AML patients, 21 patients were pretreated with remission induction therapy. In the clinical course of 21 patients, timely therapeutic approaches could be started for relapse by the early detection of WT1 mRNA overexpression before the morphological findings were apparent. Monitoring WT1 mRNA is helpful to identify patients at high-risk relapse. High overall survival rate (71.2%, 15/21, median: 24.6 months, range 1.1-35.6 months) was achieved in 3 years. The overall survival rate of 34 post-treatment patients was 61.7% (median: 23.5 months, range 0.13-126.5 months after treatment start). In conclusion, the WT1 mRNA level is a sensitive biomarker for monitoring MRD.

Effect of varicocelectomy on patients with unobstructive azoospermia and severe oligospermia.

OBJECTIVES: To assess the treatment outcome and benefits after varicocelectomy in men with unobstructive azoospermia (UOA) and severe oligospermia (SO). PATIENTS AND METHODS: The records were retrospectively evaluated for six patients with UOA and 54 with SO who had microsurgical inguinal varicocele ligation (mean age 33.3 years, SD 5.3). Fifty-one (85%) had a unilateral left-sided procedure (five of six with UOA, 46 of 54 with SO), and nine (15%) had a bilateral procedure. RESULTS: After ligation, spermatogenesis was induced in two of the six men with UOA; the mean (SD) sperm concentration increased from 2.3 (1.6) to 9.2 (12.6) million/mL (P < 0.001), but the sperm motility did not change significantly. Four (7%) of the 54 men with SO achieved paternity, with unassisted pregnancies. CONCLUSIONS: Varicocelectomy resulted in the induction or enhancement of spermatogenesis for several men with UOA or SO; it seems to be a reasonable option in selected patients with UOA or SO.

The assessment of oxidative stress in infertile patients with varicocele.

OBJECTIVES: To assess oxidative stress markers, antioxidant capacity and cytokines in seminal plasma from infertile patients with varicocele, and to investigate seminal oxidative status and sperm DNA damage after varicocelectomy. PATIENTS, SUBJECTS AND METHODS: The records were retrospectively evaluated for 28 azoospermic, 30 oligospermic (15 with varicocele and 15 without) and 30 patients with normal semen characteristics (15 with varicocele and 15 without). The mean (sd) age of the men was 32.4 (5.6) years; all men with varicocele had a unilateral or bilateral microsurgical subinguinal varicocelectomy. The level of nitric oxide (NO), 8-hydroxy-2'-deoxyguanosine (8-OHdG), hexanoyl-lysine (HEL), superoxide dismutase (SOD) activity, interleukin (IL)-6, IL-8 and tumour necrosis factor-alpha in seminal plasma were measured. In addition, sperm DNA fragmentation was analysed before and 6 months after varicocelectomy. RESULTS: Azoospermic and oligospermic patients had a significantly higher HEL concentration and SOD activity in seminal plasma; those with varicocele had a significantly higher NO, HEL, and SOD activity in seminal plasma. There was a significant increase in sperm concentration and reduction in NO, HEL, 8-OHdG level and SOD activity after varicocelectomy. Oligospermic patients with varicocele had a significantly higher IL-6 level in seminal plasma, and there was a significant reduction after varicocelectomy. The percentage of apoptosis-positive sperm decreased significantly after varicocelectomy. CONCLUSIONS: This study indicates that the seminal plasma of patients with varicocele is under excessive oxidative stress, and partly even in patients with normospermia, and that varicocelectomy reduces oxidative stress in seminal plasma and ameliorates sperm DNA damage.