First Report of Neofusicoccum ribis Causing Cankers and Dieback Diseases on Apricot Trees in Canada and Worldwide.

Apricot trees (Prunus armeniaca L.) with cankers, gummosi and dieback symptoms were observed in a commercial orchard in Niagara-on-the-Lake, Ontario, Canada. In October 2018, up to 44.9% disease incidence (n = 318) was observed on 2-year-old 'Harostar™' trees grafted onto 'Haggith' rootstocks. Fungal colonies were consistently isolated and purified from small sections of wood collected from canker margins of symptomatic trunk and shoot tissue, as described by Ilyukhin et al. (2023). Purified mycelial isolates sharing similar morphological characteristics were categorized into five distinct morphotypes. One representative isolate from each morphotype was used to inoculate excised apricot shoots as described by Ilyukhin and Ellouze (2023). One morphotype displayed necrotic lesions on the shoots consistently yielded abundant white aerial mycelium that turned grey-brown on PDA after 7 days (Figure S1) and produced black pycnidia three weeks following incubtion at 22°C in the dark. Conidia were hyaline, one-celled, fusiform, with dimensions of 19.7 - 24.2 × 3.6 - 4.8 µm (average 22.1 × 4.3 µm, n = 30), the typical morphology of a Neofusicoccum sp. (Crous et al. 2006). Species identification was verified by extracting genomic DNA of the representative isolate M1-105, amplifying and sequencing the internal transcribed spacer (ITS), translation elongation factor 1-α (EF1-α) and ß-tubulin (TUB2) gene regions with primers ITS1/ITS4, EF1-728F/EF1-986R and Bt2a/Bt2b. Nucleotide sequences (GenBank Accession No. ITS: OK287034; EF1-α: OK346636; TUB2: OK346633) have 100%, 99.61% and 99.55% identity with Neofusicoccum ribis isolates from different hosts and countries (MT587514, DQ235142, OL455952, respectively). Randomized accelerated maximum likelihood analysis (Stamatakis et al. 2008), using ITS, EF1-α and TUB2 sequence data, clustered M1-105 with the highest bootstrap support values with the N. ribis ex-epitype CBS 115475 (Figure S2). A living culture of M1-105 was deposited in the Canadian Collection of Fungal Cultures (DAOMC 252247). Pathogenicity was verified using 5 potted healthy 1-year-old 'Haroblush™' apricot cultivar grafted onto 'Krymsk® 86' rootstocks. Trunks and shoots were inoculated in a shallow wound made by a scalpel with mycelial plugs from a 5-day-old culture of M1-105. Five control trees were inoculated with sterile plugs. Trees were put in an open-air area and watered as needed. Canker symptoms appeared 7 days after inoculation, and spread above and below the inoculation point. Fifteen days post-inoculation, the upper portion of inoculated shoots showed necrosis, gummosis and wilt (Fig. S1). Neofusicoccum ribis was re-isolated from all infected trees and species identity was confirmed by sequencing as described above. Controls remained symptom-free and no fungi were isolated from the wood. Therefore, Koch's postulates were completed. Neofusicoccum ribis was reported to cause branch dieback of olive trees in Spain (Romero et al. 2005) and pistachio in Italy (Corazza et al. 1986), stem blight and dieback of blueberry in Michigan (Heger et al. 2023) and Florida (Wright and Harmon 2010) and postharvest decay of apple fruit from cold storage in Pennsylvania (Jurick et al. 2013). To the best of our knowledge, this is the first report of N. ribis causing canker and shoot dieback of apricot trees in Canada and worldwide. This report reveals N. ribis as a potential threat, causing canker and dieback. Without proper management, it could lead to significant losses in apricot orchards and the stone fruit industry. This study paves the way for crucial research on N. ribis outbreaks and effective disease control.

First Report of Diplodia intermedia Causing Canker and Dieback Diseases on Apple Trees in Canada.

A dieback of apple trees (Malus domestica (Suckow) Borkh.) associated with cankers was observed in commercial orchards in southwestern Ontario, Canada, in 2019. Fifteen 2 to 10-year-old symptomatic trees were collected from three orchards exhibiting up to 37% disease incidence. Small sections of diseased wood (1 cm long) were surface sterilized with 70% ethanol for 30 sec and 1% NaClO for 20 min, rinsed thrice in sterile water, placed on 2% PDA (Difco) amended with kanamycin (50 mg liter-1), and incubated at 22°C for 5 days in the dark (Ilyukhin et al. 2023). Fungal colonies that were consistently isolated were hyphal-tipped, transferred to individual PDA plates and incubated at 22°C for 7 days in the dark. Purified isolates with same characteristics were classed into morphotypes. One morphotype was initially white and turned dark olivaceous with dense aerial mycelium. Pycnidia were produced on pine needles on PDA (Fig. S2) after incubation at 22°C for 17 days in the dark. Conidia were brown, aseptate, ovoid, and measured 27.9 to 31.3 µm x 12.1 to 14.2 µm (mean ± S.D. of 15 conidia = 29.9 ± 0.9 µm × 13.2 ± 0.6 µm), the typical morphology of a Diplodia sp. (Phillips et al. 2012). Genomic DNA was extracted from a 7-day-old culture of a representative isolate M45-28, using the Plant/Fungi DNA Isolation Kit (Norgen Biotech, Canada). The internal transcribed spacer (ITS), translation elongation factor 1-α (EF1-α) and ß-tubulin gene regions were amplified and sequenced with primers ITS1/ITS4, EF1-728F/EF1-986R and Bt2a/Bt2b and deposited in GenBank with accession numbers MZ970605, MZ995430 and MZ995431, respectively. Based on the sequence, the fungus was identified as Diplodia intermedia A.J.L. Phillips et al. and matched isolates from different hosts and countries (ITS: 100%, MG220378; EF1-α: 100%, MG220385; ß-tubulin: 99.24%, MT592502). The maximum likelihood-based phylogenetic analysis of ITS, EF1-α and ß-tubulin concatenated sequences was performed using IQ-Tree 2.2.2.7 (Minh et al. 2020). M45-28 was clustered with high bootstrap support values with D. intermedia isolates from the Westerdijk Fungal Biodiversity Institute collection, including the ex-holotype (CBS 124462) (Fig. S1). A living culture of M45-28 was deposited in the Canadian Collection of Fungal Cultures (DAOMC 252253). Pathogenicity assay was conducted by inoculating mycelial plugs from a 7-day-old culture of M45-28 into wounds made on the trunk of 5 eight-month-old potted healthy 'Royal Gala' apple seedlings. Five control seedlings were inoculated with sterile plugs. Canker symptoms appeared 15 days after inoculation, spread around, up and down the main stem from the inoculation point, and by 7 weeks the upper portion of the seedling was dead (Fig. S2). Diplodia intermedia was re-isolated from all inoculated seedlings and species identity was confirmed by sequencing as described above, fulfilling Koch's postulates. Control seedlings remained symptomless and the fungus was not isolated from the wood. Diplodia intermedia was reported to cause cankers on apple in Uruguay (Delgado-Cerrone et al. 2016), wild apple (Malus sylvestris) in Portugal (Phillips et al. 2012), grapevines in France (Comont et al. 2016) and forest trees in Iran (Kazemzadeh Chakusary et al. 2019). To the best of our knowledge, this is the first report of D. intermedia causing canker and dieback diseases on apple trees in Canada. Further studies are required to better understand the epidemiology involved in the dynamic spread of the disease in order to recommend an adequate phytosanitary program for its control.

Cytospora paraplurivora sp. nov. isolated from orchards with fruit tree decline syndrome in Ontario, Canada.

A new species of Cytospora was isolated from cankered wood of Prunus spp. during a survey of orchards exhibiting symptoms of fruit tree decline syndrome in southern Ontario, Canada. We found isolates that are morphologically similar to species in the Cytosporaceae family, which is characterized by single or labyrinthine locules, filamentous conidiophores or clavate to elongate obovoid asci and allantoid, hyaline conidia. Multi-gene phylogenetic analysis of ITS, LSU, act and tef1- α showed that the isolates form a distinct clade, sister to Cytospora plurivora. Morphologically, our isolates showed differences in the length of conidia and culture characteristics compared to C. plurivora, suggesting the establishment of a new species. The species is described as Cytospora paraplurivora sp. nov. and placed in the family Cytosporaceae of Diaporthales. Additionally, we sequenced, assembled and characterized the genome of the representative isolate for this new species. The phylogenomic analysis confirms the species order and family level classification. C. paraplurivora sp. nov. has the potential to severely affect stone fruits production, causing cankers and dieback in stressed trees, and eventually leads to tree decline. Pathogenicity tests show that the species is pathogenic to Prunus persica var. persica.

Genomic Characteristics and Comparative Genomics Analysis of Parafenestella ontariensis sp. nov.

A new ascomycetous species of Parafenestella was isolated from Acer negundo during the survey of diseased trees in Southern Ontario, Canada. The species is morphologically similar to other taxa of Cucurbitariacea (Pleosporales). The new species is different from the extant species in the morphology of ascospores, culture characteristics and molecular data. The novel species is described as Parafenestella ontariensis sp. nov. based on morphological and multi-gene phylogenetic analyses using a combined set of ITS, LSU, tef1 and tub2 loci. Additionally, the genome of P. ontariensis was sequenced and analyzed. The phylogenomic analysis confirmed the close relationship of the species to the fenestelloid clades of Cucurbitariaceae. The comparative genomics analysis revealed that the species lifestyle appears to be multitrophic (necrotrophic or hemi-biotrophic) with a capability to turn pathogenic on a corresponding plant host.

First Report of Botryosphaeria dothidea Causing Stem Canker and Dieback of Apple Trees in Ontario, Canada.

Apple trees (Malus domestica L. Borkh.) exhibiting extensive and unknown tree fruit decline symptoms were observed in commercial orchards over the past 5 years in Ontario, Canada. The trees exhibited shoot dieback, attached wilted leaves and cankers on the main trunk. Trees with rapid development of cankers upward from the graft union developed extensive vascular discoloration that resulted in sudden collapse of the entire tree. In 2018-19, up to 42% mortality was observed on 2- to 8-year old apple trees. Nine symptomatic trees were collected from two orchards located in southwestern and one in southcentral Ontario. Samples (1 cm length) were collected from symptomatic trunk and shoot tissue, surface sterilized with 70% ethanol for 30 sec, followed by 1% NaClO for 20 min and three rinses in sterile water. The samples were air-dried and placed on a 2% potato dextrose agar (PDA, Difco) culture media supplemented with kanamycin (50 mg L-1). The PDA plates were incubated at 22°C for 5 days in the dark. All fungal colony-forming units that developed were hyphal-tip transferred to individual PDA plates and incubated at 22°C for 7 days in the dark. Purified mycelial isolates were classified into morphotypes prior to molecular identification. One morphotype showed white to olive-green colonies that developed on a moderately dense mycelial mat with aerial hyphae. Several solitary and globose black pycnidia that contained a single ostiole were produced on pine needles on PDA after incubation at 22°C for 14 days in the dark. Conidia were hyaline, fusiform, aseptate with an average size of 3.9 - 5.2 x 21.4 - 26.6 µm (N=50). Genomic DNA was extracted from 5-day old culture of a representative isolate, #M68-17, grown on PDA using the Plant/Fungi DNA Isolation Kit (Norgen Biotech Corp., Thorold, ON, Canada). The rDNA internal transcribed spacer (ITS), translation elongation factor 1- alpha gene (TEF-1α), and ß-tubulin gene (TUB2) were amplified using the primer pairs ITS1/ITS4 (White et al. 1990), EF1-728F/EF1-986R (Carbone and Kohn 1999), and Bt2a/Bt2b (O'Donnell et al. 1998), respectively, and sequenced. The nucleotide sequences obtained (GenBank # MZ926850, MZ934654, MZ934655) were 100.00% similar to those of Botyrosphaeria dothidea (Moug. ex Fr.) Ces. & De Not. isolates from other hosts in several countries in the NCBI database (MT111097, MT309401, MN515421, respectively). Randomized Accelerated Maximum Likelihood (RAxML) analysis using the three gene sequence data was completed (Stamatakis et al. 2008). The isolate #M68-17 was clustered with high bootstrap support values with the B. dothidea isolates from the fungal biodiversity centre (CBS) collection, including the ex-epitype (CBS 115476, CBS 110302) (Fig. 1). A living culture of the representative isolate, #M68-17, was deposited in the Canadian Collection of Fungal Cultures (DAOMC 252246). Pathogenicity tests were conducted in the lab on wood cuttings and in planta. Ten 20 cm apple cuttings and five potted one year old apple seedlings were surface sterilized, wounded and inoculated with 4 mm mycelium agar plug from a 5-day old culture of isolate #M68-17 and wrapped with Parafilm. Three control apple cuttings and seedlings were inoculated with PDA plugs and incubated in the same environment. Cuttings were placed inside a clear plastic container with moist paper towels and incubated at room temperature in the dark. Seedlings were placed in an open-air area between two greenhouses and watered as needed. Twelve days post-inoculation, the average length of the developed necrotic lesions on cuttings was 8.8 ± 0.4 cm. Necrotic and sunken canker symptoms appeared at 10 days, spread upward from the inoculation point and by 6 weeks the upper portion of the seedling was dead (Fig. 2). B. dothidea was isolated from all the inoculated cuttings and seedlings, thus fulfilling Koch's postulates. Control cuttings and seedlings showed no symptoms, and the fungus was not isolated from the wood. B. dothidea was reported as a pathogen causing cankers on a wide range of woody crop plants including apples, almonds, pistachios, hazelnut, walnut, olive and grapes in the United States, China, Uruguay, Spain, Tunisia and Turkey (Chebil et al. 2014; Delgado-Cerrone et al. 2016; Moral et al. 2019; Tang et al. 2012; Türkölmez et al. 2016). However, this is the first report of B. dothidea causing stem canker and death of young apple seedlings in Ontario, Canada. The findings suggest that B. dothidea has the potential to severely affect apple production in Ontario. Accurate identification of pathogen(s) associated with apple decline will support management of the disease.

A randomized trial of class II compression sleeves for full legs versus stockings after thermal ablation with phlebectomy.

OBJECTIVE: Compression stockings and bandages are widely used after invasive treatment of varicose veins. The goals of compression after venous interventions are to reduce pain, bruising, and ecchymosis. Nevertheless, patients often report discomfort with the compression. To make postprocedural compression more tolerable, foot-sparing bandages were tested in a randomized clinical trial of noninferiority. METHODS: A total of 187 patients were randomized to use class II foot-sparing compression sleeves for the full leg or class II stockings after radiofrequency ablation with concomitant phlebectomy. The primary endpoint was the quality of life, measured using the Chronic Venous Disease Quality of Life Questionnaire 20-item scale 30 days after intervention. The secondary endpoints were pain in the leg and discomfort related to the compression garment, which were assessed using the visual analog scale (VAS) at 2, 7, 14, and 30 days. RESULTS: The global index score of the questionnaire was 66.1 and 70.6 and 83.8 and 87.7 for the sleeve and stocking groups before and 30 days after intervention, respectively (P = .542 and P = .150, respectively). The VAS for pain score in the operated leg was slightly higher in the sleeve group the day after the intervention (score, 2.1 vs 1.6; P = .03). At 7, 14, and 30 days, the VAS for pain scores did not differ significantly (score, 0.7 vs 0.5; 0.5 vs 0.3; and 0.1 vs 0.1, respectively; P = NS for all). The VAS for discomfort score was not significantly different statistically in the study group at 2 days (sleeve, 1.9; vs stocking, 1.4; P = .08) but was higher after 7 days (sleeve, 0.9; vs stocking, 0.6; P = .008). No difference in discomfort was found between the study and control groups at 14 or 30 days (sleeve, 0.6; vs stocking, 0.4; and sleeve, 0.4; vs stocking, 0.4, respectively; P = NS for both). CONCLUSIONS: Quality of life after thermal ablation with phlebectomy improved equivalently in patients who had used class II compression sleeves for full legs and those who had used class II compression stockings. Pain and discomfort were slightly higher in the sleeve group.

Validation of two Amanita species from eastern North America: A.rhacopus sp. nov. and A.variicolor sp. nov.

Members of the mushroom genus Amanita usually can easily be identified to the genus in the field, however, species circumscription and identification are often problematic. Several names have been misapplied and cryptic species exist. Here, we formally describe and validate two new species of Amanitasect.Vaginatae from eastern North America that were recognised under the umbrella European names A.ceciliae by past authors: Amanitarhacopus sp. nov. and Amanitavariicolor sp. nov.

Genome-wide association study in ethnic Russians suggests an association of the MHC class III genomic region with the risk of primary varicose veins.

Heredity is a well-known risk factor for varicose veins, but genetic basis of this condition remains poorly studied. Our aim was to conduct a large-scale genetic association study for primary varicose veins (PVVs) in the population of ethnic Russians. An initial scan using Illumina HumanExome-12 v1.0 BeadChip was performed for 273 patients with PVVs and 250 controls without a history of chronic venous disease and other venous disorders. After quality control and removal of monomorphic markers, 25,424 common and 48,232 rare variants were included in the analysis. 42 single nucleotide polymorphisms (SNPs) were genotyped in the independent replication cohort of 447 PVVs patients and 443 controls. Association of common variants with PVVs was investigated by logistic regression, and the impact of rare variants was analyzed using sequence kernel association test. No effect of low frequency alleles has been revealed in our study. Common variant analysis identified a promising signal at chromosome 6 within classical major histocompatibility complex (MHC) class III subregion. The most strongly associated SNP in a combined analysis that reached a suggestive significance level of 3.2e-05 was polymorphism rs4151657 in the complement factor B gene. Testing for potential pleiotropy with other traits indicated that the same causal variant in this region increases the risk of rheumatoid arthritis and has a negative impact on human height. Our results provide suggestive evidence for the involvement of the MHC class III genes in the pathogenesis of PVVs. Further independent studies are needed to confirm our pilot findings.

Allele rs2010963 C of the VEGFA gene is associated with the decreased risk of primary varicose veins in ethnic Russians.

Objective To study the association of polymorphisms rs699947, rs2010963, rs3025039 in the VEGFA gene region and rs1870377, rs2305949, rs2071559 in the VEGFR2 gene region with the risk of primary varicose veins in ethnic Russians. Methods Genotypes were determined by real-time PCR allelic discrimination. The case group consisted of 448 patients with primary varicose veins and the control group comprised 609 individuals without a history of chronic venous disease. Association was studied by logistic regression analysis. Results Allele rs2010963 C was associated with the decreased risk of varicose veins (additive model of inheritance: odds ratio = 0.73, 95% confidence interval = 0.59-0.91, P = 0.004). Conclusions Our results provide evidence that polymorphism rs2010963 located in the 5' untranslated region of the VEGFA gene can influence genetic susceptibility to primary varicose veins in Russians. Otherwise, it can be in linkage disequilibrium with another functional single nucleotide polymorphism that can alter the level of vascular endothelial growth factor A protein.

Polymorphisms in inflammation-related genes and the risk of primary varicose veins in ethnic Russians.

Inflammation was shown to be activated in varicose veins, although its role in the development of vein wall transformation remains inconclusive. We aimed to investigate the influence of 13 inflammation-related single nucleotide polymorphisms (SNPs) TNF rs1800629 and rs3093661, IL1A rs1800587, IL1RN rs4251961, IL6 rs1800795 and rs1800796, IFNG rs2430561, IL10 rs1800896, TGFB1 rs1800469, HIF1A rs11549465, NFKB1 rs28362491, and rs4648068 on the risk of primary varicose veins (PVVs) in ethnic Russians. We genotyped 709 patients with PVVs and 278 individuals without a history of chronic venous disease and performed a single SNP and a haplotype analysis. Several associations with P < 0.05 were revealed in our study. Variant allele HIF1A rs11549465 T, TNF rs3093661 A, and NFKB1 rs28362491 ATTG deletion showed the reverse association with PVV risk, and allele IL6 rs1800795 C was associated with the increased risk of the studied pathology. Haplotype analysis revealed associations of TNF haplotypes rs3093661 A-rs1800629 G and IL6 rs1800795 C-rs1800796 G with the decreased and the increased risk of PVVs, correspondingly. However, all the observed associations failed to reach statistical significance after the correction for multiple testing, which was set at a level of 10-3 due to many tests performed. Our study therefore provides evidence that investigated polymorphisms do not play a major role in susceptibility to PVVs.

Functional polymorphism rs1024611 in the MCP1 gene is associated with the risk of varicose veins of lower extremities.

OBJECTIVE: Monocyte chemoattractant protein 1 (MCP-1) is a chemokine responsible for monocyte, basophil, and T-lymphocyte attraction. Polymorphism rs1024611 located in the regulatory region of the MCP1 gene has previously been shown to be associated with increased MCP-1 production. In our study, we aimed to examine the association of rs1024611 with the risk of primary varicose veins (PVVs) of lower extremities. METHODS: The case group comprised 470 patients with PVVs, and the control group included 269 individuals without a history of chronic venous disease. All cases and controls were ethnic Russians. Genotypes were determined by real-time polymerase chain reaction allelic discrimination. Association was studied by logistic regression analysis. RESULTS: We revealed the association of genotype G/G with the increased risk of PVVs (odds ratio [OR], 1.87; 95% confidence interval [CI], 1.02-3.44; P = .04). In the subgroup analysis, association was revealed only in patients with C2 Clinical, Etiology, Anatomy, and Pathophysiology class (allele G: OR, 1.62 [95% CI, 1.13-2.33; P = .008]; genotype G/G: OR, 3.22 [95% CI, 1.43-7.27; P = .005]), in patients with age at onset of PVVs before 30 years (allele G: OR, 1.41 [95% CI, 1.08-1.85; P = .01]; genotype G/G: OR, 2.35 [95% CI, 1.22-4.55; P = .01]), and in patients who declared no family history (allele G: OR, 1.46 [95% CI, 1.02-2.09; P = .04]; genotype G/G: OR, 2.50 [95% CI, 1.11-5.63; P = .03]). CONCLUSIONS: Our results provide evidence for MCP-1 involvement in the development of PVVs and indicate that inflammation could be implicated in the pathogenesis of this condition.

Radiofrequency ablation or stripping of large-diameter incompetent great saphenous varicose veins with C2 or C3 disease.

OBJECTIVE: The objectives of this study were to compare the results of radiofrequency ablation (RFA) and stripping for large-diameter varicose target veins for the period of 1 year, based on a composite end point; to analyze the pain severity on a digital rating scale for 7 days after RFA and stripping; and to detect the factors affecting the level of postoperative pain using the cluster analysis. METHODS: This was a multicenter retrospective cohort study. Two groups, stripping ≥14 mm and RFA ≥14 mm, of 129 varicose vein disease patients underwent surgical treatment in three specialized clinics. We eliminated symptomatic pathologic reflux with RFA in 64 patients and with stripping in 65 patients. In the postoperative phase, we evaluated the pain level, subcutaneous hemorrhage, and paresthesia. A composite end point with four components was used to analyze the results. These were three clinical adverse effects of the intervention (pain, hemorrhage, and paresthesia) and the technical outcome 1 year after the surgical intervention. RESULTS: The frequency of favorable outcomes was 20 (30.8%) in the stripping ≥14 mm group and 61 (95.3%) in the RFA ≥14 mm group (P < .0001). The odds ratio for a favorable outcome between the RFA and the stripping groups was 45.8 (95% confidence interval, 44.5-47.0). The pain clusters that were moderate were created by patients after stripping. These clusters show a link between the pain level on the one hand and an increased body mass index and large vein diameter on the other hand. CONCLUSIONS: For large-diameter veins, RFA is superior to stripping in terms of favorable outcomes according to the composite end point chosen. Significant pain after stripping was linked to a large vein diameter and excess weight or adiposis.

Morphologic changes in the vein after different numbers of radiofrequency ablation cycles.

OBJECTIVE: It has not yet been clarified whether it is possible to decrease the percentage of recurrences after radiofrequency (RF) ablation by way of increasing the number of RF ablation cycles. The aim of this study was to assess the morphologic changes in excised vein fragments after different durations of RF ablation exposure. METHODS: In the first part of the study, we performed a morphologic analysis of eight cases of great saphenous vein (GSV) recanalization 6 months after RF ablation. The second part was performed on a suprafascial segment of the GSV with a length of >22 cm and a minimum diameter of 5 mm in 10 patients, who had given their consent to intraoperative excision of suprafascial GSV segments after RF ablation treatment through four 1-cm-long diametrical cuts. Prior ultrasound analysis had shown an average 6.9-mm diameter of the suprafascial segments. The segment was divided into three 7-cm-long subsegments and one control segment. The first, second, and third segments were treated with three, two, and one RF ablation cycles (ClosureFast; Covidien, Mansfield, Mass), respectively; the control segment was not exposed to RF ablation at all. Morphologic study of 160 sections of the vein (five sections of each segment and 10 control specimens) was carried out. The specimens were dyed with hematoxylin and orcein. The ensuing analysis was performed by an experienced expert with the blind study method (the specimens were numbered without any hint as to the quantity of RF ablation cycles performed on them). The intergroup comparison of the depth of venous wall damage was based on comparison of the coefficient of alteration, which is calculated as the relation of damage depth to thickness of the vein. RESULTS: After one RF ablation cycle, the depth of blurring of the structural elements only on some portions reached the middle of the muscle layer of the wall (coefficient of alteration, α = 26%). After two cycles, blurring of the structural elements on some portions extended to the adventitia (α = 53%). After three cycles, uniform blurring of the structural elements of all layers of the venous wall up to the adventitia was seen (α = 92%). The statistically significant difference in the alteration coefficient, depending on the number of cycles of RF ablation (P < .005), was established. CONCLUSIONS: The number of RF ablation cycles has an impact on the depth of vein wall damage. One and two cycles do not cause damage to all layers of the vein wall. Three cycles cause damage to all vein wall layers.