The time aspect in storing vitrified blastocysts: its impact on survival rate, implantation potential and babies born.

STUDY QUESTION: Does the storage time of vitrified human blastocysts negatively impact their survival, the implantation potential of embryos or the malformation rate of babies born? SUMMARY ANSWER: There was no evidence that storage times of up to 6 years after vitrification (VIT) had a negative impact on blastocyst survival, the implantation potential of embryos or the malformation rate of babies born. WHAT IS KNOWN ALREADY: Although several thousand children have been born after blastocyst VIT, many aspects of this technique remain to be elucidated. New applications, such as fertility preservation, lead to long storage times of vitrified gametes or embryos but it remains to be determined if these vitrified embryos are stable over time. STUDY DESIGN, SIZE, DURATION: A retrospective study including 603 transfers was conducted between January 2009 and April 2012. Blastocysts were vitrified using a closed system. PARTICIPANTS/MATERIALS, SETTING, METHODS: All patients underwent the transfer of aseptically vitrified/warmed blastocysts in a cryo-cycle. A total of 1077 blastocysts were transferred. Survival rates (SRs), implantation potential, birth rates and characteristics of the children born were evaluated. MAIN RESULTS AND THE ROLE OF CHANCE: We found that the storage of vitrified blastocysts in aseptic conditions neither impaired blastocyst viability (SR after warming during the first year of storage was 83.0% compared with 83.1% after 5-6 years of storage: NS) nor decreased pregnancy rates (clinical pregnancy rate after 1 year of storage was 40.0 versus 38.5% after 6 years: NS). In addition, no increase in the malformation rate over time was observed. LIMITATIONS, REASONS FOR CAUTION: Our study only included the transfer of blastocysts which had been vitrified aseptically (i.e. using a closed system). Therefore, our results might not be applicable to 'open' VIT systems. The long-term follow-up of children born will be necessary to confirm our findings. WIDER IMPLICATIONS OF THE FINDINGS: The results suggest that vitrified human blastocysts can be stored for long periods of time without significant negative consequences for the offspring. Therefore, the method should be of benefit to those patients who need to consider taking measures for fertility preservation. STUDY FUNDING/COMPETING INTEREST(S): No external funding was sought for this study and the authors have no conflict of interest to declare.

Direct volumetric flow cytometric quantitation of CD34+ stem and progenitor cells.

OBJECTIVES: In this study, we compared a classic single-platform (SP) method applying beads for enumeration of CD45+ or CD34+ cells with a new device allowing direct volumetric measurements of stem and progenitor cells. BACKGROUND: Following apheresis and cyropreservation, the precise enumeration of CD34+ cells as key parameter of graft quality is mandatory for the clinical course after transplantation. Currently, flow cytometry with SP technique represents the 'gold standard' for such determinations. METHODS/MATERIALS: Fresh samples, 14 from mobilised peripheral blood (PB), 9 from apheresis products (AP) and 13 samples from frozen-thawed (FT) haematopoietic progenitor cell grafts, were analysed for CD34+ cells, CD45+ cells, and in frozen-thawed samples for viability by a bead-based flow cytometric method and in parallel by a direct, volumetric flow cytometric method. RESULTS: Comparison of CD34+ analyses revealed a significant correlation (P < 0·01) for each material between both techniques with r = 0·95 (PB), r = 0·933 (AP) and r = 0·929 (FT). Also, for analysis of CD45+ cells µL(-1) , the measured numbers evaluated with the different techniques did not significantly differ for all three materials analysed. In frozen-thawed samples, the analysis of viability was comparable for both techniques. CONCLUSIONS: The results of this study demonstrate that a direct volumetric analysis of CD34+ cells µL(-1) or CD45+ cells µL(-1) is feasible. This technique represents a simple and economical approach for standardisation of progenitor and stem cell analyses.

Sunitinib in metastatic thymic carcinomas: laboratory findings and initial clinical experience.

BACKGROUND: Thymic carcinoma (TC) is a rare aggressive tumour. Median survival with current treatments is only 2 years. Sunitinib is a multi-targeted tyrosine kinase inhibitor that has shown benefit in various other cancers. METHODS: Laboratory analyses of snap-frozen tumour tissues were performed to detect activation and genetic mutations of receptor tyrosine kinases (RTKs) in TC samples. On the basis of molecular analyses showing activation of multiple RTKs in their tumour, four patients with metastatic TCs refractory to conventional therapies were treated with sunitinib according to standard protocols. RESULTS: RTK analysis in three of the patients showed activation of multiple RTKs, including platelet-derived growth factor-beta and vascular endothelial growth factor 3. Mutations of EGFR, c-KIT, KRAS, and BRAF genes were not found. Administration of sunitinib yielded a partial remission (lasting 2 to 18+ months) according to the RECIST criteria in three patients and stable disease with excellent metabolic response in 18F-FDG-PET in another one. The overall survival with sunitinib treatment ranges from 4 to 40+ months. Withdrawal of the drug in one patient prompted rapid tumour progression that could be controlled by re-administration of sunitinib. CONCLUSIONS: Sunitinib is an active treatment for metastatic TC. A panel of molecular analyses may be warranted for optimal patient selection.

Optical limiting properties of surface functionalized nanodiamonds probed by the Z-scan method.

This work focuses on the optical limiting behavior of surface modified nanodiamonds (DNDs) namely, amino-terminated DNDs (DND-NH2) and hydrogen-terminated DNDs (DND-H). Their relevant nonlinear optical properties for optical limiting are compared to those of unfunctionalized DNDs. The optical limitation is characterized by means of nonlinear transmittance, Z-scan, and scattered intensity assessments when submitted to a nanosecond pulsed Nd:YAG laser operating at a wavelength of 532 nm. It is stated that the largest nonlinear attenuation is attributed to the DND-H system, whereas the exceedingly low threshold values for optical limiting for the DND-H and the DND-NH2 systems is attributed to their negative electron affinity character (NEA). Using Z-scan experiments, it is shown that nonlinear refraction combined with a significant nonlinear absorption predominates in the DND-H suspension, while the pure thermal origin of the nonlinear refractive index change is conjectured in the case of the DNDs. Besides, an amazing valley to peak profile was measured on DND - NH2indicating an unexpected positive sign of the nonlinear refraction coefficient. In addition, a stronger backscattered intensity signal is highlighted for the unfunctionalized DNDs through nonlinear scattering measurements.

The decay accelerating factor mutation I197V found in hemolytic uraemic syndrome does not impair complement regulation.

Hemolytic uremic syndrome is the clinical triad of thrombocytopenia, microangiopathic hemolytic anaemia and acute renal failure. Cases not associated with a preceding Shiga-like toxin producing Escherichia coli are described as atypical HUS (aHUS). Approximately 50% of patients with aHUS have mutations in one of three complement regulatory proteins, Factor H (CFH), membrane cofactor protein (MCP;CD46) or factor I (IF). A common feature of these three proteins is that they regulate complement by cofactor activity. Decay accelerating factor (DAF; CD55) regulates the complement system by disassociating the alternative and classical pathway convertases. Like CFH and MCP, the gene for DAF lies within the regulators of complement activation (RCA) gene cluster at 1q32. In 1998, we described linkage to this region in families with aHUS which led to the discovery of mutations in CFH and MCP. We therefore genotyped DAF in a panel of 46 aHUS patients including families with linkage to the RCA cluster. A mutation, I197V, was identified in one patient with familial HUS which was not found in 100 healthy controls. Molecular modelling of this mutation shows that the I197V mutation does not reside in an area which would be predicted to be important in decay accelerating activity. The expression of I197V on EBV-transformed B lymphocytes was equivalent to that of wild type controls. There was no significant decrease in decay acceleration activity of the recombinantly produced I197V mutant compared with wild type, as measured by a complement-mediated lytic assay. In conclusion, this study, identifies only one mutation in DAF in 46 patients with aHUS. This mutation, I197V, does not impair complement regulation and cannot be implicated in the pathogenesis of aHUS in this patient. This suggests that the complement regulatory abnormality in aHUS is principally one of deficient cofactor activity rather than of decay acceleration activity.

Folding induced supramolecular assembly into pH-responsive nanorods with a protein repellent shell.

We report the synthesis of ABA' triblock peptide-polysarcosine-peptide conjugates featuring two complementary phenylalanine-histidine pentapeptide strands A/A'. These sequences encode for antiparallel ß-sheet formation into folded conjugates, which promote the self-assembly into polysarcosine-shielded core-shell nanorods. These do not cause aggregation of serum proteins in human blood plasma underlining an enhanced stability.

A clickable NHC-Au(i)-complex for the preparation of stimulus-responsive metallopeptide amphiphiles.

We report the synthesis of an alkyne functionalised NHC-Au(i)-complex which is conjugated with amphiphilic oligopeptides using a copper(i) catalysed cycloaddition. The resulting Au(i)-metalloamphiphiles are shown to self-assemble into charge-regulated stimulus-responsive supramolecular polymers in water via a weakly cooperative polymerisation mechanism.

Optical properties of functionalized nanodiamonds.

Detonation nanodiamonds exhibit strong nonlinear optical properties depending on their electronic properties. In the present paper, the nanodiamond functional groups are chemically modified to obtain nanodiamonds with primary amines on their surface. The optical properties of such nanodiamonds placed in water suspensions are studied and compared with the one of classical detonation nanodiamonds. Transmission, scattering and Z-scan experiments are performed for two different wavelengths (532 nm and 1064 nm). A lower threshold for optical limiting associated to more pronounce non-linear optical effects is detected at the wavelength of 1064 nm compared to the one at 532 nm. This effect may be due to a stronger nonlinear backscattering behavior at 1064 nm. Moreover, a striking result obtained from the Z-scan experiments reveals a completely different behavior of the functionalized nanodiamonds for both wavelengths. This result is discussed in regard to the electronic properties of the material and possible charge transfer.

Closing the Gap: Mouse Models to Study Adhesion in Secondary Palatogenesis.

Secondary palatogenesis occurs when the bilateral palatal shelves (PS), arising from maxillary prominences, fuse at the midline, forming the hard and soft palate. This embryonic phenomenon involves a complex array of morphogenetic events that require coordinated proliferation, apoptosis, migration, and adhesion in the PS epithelia and underlying mesenchyme. When the delicate process of craniofacial morphogenesis is disrupted, the result is orofacial clefting, including cleft lip and cleft palate (CL/P). Through human genetic and animal studies, there are now hundreds of known genetic alternations associated with orofacial clefts; so, it is not surprising that CL/P is among the most common of all birth defects. In recent years, in vitro cell-based assays, ex vivo palate cultures, and genetically engineered animal models have advanced our understanding of the developmental and cell biological pathways that contribute to palate closure. This is particularly true for the areas of PS patterning and growth as well as medial epithelial seam dissolution during palatal fusion. Here, we focus on epithelial cell-cell adhesion, a critical but understudied process in secondary palatogenesis, and provide a review of the available tools and mouse models to better understand this phenomenon.

Immigrant women family caregivers in Canada: implications for policies and programmes in health and social sectors.

Migration has become a profound global phenomenon in this century. In Canada, uncoordinated policies, including those related to immigration, resettlement, employment, and government funding for health and social services, present barriers to immigrant women caregivers. The purpose of this paper is to share relevant insights from individual and group interviews with immigrant women family caregivers, service providers and policy influencers, and discuss these in relation to immigration, health and social policy, and programme trends in Canada. The present authors conducted individual interviews with immigrant women family caregivers (n = 29) in phase 1, followed by two group interviews with women family caregivers (n = 7), and two group interviews with service providers and policy-makers (n = 15) in phase 2. Using an inductive approach, the authors employed thematic content data analysis. Immigrant women experienced barriers to health and social services similar to Canadian-born family caregivers, particularly those who have low incomes, jobs with limited flexibility and heavy caregiving demands. These immigrant women family caregivers avoided certain formal services for a variety of reasons, including lack of cultural sensitivity. However, their challenges were compounded by language, immigration and separation from family in the home country. The identified barriers to support reinforce the importance of modifying and expanding policies and programmes affecting immigrant women's ability to care for family members with illnesses or disabilities within the context of Canadian society. Participants recommended changes to policies and programmes to deal with information, transportation, language, attitudinal and network barriers. The various barriers to services and programmes which were experienced by immigrant women caregivers underscore the importance of reviewing policies affecting immigration, caregiving, and access to health and social services. Intersectoral collaboration among agencies is essential to reduce the barriers identified in the present study, and to establish services which are linguistically and culturally appropriate.

[Gas in the portal and mesenteric venous bed in a vascular ileus]. / Plyn v portálním a mezenteriálním venózním recisti pri cévním ileu.

Mesenteric artery embolisation is serious complication of heart diseases. Its mortality is almost 95%. Diagnostics of mesenteric artery embolisation is based not only on clinical examination, but also on CT scan. Presence of the gas in portal and mesenteric venous basin is very suspected from vascular ileus. This sign can be very helpful in diagnostics and consequently in treatment of this disease. Late diagnostics and treatment of vascular ileus has fatal prognosis.

Karyotypic dissection of Hodgkin's disease cell lines reveals ectopic subtelomeres and ribosomal DNA at sites of multiple jumping translocations and genomic amplification.

Although the neoplastic significance of the chromosome changes widespread in Hodgkin's disease (HD) remains obscure, a distinct cytogenetic picture has emerged combining aneuploidy with structural rearrangements clustered at certain breakpoints. Notably absent are the recurrent chromosome translocations which distinguish other hematopoietic neoplasms and serve as clues to underlying oncogene alterations. The paucity of neoplastic cells in HD biopsies hinders detailed chromosome analysis. As an alternative, we investigated a panel of well characterized cell lines by classical and molecular cytogenetics, using single-gene and subtelomeric probes, including three autologous HD examples (HDLM-1/2/3) analyzed by 'spectral karyotyping' - the first complete HD karyotype to be documented. Although complex, most rearrangements in HDLM cells arose in vivo and included few rare but many typical HD breakpoints, notably at the r(ibosomal)DNA regions. Two types of genomic rearrangement involving DNA repeats were conspicuous: insertion and genomic amplification/coamplification of rDNA-the first genomic rDNA rearrangements to be reported in a tumor cell, and the first example of multiple 'jumping translocations' (JT). Of four subtelomeric microsatellite repeats tested in HDLM cells, three exhibited interstitial sites at JT, of which two (at 5qter and 9pter) were respectively associated with deletion of the 5q31-32 myeloid region, and coamplification of a recently described HD-recurrent amplicon at 9p2 together with transcriptionally silent rDNA. Altogether, three out of four HD cell lines carried interstitial 9p subtelomeres and rDNA rearrangements. Taken together, these data suggest tumorigenic rearrangements may be facilitated by 'hitchhiking' along with mobile DNA repeat sequences which may target gene rearrangement at 9p in HD. Southern analysis of parallel rearrangements within rDNA intergenic spacers in HDLM cells highlighted several at, or near, retroposons. As well as validating HD cell lines as cytogenetic models, and resources for identifying genes rearranged in HD, our findings warrant further investigation of the roles of DNA repeat sequences, notably subtelomeric microsatellites, rDNA spacer sequences and retroposons as facilitators and markers of tumor-gene rearrangement.

Association of HELLP syndrome with autoimmune antibodies and glucose intolerance.

OBJECTIVE: HELLP syndrome is a severe form of preeclampsia, characterized by hemolysis (H), elevated liver enzymes (EL), and low platelets (LP), whose pathogenesis is unclear. Autoimmunity is thought to play an important role. After the observation of development of type 1 diabetes in a patient with HELLP syndrome, we assumed a possible disease association based on autoimmune reactions. RESEARCH DESIGN AND METHODS: We examined 70 women with HELLP syndrome for the presence of autoimmune markers and glucose intolerance. Free thyroxine, triiodothyronine, thyroid-stimulating hormone, anti-thyroglobulin antibodies, thyroperoxidase antibodies, thyrotropin receptor antibodies, antinuclear antibodies (ANAs) and anti-DNA, islet cell antibodies, GADA, an oral glucose tolerance test, and HbA1c were determined postpartum. Patients with positive autoimmune markers or glucose intolerance were prospectively followed and repeated testing was performed. There were 60 women with a normal course of pregnancy matched for age, BMI, and number of pregnancies, which served as a control group. RESULTS: From the HELLP patients, 22 (31%) compared with only 6 (10%) control subjects had autoimmune antibodies (P < 0.01). There were 16 HELLP patients (23%) who exhibited only 1 kind of autoantibody (5 ANA, 9 thyroid antibodies, and 2 GADA), whereas in 6 HELLP patients (8.5%) 2 different antibodies were found. In all but 4 patients of the study group, these antibodies disappeared during 3 +/- 1.5 years of follow-up. Glucose intolerance was detected in 22 (31%) of the HELLP patients, 17 of them had impaired glucose tolerance (IGT), and 5 had diabetes, whereas only 4 subjects (6.5%) with IGT at postpartum were found in the control group (P < 0.01). During the follow-up, 2 HELLP patients were still diabetic and another 2 HELLP patients (1 GADA positive) had IGT versus 1 control subject. CONCLUSIONS: Our data give evidence that HELLP syndrome is associated with various autoimmune antibodies and glucose intolerance. Because glucose intolerance and/or autoimmune markers persisted during long-term follow-up in 6 patients with HELLP syndrome versus 1 in the control group, it may become advisable to reexamine patients with HELLP syndrome for detection of diabetes and autoimmune disorders.

Ovarian stimulation in ART - Unwinding pressing issues.

Conventional controlled ovarian stimulation (cCOS) can cause significant discomfort, including ovarian hyperstimulation syndrome (OHSS). Clearly, management of OHSS and poor responder patients requires new strategies to overcome these problems and facilitate the birth of a healthy child with the fewest stimulation cycles. Several alternative methods have been developed. Non-conventional controlled ovarian stimulation (non-cCOS) is based on low-dose stimulation regimens and is often termed "light", "soft", "mini", "minimal", "mild", "low cost", or "low dose IVF". Non-controlled ovarian stimulation therapies (non-COS) include natural cycle IVF or a mixture between non-controlled and non-cCOS, termed "modified natural IVF" or "antiestrogen/aromatase inhibitor/low dose FSH-cycles", in which cycles are monitored but not controlled. These approaches promise to reduce the physical, emotional, and financial burden of IVF therapy while maintaining acceptable pregnancy rates. Such approaches might reduce the risk of OHSS. However, the overall cost per baby increases due to the higher number of stimulation cycles required, and the inconvenience of ovum pick-up still remains. The primary focus should be to obtain several good quality blastocysts after a single cCOS cycle. Thus, adequate numbers of mature oocytes are mandatory. What is more difficult and expensive for patients: several non-COS/non-cCOS cycles to obtain a baby or a single cCOS cycle with a high probability to obtain more than one child? Classic cCOS using the GnRH agonist long protocol followed by single embryo transfer (SET) at the blastocyst stage and aseptic vitrification of surplus embryos optimizes the IVF outcome. This strategy, combined with outpatient management in the case of OHSS, minimizes inconvenience and risks of OHSS. Accumulation cycles (AC) by repeated COS with subsequent freezing of blastocysts, combined with preimplantation genetic screening (PGS), is a promising new approach for low responders, especially in cases of advanced maternal age (AMA).

Complement-protected amphotropic retroviruses from murine packaging cells.

The application of retroviruses generated from murine cells for in vivo gene therapy is restricted primarily because of the rapid inactivation of these viruses by the human complement system. To circumvent this disadvantageous property of murine retroviruses we have generated infectious amphotropic retroviruses that exhibit strong protection against human complement attack. The membrane of these viruses contains a fusion protein, DAFF2A, that is composed of the catalytic domain of the human complement regulatory protein (CRP) decay-accelerating factor (DAF) and the envelope protein of the amphotropic murine leukemia virus (MuLV) 4070A (EnvA). The fusion of two other CRPs, MCP and CD59, to the same amphotropic Env moiety did not lead to equivalent results. The fusion protein DAFF2A was stably expressed in mouse NIH 3T3-based helper cells and independently identified with either alpha-DAF MAb or alpha-Env PAb on the cell membrane. Western blot analysis confirmed the expected molecular weight of the fusion protein. Viral titers obtained from NIH 3T3 helper cell pools were 5 x 10(5) CFU for wild-type amphotropic EnvA virus and 1 x 10(5) CFU for DAFF2A virus, respectively. By blocking the catalytic domain of DAF by pretreatment with alpha-DAF MAb DAFF2A, recombinant virions could be converted to wild-type with respect to sensitivity against human serum. Since the method for producing virions that are protected against human serum should be applicable to any cell type it offers a novel tool for human in vivo gene therapy.

The relationship between peak velocity in the fetal descending aorta and hematocrit in rhesus isoimmunization.

OBJECTIVE: To correlate the peak velocity in the fetal descending aorta, as measured by pulsed Doppler ultrasound, with fetal hematocrit values assessed by funipuncture in pregnancies complicated by rhesus isoimmunization. METHODS: One hundred twelve consecutive funipunctures were performed on 33 rhesus-negative gravidas of 21-36 weeks' gestation (median 30). Doppler flow, corrected for angle, was measured on the fetal descending aorta with pulsed Doppler equipment immediately before funipuncture. Differences between observed peak velocities and the calculated gestational age-dependent upper confidence limits (delta peak velocities) were compared with corresponding differences between observed hematocrits and the calculated lower confidence limits (delta hematocrits), and a regression analysis on the above paired difference values was performed. In addition, the correlation coefficient between delta peak velocities and delta hematocrits was calculated for the first procedure per pregnancy only. RESULTS: The mean peak aortic velocity of anemic fetuses was higher than that of unaffected fetuses (P < .001); delta peak aortic velocities correlated negatively with delta hematocrits (r = -0.66, P < .001). The correlation coefficient between delta peak aortic velocities and delta hematocrits for the first procedure peer pregnancy only was r = -0.72 (P < .001). Prediction of fetal anemia by Doppler using gestational age-dependent 95% confidence limits was possible with positive and negative predictive values of 73 and 66%, respectively. CONCLUSION: Peak aortic velocity, a noninvasive assessment of fetal anemia, may be used as an additional test for monitoring pregnancies complicated by rhesus isoimmunization. However, the limited predictive capacity hampers its clinical usefulness.

An evaluation of the Filshie clip for postpartum sterilization in Austria.

Voluntary sterilization is a popular method of family size limitation. Among other techniques for surgical induction of female sterility, the application of various kinds of clips to the Fallopian tubes has been introduced. The Filshie clips consist of rubber-lined titanium and their use for interval sterilization has been repeatedly published. So far, there are only a few reports regarding the use of Filshie clips during the postpartum period, when tubes are edematous and more friable. Therefore, 300 women voluntarily requesting postpartum surgical sterilization for the purpose of family size limitation were enrolled into a prospective trial. Within 72 h of delivery, 282 women were sterilized under general anesthesia using a subumbilical minilaparotomy approach and Filshie clip application. Of these women, 251 were available for follow-up examination at 6 weeks, 240 at 6 months, 234 at 12 months, and 209 at 24 months after the sterilization procedure. Complication rates were low, and there were no pregnancies during the follow-up period. These results indicate that the application of Filshie clips is a safe and efficacious method of surgical female sterilization in the postpartum period.

Growth deficient fetuses with absent or reversed umbilical artery end-diastolic flow are metabolically compromised.

Controversy continues regarding the clinical relevance of absent or reversed umbilical artery blood flow during diastole. The purpose of this study was to characterize the blood gas and lactate measurements of growth deficient fetuses with absent (ADF) or reversed (RDF) umbilical artery (UA) diastolic flow. In a descriptive study from February 1988 through October 1991, 42 consecutive structurally and karyotypically normal growth deficient fetuses identified to have either ADF or RDF diastolic flow in the UA were studied. Heparinized blood specimens were obtained from them and the pH, PCO2, PO2 and lactate measured. Fourteen of these specimens were obtained from the umbilical vein by cordocentesis and 28 at the caesarean delivery of non-labouring patients. Statistical analyses were performed using Fisher's exact test, Student t-test and linear correlation. All measured parameters in fetuses with ADF or RDF undergoing cordocentesis were significantly abnormal compared to gestational age corrected norms. Both the mean venous and arterial pH of fetuses with RDF were significantly lower than that of fetuses with ADF. With few exceptions, preoperative maternal oxygenation failed to correct the fetal hypoxaemia associated with either ADF or RDF. In the setting of severe fetal growth deficiency secondary to uteroplacental dysfunction, ADF and RDF are clinically reliable indicators of fetal compromise as determined by the umbilical blood gases. RDF is associated with a greater impairment of placental gas exchange than ADF.

Therapy for a patient with oligodontia: case report.

Oligodontia, the congenital absence of numerous teeth, presents many problems for both patient and therapist. Chief among these is the sequence and timing of orthodontic therapy, implant placement, and restorative dentistry. The absence of teeth can pose not only functional but psychosocial problems for young people. This report details a young woman who was diagnosed with oligodontia at age 9, treated using a multidisciplinary approach, and followed for 12 years.