Environmental pollution due to cadmium: measure of semen quality as a marker of exposure and correlation with reproductive potential.

PURPOSE: Contradictory reports exist in the literature regarding an association of cadmium with parameters of semen quality. The aim of the study was to assess cadmium levels in both blood and seminal plasma and to analyze the relationships between cadmium concentrations and lifestyle and semen parameters. MATERIAL AND METHODS: Fifty healthy male volunteers were recruited to provide semen and blood samples. Each patient completed an extensive questionnaire regarding his occupation, residence, social status, diet, water source, smoking habits, and medical and surgical history. Semen analysis was carried out according to WHO guidelines. Detection of cadmium in both semen and blood samples was carried out by means of atomic absorption spectrophotometer. RESULTS: Mean concentrations of cadmium were 8.18 ± 1.6 ng/ml in blood samples and 2.56 ± 0.9 ng/ml in semen samples. Cadmium blood levels were significantly higher in men from industrialized areas and in current smokers, but were not correlated with semen levels. A significant positive correlation was found between cadmium blood levels, number of immotile spermatozoa, and teratozoospermia index (TZI). Significant inverse relationships between cadmium blood concentration and type-a and type a + b motility were found. CONCLUSIONS: The present data show a significant correlation between blood cadmium concentrations, cigarette smoking, occupational exposure, and parameters of semen quality. Such a reduction in spermiogenetic function could be an early marker of a toxic effect by cadmium pollution.

Low-dose estrogen and drospirenone combination: effects on metabolism and endothelial function in postmenopausal women with metabolic syndrome.

PURPOSE: Metabolic syndrome and endothelial dysfunction play a relevant role in the cardiovascular risk in post-menopause. The aim of the study was to assess the effects of a low-dose hemihydrate estradiol and drospirenone combination on cardiovascular risk parameters in postmenopausal women with metabolic syndrome. MATERIALS AND METHODS: Twenty-eight healthy women (group A) and 28 women with metabolic syndrome (group B) were treated with hemihydrate estradiol one mg + drospirenone two mg. At recruitment and after six months, clinical and laboratory parameters of metabolic syndrome were evaluated. Endothelial function was assessed measuring the flow-mediated dilatation of the brachial artery and the intima-media thickness of the common carotid artery. RESULTS: After six months an overall improvement of metabolism was observed in both groups reaching statistical significance for triglycerides, total cholesterolemia, and systolic pressure in group B. A trend to lower baseline flow-mediated dilatation was also found in group B. CONCLUSIONS: Drospirenone improves cardiovascular risk factors and does not impair endothelial function in menopausal women with metabolic syndrome.

Screening for trisomy 21 by maternal age fetal nuchal translucency thickness and maternal serum sample.

AIM: The aim of this paper was to examine the performance of two-stage first-trimester combined screening based on maternal age, fetal nuchal translucency (NT) thickness and maternal serum sample "free beta-human chorionic gonadotropin (ß-hCG) and pregnancy-associated plasma protein-A (PAPP-A)". METHODS: A combined screening for chromosomal anomalies was performed in 713 singleton pregnancies. We performed a two-stage screening with the blood taken at 8+0 to 10+6 weeks and the measurement of NT performed at 12+0 to 12+6 weeks. The maternal age related risk for trisomy 21 was calculated and adjusted according to the gestational age at the time of screening to derive the a-priori risk. The measured free beta-human chorionic gonadotropin (ß-hCG) and pregnancy-associated plasma protein-A (PAPP-A) were converted into a multiple of the median (MoM) for gestational age, adjusted for maternal weight, smoking status, ethnicity, method of conception (spontaneous or IVF) and parity. The measured NT was assessed in relationship of mesasure of CRL. Finally, the risk resulting by NT thickness and biochemical markers were multiplied by the a-priori risk to derive the patient-specific risk. RESULTS: The ultrascreen was considered positive in the case where the risk was greater than 1:250. In this case it was suggested the study of the fetal karyotype through an invasive test. In our study we had 23 positive cases after the combined screening: all patients have opted for the study of fetal karyotype, and in 5 cases the result was abnormal (trisomy 21). We had 1 case where the test was negative but the fetal karyotype was abnormal (trisomy 21). We have calculated sensitivity and false positive rate of the test. CONCLUSION: In our study there were 707 cases with a normal karyotype or delivery of a phenotypically normal baby and 6 cases with trisomy 21. The detection rate of the first trimester screening for chromosomal anomalies was 83% with a false positive rate of 3,2%. The aim of the study was estimated the performance of two-step strategy screening. In our study, the performance of the screening model, based on the two-stage, was not higher than the performance of screening based on a single-step reported in literature. In our opinion, there is no potential advantage in terms of detection rate.

[Phytoestrogens and menopause]. / Fitoestrogeni e menopausa.

Menopause is the interruption of menstrual and reproductive capacity, therefore, that occurs naturally in all women between 48 and 55 years, due to a lower production of gonadal steroids. The period becomes progressively irregular and lack of ovulation and menstrual flow decrease, and finally disappears. The time between the first symptoms and the cessation of the menstrual cycle is called menopause. With the onset of menopause the woman undergoes a series of changes related to estrogen deficiency, which occur in all tissues of the body. In this period one can distinguish an early stage, characterized by hot flashes, mood swings, night sweats and insomnia, and a late phase in which we highlight more symptoms related to the interruption of hormonal such as osteoporosis, obesity, at urogenital and increased incidence of cardiovascular disease. In Italy, only 5.2% of women aged 45-64 years used hormone replacement therapy, and only 20-30% follow a therapy for more than two years, both for psychological reasons, and for fear of side effects. Not surprisingly, therefore, phytoestrogens are given a high importance, as they are considered a natural alternative tank to to their plant origin. Interest in phytoestrogens was born from the observation that postmenopausal women who live in the East have a lower incidence of symptoms, cardiovascular disease, cancer and osteoporosis hormone use, compared to Western women.

Betulinic acid and possible influence on the clearance of Human Papilloma Virus: cytological and virological follow-up.

AIM: Persistent infection with high-risk Human Papilloma Virus (HPV) types is considered necessary for the development of cervical cancer. No such efficient antiviral agent exists at the present time. The aim of this study was to assess whether the use of topical products, vaginal ovules, containing betulinic acid and betulin may influence viral replication in vivo by promoting its negativity. METHODS: We enrolled 62 patients at the colposcopy ambulatory with negative colposcopy or with congenital ectopic or normal transformation zone, but with HPV-test positive for high risk. We divided the patients into two groups: group A had no kind of treatment, group B has undergone a treatment with vaginal ovules containing betulinic acid and betulin. All patients have taken a new HPV-test six months after the first control. RESULTS: After six months, we compared the percentage of negative HPV-test. We observed a percentage of negativity of 93% in group B versus 68% in group A. The data was statistically significant at χ2 test (P<0,05). CONCLUSION: We believe that the results should be investigated further to confirm if there is a correlation between these substances and the increase in the rate of viral negativity.

Pregnancy in a methylmalonic acidemia patient with kidney transplantation: a case report.

Presently pregnancy is no more exceptional in women with metabolic diseases. However, it still poses significant medical problems both before and after childbirth. The challenge is even greater if the mother has undergone organ transplantation, because of her metabolic disease. We report on a case of pregnancy in a patient 29-year-old with methylmalonic acidemia cblA type (OMIM 251100) who received a renal transplantation at the age of 17 for end-stage renal disease (ESRD) caused by her primary disease. During pregnancy neither metabolic crises nor renal function changes were observed in the mother, with the only exception of a mild increase of her systemic blood pressure. To the fetus pregnancy was uneventful and during the first 30 months after birth the baby's neuropsychomotor development was normal and there were no episodes of metabolic derangement. This is evidence that methylmalonicacidemia cblA, even when treated with renal transplantation for inherent ESRD, is no contraindication to pregnancy. It is even possible that a functioning transplanted kidney contributes to improve metabolic parameters.

[The use of transvaginal ultrasound following voluntary interruption of pregnancy to reduce complications due to incomplete curettage]. / L'ecografia transvaginale dopo svuotamento cavitario durante il I trimestre di gestazione e riduzione delle complicanze da incompleta revisione cavitaria strumentale.

AIM: The aim of the study was to assess by what percentage the use of transvaginal ultrasound (TV, curettage) following voluntary interruption of pregnancy (IVG) reduces the incidence of haemorrhagic events resulting from incomplete drainage of the cavity during the first trimester of gestation. METHODS: The present is a randomized prospective study involving 720 women who in the period between January 2005 and January 2007 underwent drainage of the cavity during the first trimester of gestation. The study group involved 360 women who were submitted to TV ultrasound using the Toshiba JustVision 400 system at the end of the surgical procedure. The control group also numbered 360 women; they, by contrast, did not undergo ultrasound examination at the end of surgery. In the study group, in those cases in which the ultrasound examination carried out with a TV probe highlighted an endometrial rima of thickness 8 mm, recurettage was carried out. Patients then underwent further gynecological and ultrasound control examination, 5-8 days after the surgical procedure. RESULTS: The index of complications overall was 3.2%. Retained products of conception were encountered in 2 women in the study group (0.5%) and in 13 women from the control group (3.6%, P<0.05). Vaginal bleeding requiring hospitalization was observed in 2 women in the study group (0,5%) against 6 in the control group (1.6%, P=0.2). Cases of endometritis were diagnosed in only one woman in the study group (0.3%) against 5 in the control group (1.4%) and uterine perforation occurred in only one woman in the control group versus no case in the study group. There were no cases of retained products of conception in women presenting endometrial thickness <8 mm, a dimension obtained with the aid of ultrasound at the end of the surgical procedure. CONCLUSION: The use of ultrasound examination, carried out with the aid of a TV probe immediately following uterine drainage during the first trimester of pregnancy, may considerably reduce the incidence both of post-IVG haemorrhages due to incomplete curettage and of the total number of complications. Evaluation of endometrial thickness is of crucial importance. If this turns out to be =8 mm at the end of the surgical procedure, recurettage of the uterine cavity is indicated.

Female adnexal tumor of probable wolffian origin: clinicopathological, immunohistochemical and cytofluorimetric analyses of a 22-year-old virgin. case report.

The term female adnexal tumor of probable Wolffian origin "FATWO" designs this tumor wich arises by the rare persisting remnants of the mesonephric duct (Wolffian duct). About 40 cases have been reported in literature. Few cases of recurrence have been reported, FATWO usually shows no signs of hormonal activity. We report a case of the youngest patient affected by FATWO in October 2002. At laparotomy the left adnexa were deformed by a well-capsulated mass, totally removed and sent to the pathologist with a specimen of peritoneal fluid and of the omentum. The histological examination showed a prevalent tubular structure with focal retiform area, without intraluminal mucines. Immunohistochemical findings of the case reported are similar to those described by other authors, except for inhibin which has not been detected by us. The cytofluorimetry showed the low presence of aneuploid cells, with a very low prolifing component (< 1%).

Haematosalpinx of fallopian tube: isolated torsion with congenital abnormal vascularisation.

We report a case of haematosalpinx due to torsion of the Fallopian tube with congenital abnormal blood supply in a girl. The symptoms were not specific and the first diagnosis was a pelvic complex mass like a tumour. The authors discuss the possible aetiology.

Fertility after laparoscopic myomectomy of large uterine myomas: operative technique and preliminary results.

OBJECTIVE: The purpose of this study was to evaluate indications and complications of laparoscopic myomectomy with regard to the reproductive outcome of infertile women with a large leiomyomatous uterus. METHODS: From January 1997 to July 1999, 144 patients underwent laparoscopic myomectomy for a myoma measuring > or = 5 cm in diameter. Indications for surgery were increase in size of myoma in infertile patients (70.8%), pain (44.4%) or abnormal bleeding (68%). Average size of myomas were 7.8 cm with a range of 5 cm to 18 cm. The myomas were intramural/submucosal (n = 108), subserosal (n = 15), intraligamentous (n = 14) and peduncolated (n = 7). The laparotomy conversion rate was 1.39% (n = 2); one case required a blood transfusion. Operating time ranged from 58 to 180 minutes with an average of 95 minutes. Average hospital stay was 2.6 days and the overall complication rate was 2.08%. Eighteen patients (12.5%) underwent second-look laparoscopy. The rate of postoperative adhesion was 33.3%; there were no adnexal adhesions. In all cases ultrasonography was done one day after the operation and five weeks postoperatively to compare wound healing, the last control showed an irregular hypodense area in only 14 patients (9.7%). RESULTS: Twenty-six patients operated on in 1997 went on to conceive: nine vaginal deliveries, 12 Caesarean sections, four miscarriages and one ectopic pregnancy. No uterine rupture was observed. The pregnancy rate for patients submitted to laparoscopic myomectomy in 1997 (n = 38) was 34.21% at six months (n = 13) and 55.26% (n = 21) at 12 months after the procedures. CONCLUSIONS: Our preliminary results confirm that conception occurs in the majority of infertile women with a large leiomyomatous uterus who undergo myomectomy and second-look laparoscopy leads to a low complication rate.

Antenatal diagnosis and obstetric management of cystic hygroma occurring in twin pregnancies. A case report.

The lymphatic system has an early development in the embryo. Usually, its development is complete by the 40th postconceptional day. If this connection fails to develop, lymphatic stasis and dilatation of the lymphatic channels may occur, causing a number of pathologies such as: lymphangiomas, lymphangiectasis and cystic hygromas. Prenatal diagnosis can be made during the first trimester of pregnancy by ultrasonographic examination. A case of a twin pregnancy associated with cystic hygroma and bilateral hypoplasia of lower and upper limbs of both foetuses without chromosomal abnormalities is reported.

Depot leuprorelin acetate versus danazol in the treatment of infertile women with symptomatic endometriosis.

PURPOSE OF INVESTIGATION: Endometriosis is a common finding in women with infertility, but the mechanism by which it renders a woman infertile remains unclear. The medical treatment of pelvic endometriosis includes hormonal therapy that directly attacks endometriosis lesions or indirectly by inhibiting endometrial proliferation through estrogenic deprivation. The aim of this study was to compare the efficacy and safety of leuprorelin acetate depot and danazol for endometriosis in infertile women. METHODS: This randomized trial involved 81 women 19-41 years old with regular menses and known pelvic endometriosis who were recruited from the Fertility Center of the Second University of Naples between 1992 and 1999. Fifty-four women were given 3.75 mg of leuprolide acetate depot every 28 days for 24 weeks and the remaining 27 took 200 mg of danazol three times daily for 24 weeks. Efficacy assessments were based on pre-admission and end-of-treatment laparoscopic scores and subjective symptoms scores at 4-week intervals during and after treatment. Safety was evaluated by adverse events and clinical laboratory tests. RESULTS: In each group, endometriosis growth and symptoms significantly improved during treatment (p < 0.001). Significantly fewer patients randomized to leuprorelin acetate (5.5%) withdrew during treatment compared with 18.5% randomized to danazol (p < 0.05). After treatment symptoms returned in each group, but severity was less than at admission at all time points (p < 0.02). Hypoestrogenic side-effects were more common in those receiving leuprorelin, particularly hot flushes, but anabolic/androgenic side-effects of weight gain and acne were more common in those receiving danazol. CONCLUSION: Both leuprorelin acetate depot and danazol are effective in the treatment of endometriosis in infertile patients. The hypoestrogenic side-effects of leuprorelin may be better tolerated than the androgenic, anabolic effects of danazol.

A syndrome characterized by psychiatric disorders, recurrent mucosal infections and natural immunity deficits: clinical approach.

The authors summarize their own previous work on the identification of a subset of patients characterized by psychiatric disorders, recurrency of mucosal infections and impaired natural immunity. The diagnostic approach to these patients based on the close collaboration between infectivologists, immunologists and psychiatrists is described with the aim to find out combined treatments for the amelioration of clinical manifestations.

Therapy with efavirenz plus indinavir in patients with extensive prior nucleoside reverse-transcriptase inhibitor experience: a randomized, double-blind, placebo-controlled trial.

A randomized, double-blind, placebo-controlled trial compared efavirenz (600 mg every 24 h) plus indinavir (1000 mg every 8 h) with placebo (every 24 h) plus indinavir (800 mg every 8 h) among 327 nucleoside analogue reverse-transcriptase inhibitor (NRTI)-experienced human immunodeficiency virus (HIV)-infected adults. Patients received 50 cells/mm(3), >10,000 plasma HIV-1 RNA copies/mL, and no prior protease inhibitor or non-NRTI therapy. Patients had a mean of 2.8 years of prior NRTI therapy. At 24 weeks, plasma HIV-1 RNA level was <400 copies/mL in 68.2% of efavirenz versus 52.4% of placebo recipients (P=.004). CD4 cell count increases were 104+/-9 cells/mm(3) and 77+/-10 cells/mm(3) in efavirenz and placebo recipients, respectively (P=.023). Responses in efavirenz recipients were sustained at 48 weeks. Thus, efavirenz plus indinavir with concomitant NRTIs is effective therapy for NRTI-experienced patients.

A multicentre, randomized, double-blind, placebo-controlled trial of naltrexone in the treatment of alcohol dependence or abuse.

The opioid antagonist, naltrexone, is reported, in single centre studies, to improve the clinical outcome of individuals with alcohol dependence participating in outpatient psychosocial programmes. This is the first multicentre controlled study to evaluate the efficacy and safety of naltrexone as adjunctive treatment for alcohol dependence or abuse. Patients who met criteria for alcohol dependence (n = 169) or alcohol abuse (n = 6) were randomly assigned to receive double-blind oral naltrexone 50 mg daily (n = 90) or placebo (n = 85) for 12 weeks as an adjunct to psychosocial treatment. The primary efficacy variable was time to first episode of heavy drinking; secondary efficacy assessments included time to first drink, alcohol consumption, craving, and changes in the serum biological markers gamma-glutamyl transferase (GGT), and aspartate and alanine aminotransferases. Compliance was assessed by tablet counts and, in the naltrexone-treated group, by measurement of urinary concentrations of 6-ss-naltrexol. Forty-nine (58%) patients randomized to placebo and 53 (59%) randomized to naltrexone did not complete the study. In intention-to-treat analyses, there was no difference between groups on measures of drinking. The median reduction from baseline of serum GGT (P: < 0.05) and the reductions in alcohol craving (Obsessive and Compulsive Drinking Scale: OCDS) were greater in the naltrexone group (P: < 0.05), from approximately half-way through the study. Of 70 patients (35 placebo; 35 naltrexone) who met an a priori definition of compliance (80% tablet consumption, attendance at all follow-up appointments), those allocated to naltrexone reported consuming half the amount of alcohol (P: < 0.05), had greater median reduction in serum GGT activity (P: < 0.05), and greater reduction in alcohol craving (OCDS total score: P: < 0.05; Obsessive subscale score: P: < 0.05), compared to patients in the placebo group. Use of naltrexone raised no safety concerns. Naltrexone is effective in treating alcohol dependence/abuse in conjunction with psychosocial therapy, in patients who comply with treatment.

[Neuronal dysplasia of the colon. A case report]. / Displasia neuronale del colon. Descrizione di un caso clinico.

A clinical case of Neuronal Intestinal Dysplasia (NID) is reported. This is an important pathology which is part of congenital alterations of bowel's innervation. This disease consists of two forms (A and B) which occur during different ages causing problems in intestinal motility with consequent various symptoms. Actually NID is diagnosed easier than in the past because histopathological techniques are very refined and permit to distinguish NID from cases once classified as Hirschsprung's disease. Histology can show an increase of Ach and Ldh activity in cholinergic fibers of lamina propria and of circular layer of intestinal wall. In this report a chronic status of constipation refractory to medical therapy has been treated with a total colectomy and an ileo-rectal anastomosis, resulting in a complete resolution of symptoms.

Induced abortion and contraception: survey of 576 young women in Naples.

The aim of the study was to investigate the link between induced abortion and contraceptive methods. Five hundred and seventy-six women who underwent induced abortions at the Obstetrics and Gynecology Institute of the Second University of Naples were interviewed. They were asked about their knowledge of contraception methods; age, school attendance rate and marital status were also considered. The women were 27.8 (mean age) years old, high school educated (58.5%), married (41%), nulliparous (63%). Twenty-four percent of the women admitted a previous induced abortion. Withdrawal (176 women), condoms (104) and oral contraceptives (74 women) were the most widely used contraceptive methods. Withdrawal (37%) resulted in being the most utilized method during the cycle in which conception occurred; no method had been employed by 31% of the women. We found that 35% of the women had used their contraceptive method in a regular way but become pregnant nevertheless. Another group of 40% had forgotten to use their contraceptives for a few days and became pregnant by accident. In the last group of 25% of the women had not used any contraceptive methods. The diffusion of modern methods of fertility regulation influences the number of induced abortions as shown by its reduction since 1982. Our data confirm that induced abortion is the consequence of an insufficient use of modern contraceptives. Therefore more information is necessary to get women and men to use contraceptive methods regularly.

Efavirenz plus zidovudine and lamivudine, efavirenz plus indinavir, and indinavir plus zidovudine and lamivudine in the treatment of HIV-1 infection in adults. Study 006 Team.

BACKGROUND: Efavirenz is a nonnucleoside reverse-transcriptase inhibitor of human immunodeficiency virus type 1 (HIV-1). We compared two regimens containing efavirenz, one with a protease inhibitor and the other with two nucleoside reverse-transcriptase inhibitors, with a standard three-drug regimen. METHODS: The study subjects were 450 patients who had not previously been treated with lamivudine or any nonnucleoside reverse-transcriptase inhibitor or protease inhibitor. In this open-label study, patients were randomly assigned to one of three regimens: efavirenz (600 mg daily) plus zidovudine (300 mg twice daily) and lamivudine (150 mg twice daily); the protease inhibitor indinavir (800 mg every eight hours) plus zidovudine and lamivudine; or efavirenz plus indinavir (1000 mg every eight hours). RESULTS: Suppression of plasma HIV-1 RNA to undetectable levels was achieved in more patients in the group given efavirenz plus nucleoside reverse-transcriptase inhibitors than in the group given indinavir plus nucleoside reverse-transcriptase inhibitors (70 percent vs. 48 percent, P<0.001). The efficacy of the regimen of efavirenz plus indinavir was similar (53 percent) to that of the regimen of indinavir, zidovudine, and lamivudine. CD4 cell counts increased significantly with all combinations (range of increases, 180 to 201 cells per cubic millimeter). More patients discontinued treatment because of adverse events in the group given indinavir and two nucleoside reverse-transcriptase inhibitors than in the group given efavirenz and two nucleoside reverse-transcriptase inhibitors (43 percent vs. 27 percent, P=0.005). CONCLUSIONS: As antiretroviral therapy in HIV-1-infected adults, the combination of efavirenz, zidovudine, and lamivudine has greater antiviral activity and is better tolerated than the combination of indinavir, zidovudine, and lamivudine.

Possible interactions between dietary antioxidants and chemotherapy.

Many patients treat themselves with oral antioxidants and other alternative therapies during chemotherapy, frequently without advising their conventional health care provider. No definitive studies have demonstrated the long-term effects of combining chemotherapeutic agents and oral antioxidants in humans. However, there is sufficient understanding of the mechanisms of action of both chemotherapeutic agents and antioxidants to predict the obvious interactions and to suggest where caution should be exercised with respect to both clinical decisions and study interpretation. This article will describe these potential interactions and areas of concern, based on the available data. It will also suggest several potential courses of action that clinicians may take when patients indicate that they are taking or plan to use alternative therapies.

The safety profile of naltrexone in the treatment of alcoholism. Results from a multicenter usage study. The Naltrexone Usage Study Group.

BACKGROUND: Naltrexone hydrochloride is the first medication approved in the United States for the treatment of alcohol dependence in almost 50 years. This study was designed to collect safety data in a setting that reflected the expected clinical use of naltrexone. METHODS: This was a 12-week, nonrandomized, open-label usage study conducted in 40 alcoholism treatment centers throughout the United States, including free-standing alcoholism treatment programs, university clinics, Veterans Administration hospitals, and office-based primary care practices. Eligible patients were assigned, at the investigators' discretion, to a naltrexone treatment group or to a reference group that did not receive study medication. At study entry, patients must have been abstinent from alcohol for 1 to 6 weeks and enrolled in a psychosocial treatment program for alcoholism. Patients often underrepresented in controlled clinical trials, including women and patients with comorbid medical and psychiatric illness, were eligible. Patients with polysubstance abuse or infection with the human immunodeficiency virus were not excluded. RESULTS: Of 865 patients enrolled, 570 received naltrexone and 295 were in a reference group. The most common new-onset adverse clinical events in the naltrexone group were nausea (9.8%) and headache (6.6%). Naltrexone was discontinued in 15.0% of patients because of adverse events, most frequently nausea. The results of liver function tests in the naltrexone group were similar to those in the reference group. No death occurred during the study. CONCLUSIONS: This is the largest study to date describing the safety of naltrexone in a heterogeneous population of persons with alcoholism. No new safety concerns were identified.