Effect of ejaculatory abstinence period on fertilization and clinical outcomes in ICSI cycles: a retrospective analysis.

RESEARCH QUESTION: Does ejaculatory abstinence impact fertilization outcomes in intracytoplasmic sperm injection (ICSI) cycles in infertile couples? DESIGN: This single-centre retrospective observational study included 6919 ICSI cycles from 2013 to 2022. The primary outcome was the assessment of oocyte fertilization, measured in terms of the rate of formation of two-pronuclear (2PN), 3PN and 1PN zygotes. Secondary outcomes were blastulation, cumulative positive ß-human chorionic gonadotrophin test and clinical pregnancy rates. Relationships between ejaculatory abstinence and fertilization outcomes, and ejaculatory abstinence and clinical outcomes were evaluated with multivariable analysis, including possible confounders. RESULTS: A positive association was observed between ejaculatory abstinence and semen sample volume (P < 0.001), sperm concentration (P < 0.001) and total motile sperm count (P < 0.001). No association was found between the 1PN zygote rate and ejaculatory abstinence (P = 0.97). Conversely, for each additional day of ejaculatory abstinence, the likelihood of obtaining 2PN zygotes from all inseminated oocytes decreased by 3% [adjusted odds ratio (aOR) 0.97, 95% CI 0.94-0.99], whilst the likelihood of obtaining 3PN zygotes from all inseminated oocytes increased significantly by 14% (aOR 1.14, 95% CI 1.07-1.22). No significant associations were found between ejaculatory abstinence and blastulation, cumulative pregnancy or miscarriage rates. CONCLUSIONS: A longer ejaculatory abstinence period significantly decreases the rate of 2PN zygotes, and increases the rate of 3PN zygotes without directly affect blastulation and pregnancy rates.

Associations between paternal versus maternal parenting behaviors and child and adolescent internalizing problems: A systematic review and meta-analysis.

There is a large evidence base supporting an important role of parenting behavior in influencing youth mental health; however, this literature often fails to capture the potentially unique and interactive role of mothers and fathers. This systematic review and meta-analysis aimed to investigate the role of maternal and paternal parenting behaviors in relation to child and adolescent internalizing problems. Following PRISMA (2020) guidelines, 88 studies were identified. Of these, 47 studies and upward of 65 parent-behavior and child-outcome combinations were examined. Findings indicated a unique role of maternal overprotection in the prediction of child anxiety symptoms. For other parenting behaviors, largely similar associations were found for maternal and paternal parenting behaviors with child and adolescent anxiety, depressive symptoms and broader internalizing problems. There was preliminary support for the interaction of maternal and paternal parenting being important in predicting youth symptoms. Although findings did not strongly substantiate differences in the effects of maternal and paternal parenting practices, with only one significant difference identified, further research would benefit from stronger representation of fathers, to enable a more rigorous and comprehensive understanding of each parent's role, and their interactive influence on internalizing outcomes of their children.

Not only for caregivers: intranasal glucagon for severe hypoglycaemia in a simulation study.

AIMS: To evaluate: (i) the propensity of paediatrics and emergency medicine residents to select different therapeutic options and (ii) the speed and administration success in a high-fidelity simulation of severe hypoglycaemia in a child with type 1 diabetes (T1DM). METHODS: In this single-centre high-fidelity simulation study, 51 paediatrics or emergency medicine residents were exposed to a scenario of severe hypoglycaemia in a T1DM child attending an ambulatory setting, before and after a training on the preparation and administration of both injectable and IN glucagon. Time for drug delivery and its effectiveness were collected. RESULTS: Before training, 45.1% of participants chose to administer injectable glucagon, 43.1% intravenous glucose solution, 5.9% intranasal (IN) glucagon, and 5.9% took no action. Administration was successful in 74% of injectable glucagon, 33.3% intravenous glucose solution, and 22.7% IN glucagon. After training, 58.8% of participants chose IN and 41.2% injectable glucagon, with 100% of successful administrations for IN glucagon and 90.5% for injectable glucagon. Time to successful administration was shorter for IN than injectable glucagon (23 ± 10 vs. 38 ± 7 s, p < 0.0001). CONCLUSIONS: IN glucagon is an easy and effective option for severe hypoglycaemia treatment, with an almost zero possibility of failure provided that adequate training is imparted.

Impact of time from diagnosis to treatment on erectile function outcomes after radical prostatectomy.

BACKGROUND: Concerns exist about the effect of delaying treatment for prostate cancer (PCa) regarding both oncological and functional outcomes after radical prostatectomy (RP). OBJECTIVE: To assess the impact of time from diagnosis to RP on post-operative erectile function (EF) outcomes. MATERIALS AND METHODS: We analyzed data for 827 patients treated with RP at a single center from 2002 to 2017. The International Index of Erectile Function-EF (IIEF-EF) was compiled by every patient (EF recovery equal to IIEF-EF ≥ 22). Time from diagnosis to treatment was defined as the interval between biopsy and RP. Cox regression analysis was used to test the impact of time to surgery on the probability of EF recovery. Kaplan-Meier analysis compared the cumulative incidence of EF recovery according to time from diagnosis to surgery. The impact of time to RP on EF was tested also in a sub-cohort of patients eligible for active surveillance (AS). RESULTS: Overall, low-, intermediate-, and high-risk PCa was found in 306 (37%), 422 (51%), and 99 (12%) patients. Of them, 148 (17.9%) would have been eligible for AS. A total of 152 (18%) and 22 (2.7%) patients were treated after 6 and 12 months from diagnosis. The overall probability of EF recovery was 32% (95% CI: 29-36) at 24 months. Cox regression analysis showed that time from biopsy to surgery was not associated with a different chance of EF recovery (HR: 1.01; 95% CI: 0.97-1.05; p = 0.7). At Kaplan-Meier analysis, the cumulative incidence of EF recovery did not differ between patients treated within 6 months, from 6 to 12 months and after 12 months from diagnosis. Similar findings were obtained for patients eligible for AS. DISCUSSION: Patients may be reassured regarding their chance of post-operative EF recovery in the case of a delayed surgical treatment. CONCLUSIONS: Delaying surgery after PCa diagnosis does not affect post-operative EF recovery outcomes regardless of oncological risk.

Prevalence and phenotype of the c.1529C>T SPG7 variant in adult-onset cerebellar ataxia in Italy.

BACKGROUND AND PURPOSE: Hereditary ataxias are heterogeneous groups of neurodegenerative disorders, characterized by cerebellar syndromes associated with dysarthria, oculomotor and corticospinal signs, neuropathy and cognitive impairment. Recent reports have suggested mutations in the SPG7 gene, causing the most common form of autosomal recessive spastic paraplegia (MIM#607259), as a main cause of ataxias. The majority of described patients were homozygotes or compound heterozygotes for the c.1529C>T (p.Ala510Val) change. We screened a cohort of 895 Italian patients with ataxia for p.Ala510Val in order to define the prevalence and genotype-phenotype correlation of this variant. METHODS: We set up a rapid assay for c.1529C>T using restriction enzyme analysis after polymerase chain reaction amplification. We confirmed the diagnosis with Sanger sequencing. RESULTS: We identified eight homozygotes and 13 compound heterozygotes, including two novel variants affecting splicing. Mutated patients showed a pure cerebellar ataxia at onset, evolving in mild spastic ataxia (alternatively) associated with dysarthria (~80% of patients), urinary urgency (~30%) and pyramidal signs (~70%). Comparing homozygotes and compound heterozygotes, we noted a difference in age at onset and Scale for the Assessment and Rating of Ataxia score between the two groups, supporting an earlier and more severe phenotype in compound heterozygotes versus homozygotes. CONCLUSIONS: The SPG7 c.1529C>T (p.Ala510Val) mutants accounted for 2.3% of cerebellar ataxia cases in Italy, suggesting that this variant should be considered as a priority test in the presence of late-onset pure ataxia. Moreover, the heterozygous/homozygous genotype appeared to predict the onset of clinical manifestation and disease progression.

Performance and stability of an expanded granular sludge bed reactor modified with zeolite addition subjected to step increases of organic loading rate (OLR) and to organic shock load (OSL).

This paper shows the effect of organic shock loads (OSLs) on the anaerobic digestion (AD) of synthetic swine wastewater using an expanded granular sludge bed (EGSB) reactor modified with zeolite. Two reactors (R1 and R2), each with an effective volume of 3.04 L, were operated for 180 days at a controlled temperature of 30 °C and hydraulic retention time of 12 h. In the case of R2, 120 g of zeolite was added. The reactors were operated with an up-flow velocity of 6 m/h. The evolution of pH, total Kjeldahl nitrogen, chemical oxygen demand (COD) and volatile fatty acids (VFAs) was monitored during the AD process with OSL and increases in the organic loading rate (OLR). In addition, the microbial composition and changes in the structure of the bacterial and archaeal communities were assessed. The principal results demonstrate that the presence of zeolite in an EGSB reactor provides a more stable process at higher OLRs and after applying OSL, based on both COD and VFA accumulation, which presented with significant differences compared to the control. Denaturing gradient gel electrophoresis band profiles indicated differences in the populations of Bacteria and Archaea between the R1 and R2 reactors, attributed to the presence of zeolite.

Identification and characterization of a new potyvirus infecting cucurbits.

A new potyvirus, tentatively named cucurbit vein banding virus (CVBV), was identified in crops of cucurbits in San Pedro (Buenos Aires, Argentina). The complete genome sequences of two isolates of CVBV were obtained by next-generation sequencing (Illumina). The genomic RNA consisted of 9968 and 9813 nucleotides, respectively, and displayed typical potyvirus organization. The percentage identity for these two genome sequences, using BLASTn, was 77% to sweet potato virus c and 73% to tomato necrotic stunt virus. BLASTx analysis of the complete polyprotein showed that the most closely related virus is plum pox virus, with 48% amino acid sequence identity for both isolates. Sequence comparisons and phylogenetic analyses indicate that CVBV belongs to a previously undescribed species in genus Potyvirus.

Photon iso-effective dose for cancer treatment with mixed field radiation based on dose-response assessment from human and an animal model: clinical application to boron neutron capture therapy for head and neck cancer.

Boron neutron capture therapy (BNCT) is a treatment modality that combines different radiation qualities. Since the severity of biological damage following irradiation depends on the radiation type, a quantity different from absorbed dose is required to explain the effects observed in the clinical BNCT in terms of outcome compared with conventional photon radiation therapy. A new approach for calculating photon iso-effective doses in BNCT was introduced previously. The present work extends this model to include information from dose-response assessments in animal models and humans. Parameters of the model were determined for tumour and precancerous tissue using dose-response curves obtained from BNCT and photon studies performed in the hamster cheek pouch in vivo models of oral cancer and/or pre-cancer, and from head and neck cancer radiotherapy data with photons. To this end, suitable expressions of the dose-limiting Normal Tissue Complication and Tumour Control Probabilities for the reference radiation and for the mixed field BNCT radiation were developed. Pearson's correlation coefficients and p-values showed that TCP and NTCP models agreed with experimental data (with r > 0.87 and p-values >0.57). The photon iso-effective dose model was applied retrospectively to evaluate the dosimetry in tumours and mucosa for head and neck cancer patients treated with BNCT in Finland. Photon iso-effective doses in tumour were lower than those obtained with the standard RBE-weighted model (between 10% to 45%). The results also suggested that the probabilities of tumour control derived from photon iso-effective doses are more adequate to explain the clinical responses than those obtained with the RBE-weighted values. The dosimetry in the mucosa revealed that the photon iso-effective doses were about 30% to 50% higher than the corresponding RBE-weighted values. While the RBE-weighted doses are unable to predict mucosa toxicity, predictions based on the proposed model are compatible with the observed clinical outcome. The extension of the photon iso-effective dose model has allowed, for the first time, the determination of the photon iso-effective dose for unacceptable complications in the dose-limiting normal tissue. Finally, the formalism developed in this work to compute photon-equivalent doses can be applied to other therapies that combine mixed radiation fields, such as hadron therapy.

Study protocol: families and childhood transitions study (FACTS) - a longitudinal investigation of the role of the family environment in brain development and risk for mental health disorders in community based children.

BACKGROUND: Extant research has demonstrated that parenting behaviour can be a significant contributor to the development of brain structure and mental health during adolescence. Nonetheless, there is limited research examining these relationships during late childhood, and particularly in the critical period of brain development occurring between 8 and 10 years of age. The effects of the family environment on the brain during late childhood may have significant implications for later functioning, and particularly mental health. The Families and Childhood Transitions Study (FACTS) is a multidisciplinary longitudinal cohort study of brain development and mental health, with two waves of data collection currently funded, occurring 18-months apart, when child participants are aged approximately 8- and 10-years old. METHODS/DESIGN: Participants are 163 children (M age [SD] = 8.44 [0.34] years, 76 males) and their mothers (M age [SD] = 40.34 [5.43] years). Of the 163 families who consented to participate, 156 completed a video-recorded and observer-coded dyadic interaction task and 153 completed a child magnetic resonance imaging brain scan at baseline. Families were recruited from lower socioeconomic status (SES) areas to maximise rates of social disadvantage and variation in parenting behaviours. All experimental measures and tasks completed at baseline are repeated at an 18-month follow-up, excluding the observer coded family interaction tasks. The baseline assessment was completed in October 2015, and the 18-month follow up will be completed May 2017. DISCUSSION: This study, by examining the neurobiological and mental health consequences of variations in parenting, has the potential to significantly advance our understanding of child development and risk processes. Recruitment of lower SES families will also allow assessment of resilience factors given the poorer outcomes often associated with this population.

Poultry slaughterhouse wastewater treatment plant for high quality effluent.

This paper assesses a wastewater treatment plant (WWTP) regarding the technology used, as well as organic matter and nutrient removal efficiencies aiming to optimize the treatment processes involved and wastewater reclamation. The WWTP consists of a dissolved air flotation (DAF) system, an upflow anaerobic sludge blanket (UASB) reactor, an aerated-facultative pond (AFP) and a chemical-DAF system. The removal efficiencies of chemical oxygen demand (COD) (97.9 ± 1.0%), biochemical oxygen demand (BOD) (98.6 ± 1.0%) and oil and grease (O&G) (91.1 ± 5.2%) at the WWTP, the nitrogen concentration of 17 ± 11 mg N-NH3 and phosphorus concentration of 1.34 ± 0.93 mg PO4(-3)/L in the final effluent indicate that the processes used are suitable to comply with discharge standards in water bodies. Nitrification and denitrification tests conducted using biomass collected at three AFP points indicated that nitrification and denitrification could take place in the pond.

Effect of Natural Mineral on Methane Production and Process Stability During Semi-Continuous Mono-Digestion of Maize Straw.

The effect of natural mineral on the mono-digestion of maize straw was evaluated in continuously stirred tank reactors (CSTRs) at 38 °C. Different strategies of mineral addition were studied. The organic loading rate (OLR) was varied from 0.5 to 2.5 g volatile solid (VS) L(-1) d(-1). A daily addition of 1 g mineral L(-1) in reactor 2 (R2) diminished the methane production by about 11 % with respect to the initial phase. However, after a gradual addition of mineral, an average methane yield of 257 NmL CH4 g VS(-1) was reached and the methane production was enhanced by 30 % with regard to R1. An increase in the frequency of mineral addition did not enhance the methane production. The archaeal community was more sensitive to the mineral than the bacterial population whose similarity stayed high between R1 and R2. Significant difference in methane yield was found for both reactors throughout the operation.

Toward a clinical application of ex situ boron neutron capture therapy for lung tumors at the RA-3 reactor in Argentina.

PURPOSE: Many types of lung tumors have a very poor prognosis due to their spread in the whole organ volume. The fact that boron neutron capture therapy (BNCT) would allow for selective targeting of all the nodules regardless of their position, prompted a preclinical feasibility study of ex situ BNCT at the thermal neutron facility of RA-3 reactor in the province of Buenos Aires, Argentina. (l)-4p-dihydroxy-borylphenylalanine fructose complex (BPA-F) biodistribution studies in an adult sheep model and computational dosimetry for a human explanted lung were performed to evaluate the feasibility and the therapeutic potential of ex situ BNCT. METHODS: Two kinds of boron biodistribution studies were carried out in the healthy sheep: a set of pharmacokinetic studies without lung excision, and a set that consisted of evaluation of boron concentration in the explanted and perfused lung. In order to assess the feasibility of the clinical application of ex situ BNCT at RA-3, a case of multiple lung metastases was analyzed. A detailed computational representation of the geometry of the lung was built based on a real collapsed human lung. Dosimetric calculations and dose limiting considerations were based on the experimental results from the adult sheep, and on the most suitable information published in the literature. In addition, a workable treatment plan was considered to assess the clinical application in a realistic scenario. RESULTS: Concentration-time profiles for the normal sheep showed that the boron kinetics in blood, lung, and skin would adequately represent the boron behavior and absolute uptake expected in human tissues. Results strongly suggest that the distribution of the boron compound is spatially homogeneous in the lung. A constant lung-to-blood ratio of 1.3 ± 0.1 was observed from 80 min after the end of BPA-F infusion. The fact that this ratio remains constant during time would allow the blood boron concentration to be used as a surrogate and indirect quantification of the estimated value in the explanted healthy lung. The proposed preclinical animal model allowed for the study of the explanted lung. As expected, the boron concentration values fell as a result of the application of the preservation protocol required to preserve the lung function. The distribution of the boron concentration retention factor was obtained for healthy lung, with a mean value of 0.46 ± 0.14 consistent with that reported for metastatic colon carcinoma model in rat perfused lung. Considering the human lung model and suitable tumor control probability for lung cancer, a promising average fraction of controlled lesions higher than 85% was obtained even for a low tumor-to-normal boron concentration ratio of 2. CONCLUSIONS: This work reports for the first time data supporting the validity of the ovine model as an adequate human surrogate in terms of boron kinetics and uptake in clinically relevant tissues. Collectively, the results and analysis presented would strongly suggest that ex situ whole lung BNCT irradiation is a feasible and highly promising technique that could greatly contribute to the treatment of metastatic lung disease in those patients without extrapulmonary spread, increasing not only the expected overall survival but also the resulting quality of life.

The influence of the buffering capacity on the production of organic acids and alcohols from wastewater in anaerobic reactor.

Some bacteria common in anaerobic digestion process can ferment a broad variety of organic compounds to organic acids, alcohols, and hydrogen, which can be used as biofuels. Researches are necessary to control the microbial interactions in favor of the alcohol production, as intermediary products of the anaerobic digestion of organic compounds. This paper reports on the effect of buffering capacity on the production of organic acids and alcohols from wastewater by a natural mixed bacterial culture. The hypothesis tested was that the increase of the buffering capacity by supplementation of sodium bicarbonate in the influent results in benefits for alcohol production by anaerobic fermentation of wastewater. When the influent was not supplemented with sodium bicarbonate, the chemical oxygen demand (COD)-ethanol and COD-methanol detected in the effluent corresponded to 22.5 and 12.7 % of the COD-sucrose consumed. Otherwise, when the reactor was fed with influent containing 0.5 g/L of sodium bicarbonate, the COD-ethanol and COD-methanol were effluents that corresponded to 39.2 and 29.6 % of the COD-sucrose consumed. Therefore, the alcohol production by supplementation of the influent with sodium bicarbonate was 33.6 % higher than the fermentation of the influent without sodium bicarbonate.

Co-treatment of landfill leachate and domestic wastewater using a submerged aerobic biofilter.

This study used a pilot-scale submerged aerobic biofilter (SAB) to evaluate the co-treatment of domestic wastewater and landfill leachate that was pre-treated by air stripping. The leachate tested volumetric ratios were 0, 2, and 5%. At a hydraulic retention time of 24 h, the SAB was best operated with a volumetric ratio of 2% and removed 98% of the biochemical oxygen demand (BOD), 80% of the chemical oxygen demand (COD) and dissolved organic carbon (DOC), and 90% of the total suspended solids (TSS). A proposed method, which we called the "equivalent in humic acid" (Eq.HA) approach, indicated that the hardly biodegradable organic matter in leachate was removed by partial degradation (71% of DOC Eq.HA removal). Adding leachate at a volumetric ratio of 5%, the concentration of the hardly biodegradable organic matter was decreased primarily as a result of dilution rather than biodegradation, which was confirmed by Fourier transform infrared (FTIR) spectroscopy. The total ammoniacal nitrogen (TAN) was mostly removed (90%) by nitrification, and the SAB performances at the volumetric ratios of 0 and 2% were equal. For the three tested volumetric ratios of leachate (0, 2, and 5%), the concentrations of heavy metals in the treated samples were below the local limits.

Design, construction and application of a neutron shield for the treatment of diffuse lung metastases in rats using BNCT.

A model of multiple lung metastases in BDIX rats is under study at CNEA (Argentina) to evaluate the feasibility of BNCT for multiple, non-surgically resectable lung metastases. A practical shielding device that comfortably houses a rat, allowing delivery of a therapeutic, uniform dose in lungs while protecting the body from the neutron beam is presented. Based on the final design obtained by numerical simulations, the shield was constructed, experimentally characterized and recently used in the first in vivo experiment at RA-3.

Biodistribution of the boron carriers boronophenylalanine (BPA) and/or decahydrodecaborate (GB-10) for Boron Neutron Capture Therapy (BNCT) in an experimental model of lung metastases.

BNCT was proposed for the treatment of diffuse, non-resectable tumors in the lung. We performed boron biodistribution studies with 5 administration protocols employing the boron carriers BPA and/or GB-10 in an experimental model of disseminated lung metastases in rats. All 5 protocols were non-toxic and showed preferential tumor boron uptake versus lung. Absolute tumor boron concentration values were therapeutically useful (25-76ppm) for 3 protocols. Dosimetric calculations indicate that BNCT at RA-3 would be potentially therapeutic without exceeding radiotolerance in the lung.

Improvement of the boron neutron capture therapy (BNCT) by the previous administration of the histone deacetylase inhibitor sodium butyrate for the treatment of thyroid carcinoma.

We have shown that boron neutron capture therapy (BNCT) could be an alternative for the treatment of poorly differentiated thyroid carcinoma (PDTC). Histone deacetylase inhibitors (HDACI) like sodium butyrate (NaB) cause hyperacetylation of histone proteins and show capacity to increase the gamma irradiation effect. The purpose of these studies was to investigate the use of the NaB as a radiosensitizer of the BNCT for PDTC. Follicular thyroid carcinoma cells (WRO) and rat thyroid epithelial cells (FRTL-5) were incubated with 1 mM NaB and then treated with boronophenylalanine ¹°BPA (10 µg ¹°B ml⁻¹) + neutrons, or with 2, 4-bis (α,ß-dihydroxyethyl)-deutero-porphyrin IX ¹°BOPP (10 µg ¹°B ml⁻¹) + neutrons, or with a neutron beam alone. The cells were irradiated in the thermal column facility of the RA-3 reactor (flux = (1.0 ± 0.1) × 10¹° n cm⁻² s⁻¹). Cell survival decreased as a function of the physical absorbed dose in both cell lines. Moreover, the addition of NaB decreased cell survival (p < 0.05) in WRO cells incubated with both boron compounds. NaB increased the percentage of necrotic and apoptotic cells in both BNCT groups (p < 0.05). An accumulation of cells in G2/M phase at 24 h was observed for all the irradiated groups and the addition of NaB increased this percentage. Biodistribution studies of BPA (350 mg kg⁻¹ body weight) 24 h after NaB injection were performed. The in vivo studies showed that NaB treatment increases the amount of boron in the tumor at 2-h post-BPA injection (p < 0.01). We conclude that NaB could be used as a radiosensitizer for the treatment of thyroid carcinoma by BNCT.

Boron neutron capture therapy for oral precancer: proof of principle in an experimental animal model.

OBJECTIVES: Field-cancerized tissue can give rise to second primary tumours, causing therapeutic failure. Boron neutron capture therapy (BNCT) is based on biological targeting and would serve to treat undetectable foci of malignant transformation. The aim of this study was to optimize BNCT for the integral treatment for oral cancer, with particular emphasis on the inhibitory effect on tumour development originating in precancerous conditions, and radiotoxicity of different BNCT protocols in a hamster cheek pouch oral precancer model. MATERIALS AND METHODS: Groups of cancerized hamsters were locally exposed to single or double (2 or 4 weeks apart) applications of BNCT at different dose levels, mediated by the boron compounds boronophenylalanine (BPA) or BPA and decahydrodecaborate (GB-10) administered jointly. Cancerized, sham-irradiated hamsters served as controls. Clinical status, tumour development from field-cancerized tissue and mucositis were followed for 8 months. RESULTS: A double application (4 weeks apart) of BNCT mediated by GB-10+ BPA at a total dose of 10 Gy in two 5-Gy doses rendered the best therapeutic advantage (63-100% inhibition of tumour development from field-cancerized tissue), minimizing dose-limiting mucositis. CONCLUSION: BNCT can be optimized for the integral treatment for head and neck cancer, considering the implications for field-cancerized tissue.