Effects of estrogen receptor α and ß on the expression of visfatin and retinol-binding protein 4 in 3T3-L1 adipocytes.

The aim of this study was to investigate the effects of estrogen and estrogen receptor α (ERα) and ß (ERß) on the expression of visfatin and retinol-binding protein 4 (RBP4) by treating 3T3-L1 adipocytes with estradiol (E2), estrogen receptor agonists and antagonists. Mature adipocytes were exposed to E2, the ERα agonist, 4,4',4''-(4-propyl-[1H]-pyrazole-1,3,5-triyl)trisphenol (PPT), the ERß agonist, 2,3-bis(4-hydroxyphenyl)-propionitrile (DPN), E2 with the ERα antagonist, 1,3-bis(4-hydroxyphenyl)-4-methyl-5-[4-(2-piperidinylethoxy)phenol]-1H-pyrazole dihydrochloride (MPP), and E2 with the ERß antagonist, (5R, 11R)-5,11-diethyl-5,6,11,12-tetrahydro-2,8-chrysenediol [(R,R)-THC], at various concentrations. To determine the effects of ER subtypes on the expression of adipokines, quantitative reverse transcriptase-polymerase chain reaction (qRT-PCR) and western blot analyses were performed. E2 concentrations of 10-5 and 10-6 mol/l induced a statistically significant increase in the expression of RBP4 (P=0.012 and P=0.011, respectively). In the cells treated with 10-5 mol/l PPT, RBP4 expression significantly increased (P<0.05) in a dose-dependent manner. Treatment with the ERα antagonist, MPP (10-5 mol/l), and E2 suppressed the expression of RBP4 (P=0.032). However, the expression of RBP4 was not significantly altered when the cells were treated with the ERß agonist or antagonist. The expression of visfatin was not affected by different concentrations of E2 and ERs. 17ß-estradiol significantly increased the secretion of RBP4 and upregulated RBP4 expression via ERα but not ERß in 3T3-L1 adipocytes. RBP4 expression was regulated by estrogen in the 3T3-L1 adipocytes and this effect was selectively mediated by ERα.

Effect of ovariectomy, 17-beta estradiol, and progesterone on histology and estrogen receptors of bladder in female partial bladder outlet obstruction rat model.

AIM: The aim of this study was to investigate the effect of bilateral ovariectomy (OVX), 17-beta estradiol (E2), and progesterone (P4) on the histology and estrogen receptor (ER) expression of the bladder using a female partial bladder outlet obstruction (pBOO) rat model. MATERIAL AND METHODS: A total of 60 female Sprague-Dawley rats were evenly assigned into six groups of 10 each. Group A served as the control. Groups B-F underwent induced pBOO. Groups C-F underwent OVX. Groups D-F were given E2 (0.1 mg/kg/day), Group E was given P4 (1 mg/kg/day), and Group F was given P4 and dehydroepiandrosterone (DHEA) (300 µg/kg/day) by an Alzet pump. Four weeks later, serum E2 and P4 levels were evaluated. Each rat was anesthetized and the urinary bladder was removed for weighing and histological study. RESULTS: Expression of ER-ß was not significantly different between the control group and the other study groups. pBOO was shown to increase both bladder weight and detrusor muscle thickness. OVX had an additive effect to BOO on increased blood vessel density in the bladder. E2 was shown to increase blood vessel density, while P4 supplementation decreased blood vessel density. DHEA did not cause any significant effects on blood vessel density. CONCLUSION: Hormone therapy did not change the expression of ER in bladder outlet obstruction. Estradiol stimulated the increased angiogenesis of the bladder detrusor but P4 decreased the angiogenesis of the bladder detrusor. DHEA had no effect on the bladder detrusor.

Anti-proliferative effect of Klimaktoplan® on human breast cancer cells.

OBJECTIVE: With the health concerns of menopausal hormone replacement therapy, alternatives have been sought. Klimaktoplan® is a homeopathic formulation consisting of four main components and has been used for relief of menopausal symptoms for a long time. The study investigated the safety of Klimaktoplan® through its effect on the proliferation of breast cancer (MCF-7) and non-malignant mammary epithelial cells (MCF-10A). METHODS: MCF-7 and MCF-10A cells were cultured in 312.5, 625, and 1,250 µg/ml Klimaktoplan®. 17-Beta estradiol (E2) and medroxyprogesterone 17-acetate (MPA) were used for comparison with Klimaktoplan®. E2 only (0.001, 0.01, and 0.1 µM), and the combination of E2 (0.001, 0.01, and 0.1 µM) and MPA (0.01, 0.1, and 1 µM) were tested. Control cells for Klimaktoplan® and E2 groups were treated with dimethylsulfoxide (DMSO), and DMSO + ethanol was used for the combination group. Cellular proliferation was evaluated by the formation of insoluble formazan after incubation of 4 days. RESULTS: Klimaktoplan® had a concentration-dependent anti-proliferative effect on breast cancer cells at 625 and 1,250 µg/ml, while not affecting proliferation of non-malignant mammary cells at any tested concentration. The effect of lactose was evaluated as lactose (the adjuvant of Klimaktoplan®) affect cell growth. E2 and lactose increased the proliferation of both malignant and non-malignant cells. The effect of E2 + MPA on the proliferation of malignant and non-malignant mammary cells was lower than estradiol only, but was higher than control. CONCLUSIONS: Klimaktoplan® has an anti-proliferative effect on breast cancer cells, but not for non-malignant mammary epithelial cells, unlike E2 and E2 + P. With further research, KP would be a good alternative or additive in women with menopausal symptoms who wish to avoid conventional E or E + P hormone therapy.

Cancer of the supernumerary ovary in Mayer-Rokitansty-Küster-Hauser Syndrome: A case report.

Mayer-Rokitansty-Küster-Hauser (MRKH) syndrome is a Müllerian anomaly that presents with varying degrees of uterovaginal aplasia and is secondarily associated with cervicothoracic, auditory and skeletal anomalies. However, MRKH syndrome patients have normal and functional ovaries. A supernumerary ovary is an extremely rare form of an ectopic ovary and there are no reported cases of MRKH syndrome with cancer of the supernumerary ovary in the current literature. A 31-year-old female with a history of MRKH syndrome that was diagnosed 4 years previously presented with abdominal pain and a suspected malignant pelvic mass was identified. During the staging surgery, both ovaries were separated from the main mass, observed and removed. A third ovary was discovered in the pelvic mass and the diagnosis of primary ovarian cancer from the third ovary was confirmed by immunohistochemistry. We report the first known case of cancer of the supernumerary ovary in a patient with MRKH syndrome. Although both ovaries were confirmed to be normal in the patient with MRKH syndrome, we propose that an ovarian neoplasm should be considered in the diagnosis of a pelvic mass.

Fertility preservation in women with cancer.

Fertility preservation (FP) is an effort to retain the fertility of cancer patients, and as an emerging discipline, it plays a central role in cancer care. Because of improvement in diagnostic and therapeutic strategies, an increasingly large number of patients are surviving with cancer. FP specialists should make an effort to spread the significance of FP among reproductive women with cancer and provide appropriate education both for associated physicians and for cancer patients who wish to preserve their fertility. Physicians who take part in the initial diagnosis and management of cancer should consider the importance of early referral of young cancer patients to FP specialists and take care of those patients by providing timely information and appropriate counseling. Individualized treatment strategies should be delivered depending on the patient's situation with appropriate team approach.

Transvaginal single-port natural orifice transluminal endoscopic surgery for benign uterine adnexal pathologies.

Transvaginal natural orifice transluminal endoscopic surgery (NOTES) with pneumoperitoneum has been used in cholecystectomies, appendectomies, and nephrectomies, but transvaginal NOTES using a single port in gynecologic procedures has not been described despite gynecologist familiarity with the vaginal approach. We performed transvaginal single-port NOTES in 10 women with benign uterine adnexal disease: oophorectomy in 3 patients, salpingostomy and salpingectomy in 2 each, and ovarian cystectomy, paratubal cystectomy, and ovarian wedge resection in 1 each. The patients were discharged at 1 or 2 days postoperatively, and were satisfied, with minimal pain, no abdominal scar, and no complications at 2-month follow-up. We conclude that transvaginal single-port NOTES to treat benign uterine adnexal disease is a feasible and attractive option.

The relationship between fat depot-specific preadipocyte differentiation and metabolic syndrome in obese women.

OBJECTIVE: Obesity is strongly associated with metabolic syndrome, but not all obese individuals display a clustering of metabolic risk factors. Recent studies have shown that in vitro subcutaneous (SC)-preadipocyte differentiation is negatively associated with obesity. These results suggest that impaired adipogenesis is an important factor linking obesity to metabolic disorders. We examined whether in vitro preadipocyte differentiation is associated with metabolic syndrome, independent of obesity. DESIGN/PATIENTS/MEASUREMENTS: Paired adipose tissue samples were obtained from the 13 nonobese women and the 65 obese women. The CD34(+)/CD31(-) cells were isolated from the stromal-vascular fraction of both SC and omental (OM) fat depots by immune magnetic separation, and the subset was cultured with a differentiation cocktail. Then, we analysed the relationship between the degree of preadipocyte differentiation and metabolic factors. RESULTS: Obese women without metabolic syndrome (n = 37) had significantly higher SC-preadipocyte differentiation than equally obese women with metabolic syndrome (n = 28); however, OM-preadipocyte differentiation was similar in both groups. SC-preadipocyte differentiation was strongly correlated with triglycerides, HDL cholesterol, homoeostasis model assessment of insulin resistance and OM-adipocyte size. However, OM-preadipocyte differentiation was not correlated with any of these parameters. CONCLUSIONS: This study identified that SC-preadipocyte differentiation is associated with metabolic syndrome independent of obesity, whereas OM-preadipocyte differentiation is not. These findings suggest that, in the setting of obesity, an enhanced adipogenic capacity of SC depots could be protective for metabolic syndrome. Our data underscores an interaction between adipose tissue homoeostasis and metabolic disorder.

The relationship between blood mercury level and osteoporosis in postmenopausal women.

OBJECTIVE: Postmenopausal women have a higher prevalence of osteoporosis compared with premenopausal women. Postmenopause status has been found to be an independent risk factor for osteoporosis. Several studies have reported that heavy metals, including lead (Pb), mercury (Hg), cadmium (Cd), and arsenic (As), have detrimental effects on bone. The aim of this study was to evaluate the association among heavy metals, including Pb, Hg, Cd, and As, bone mineral density, and osteoporosis in postmenopausal Korean women. METHODS: We conducted a cross-sectional study of 481 postmenopausal women, all of whom were enrolled in the Korean National Health and Nutrition Examination Survey in 2008. Bone mineral density was measured using dual-energy x-ray absorptiometry. Blood Pb, Hg, and Cd and urinary As levels were measured. RESULTS: Postmenopausal women with higher blood Hg levels were more likely to be younger and have higher vitamin D levels, fish consumption, and prevalence of osteoporosis. On multivariate logistic regression analysis, postmenopausal women with blood Hg levels in the fourth quartile had a 0.36-fold decreased risk of having osteoporosis compared with those with levels in the first quartile, after adjustments for age, body mass index, alcohol intake, smoking history, exercise, use of oral contraceptive pills, hormone therapy, intake of caloric energy and calcium, fish consumption, and vitamin D level. However, there was no association between other heavy metals and osteoporosis. CONCLUSIONS: High blood Hg levels were associated with a lower risk of having osteoporosis in postmenopausal women. Because biomarkers of all four metals measured in this study reflect recent exposures, further studies are necessary to clarify the association of osteoporosis with the level of heavy metals in biomarkers for long-term exposure such as hair or fingernail.

Adolescent pregnancy is associated with osteoporosis in postmenopausal women.

OBJECTIVE: Adolescence is a critical time of life to accumulate bone for peak bone mass. Factors that may interfere with bone mass accrual during this period may increase the risk of osteoporosis. Several studies have reported that pregnancy during adolescence has detrimental effects on bone mass measurements after pregnancy. However, less is known about how adolescent pregnancy affects bone mineral density (BMD) and osteoporosis after menopause. The aim of this study was to evaluate the association between adolescent pregnancy and osteoporosis in postmenopausal Korean women. METHODS: We conducted a cross-sectional study of 719 postmenopausal women, all of whom were enrolled in the Korean National Health and Nutrition Examination Survey in 2008. BMD was measured using dual-energy x-ray absorptiometry. RESULTS: Postmenopausal women with histories of adolescent pregnancy had lower BMD of the total hip, femoral neck, and lumbar spine than did women without histories of adolescent pregnancy. Multivariate logistic regression analyses revealed that postmenopausal women with history of adolescent pregnancy were at increased risk of osteoporosis (odds ratio, 2.20; 95% CI, 1.12-4.30) compared with women without history of adolescent pregnancy after adjustments for age, body mass index, marital status, education level, household income, alcohol intake, smoking history, exercise, age at menarche, age at menopause, parity, hormone therapy use, intake of energy and calcium, and vitamin D level. CONCLUSIONS: Adolescent pregnancy may be a predictor of osteoporosis in postmenopausal women.

Ovarian hyper-response to administration of an GnRH-agonist without gonadotropins.

Several case reports have indicated that a small subgroup of patients may develop ovarian hyperstimulation following the administration of gonadotropin-releasing hormone agonists (GnRHa) without gonadotropins. However, since only few such cases have been published, it is unclear what course to follow in subsequent cycles after ovarian hyperstimulation in the first cycle using only GnRHa. A 33-yr-old woman was referred to in vitro fertilization for oocyte donation. A depot preparation (3.75 mg) of tryptorelin without gonadotropins induced ovarian multifollicular enlargement with high estradiol level, and was followed by human chorionic gonadotropin administration and oocyte retrieval. In a subsequent cycle of the same patient, a low dose of tryptorelin (0.05 mg) did not induce ovarian hyperstimulation, and resulted in clinical pregnancy. This report shows potential management of ovarian hyperstimulation following the administration of GnRHa without gonadotropins.

Serum ferritin levels are associated with metabolic syndrome in postmenopausal women but not in premenopausal women.

OBJECTIVE: Ferritin, a marker of total body iron stores, is known to be associated with the risk of having metabolic syndrome and has been demonstrated to increase after the onset of menopause. Postmenopause status is an important determinant of metabolic syndrome. The aim of this study was to perform a menopause status-specific analysis of the association between ferritin levels and metabolic syndrome. METHODS: We conducted a cross-sectional study of 3,082 participants (1,691 premenopausal women and 1,391 postmenopausal women), all of whom were enrolled in the Korean National Health and Nutrition Examination Survey 2007. RESULTS: Premenopausal and postmenopausal women with metabolic syndrome had higher ferritin levels than did those without metabolic syndrome. After adjustments for age; body mass index; alcohol intake; smoking history; exercise; hormone therapy use; hemoglobin, aspartate aminotransferase, and alanine aminotransferase levels; and intake of energy and iron, multivariate logistic regression analysis revealed that postmenopausal women with ferritin levels in the third tertile had an increased risk of having metabolic syndrome (odds ratio, 1.62; 95% CI, 1.04-2.81) compared with postmenopausal women with levels in the first quartile. No such association was detected in premenopausal women. CONCLUSIONS: Increased ferritin levels may be a determinant for metabolic syndrome in postmenopausal women but not in premenopausal women.

Serum calcium level is associated with metabolic syndrome in elderly women.

OBJECTIVE: To investigate the association between serum calcium level and metabolic syndrome, defined using the National Cholesterol Education Program-Adult Treatment Panel III (NCEP-ATP III) definition, in Korean elderly women. STUDY DESIGN: We conducted a cross-sectional study of 1941 elderly women (mean age: 65.16±4.58 years) who participated in annual health examinations at Korea university Medical Center between January 1, 2006 and December 31, 2009 and had normal serum calcium levels. RESULTS: Women with metabolic syndrome had higher serum calcium levels than those without metabolic syndrome (9.26±0.35 vs. 9.20±0.33, P<0.001). In multiple logistic regression analysis, serum calcium level within normal range was positively associated with the risk of having metabolic syndrome (odds ratio 2.12, 95% confidence interval 1.50-2.98). With regard to components of metabolic syndrome, serum calcium level was also positively associated with the risk of having high triglyceride, and high glucose, whereas it was inversely associated with the risk of having low high density lipoprotein. However, there was no association of serum calcium level with abdominal obesity or high blood pressure. CONCLUSIONS: The higher was the level of calcium within normal range, the greater were the odds of metabolic syndrome in healthy and elderly women. Prospective studies are needed to investigate the role of calcium in the development of metabolic syndrome in the future.

Relationship between serum estradiol and follicle-stimulating hormone levels and urodynamic results in women with stress urinary incontinence.

INTRODUCTION AND HYPOTHESIS: The purpose of the current study was to determine the relationship between the serum estradiol (E2) and follicle-stimulating hormone (FSH) levels and the urodynamic study results in women with stress urinary incontinence (SUI). METHODS: Eighty women were selected among patients who underwent urodynamic testing for SUI. Basic demographic features were evaluated and laboratory tests were performed. Multiple linear regression analyses were performed among the serum E2 and FSH levels and urodynamic results. RESULTS: E2 had a negative correlation with the Q-tip test in post-menopause. FSH had positive correlations with the post-void residual volume in the uroflowmetry and the voiding and flow times in the pressure-flow study in all of the patients and a negative correlation with the peak flow rate in the pressure-flow study in pre-menopause. CONCLUSIONS: E2 and FSH were associated with urodynamic parameters in female patients with SUI.

Human feeder cells can support the undifferentiated growth of human and mouse embryonic stem cells using their own basic fibroblast growth factors.

In the culture system using human feeder cells, the mechanism through which these cells support undifferentiated growth of embryonic stem cells (ESCs) has not been well investigated. Here, we explored the mechanisms of 3 kinds of human feeder cells, including human placental cells from the chorionic plate, human bone marrow stromal cells, and human foreskin fibroblasts. First, we determined that undifferentiated growth of 2 kinds each of human (H1 and HSF6) and mouse (D3 and CE3) ESCs was possible in all human feeder cell types tested (human placental cells, human bone marrow stromal cells, and human foreskin fibroblasts), without the need for exogenous cytokine supplementation including basic fibroblast growth factor (bFGF) and leukemia inhibitory factor. We then prepared their corresponding endogenous bFGF-knockout feeders using siRNA and tried to maintain human and mouse ESCs in their undifferentiated state; however, neither human nor mouse ESCs could be maintained in bFGF-knockout human feeder cells. The expressions of stemness markers such as Oct-4 and Nanog were significantly decreased in the bFGF-knockout group compared with those in the controls, and differentiation had already occurred, despite the undifferentiated morphologic appearance of the ESCs. In conclusion, human feeder cells are able to support the undifferentiated growth of human and mouse ESCs via bFGF synthesis. Further, a bFGF-dependent pathway might be crucial for maintaining the undifferentiated characteristics of mouse and human ESCs.

Relationship between uric acid and metabolic syndrome according to menopausal status.

OBJECTIVE: Uric acid, the levels of which have been shown to increase after menopause, has been associated with metabolic syndrome. The prevalence of metabolic syndrome has also been determined to increase after menopause. Therefore, we surmised that menopausal status-specific analyses for the characterisation of the relationship between uric acid and the metabolic syndrome were warranted. METHODS: We included 1644 patients: 1018 premenopausal women and 626 postmenopausal women, all of whom participated in annual health examinations at Anam Hospital in Seoul, Korea, from January 2008 through December 2008. RESULTS: On the multivariate logistic regression analysis, uric acid was identified as an independent risk factor for metabolic syndrome in both premenopausal and postmenopausal women. Uric acid levels had different relationships with blood pressure based on menopausal status, however, no such relationships with fasting glucose or age were found. CONCLUSIONS: Increased uric acid levels were associated with increased risk for metabolic syndrome in both premenopausal and postmenopausal women. In studies regarding uric acid and metabolic syndrome in women, the effects of menopausal status should be considered.

Stemness evaluation of mesenchymal stem cells from placentas according to developmental stage: comparison to those from adult bone marrow.

This study was done to evaluate the stemness of human mesenchymal stem cells (hMSCs) derived from placenta according to the development stage and to compare the results to those from adult bone marrow (BM). Based on the source of hMSCs, three groups were defined: group I included term placentas, group II included first-trimester placentas, and group III included adult BM samples. The stemness was evaluated by the proliferation capacity, immunophenotypic expression, mesoderm differentiation, expression of pluripotency markers including telomerase activity. The cumulative population doubling, indicating the proliferation capacity, was significantly higher in group II (P<0.001, 31.7±5.8 vs. 15.7±6.2 with group I, 9.2±4.9 with group III). The pattern of immunophenotypic expression and mesoderm differentiation into adipocytes and osteocytes were similar in all three groups. The expression of pluripotency markers including ALP, SSEA-4, TRA-1-60, TRA-1-81, Oct-4, and telomerase were strongly positive in group II, but very faint positive in the other groups. In conclusions, hMSCs from placentas have different characteristics according to their developmental stage and express mesenchymal stemness potentials similar to those from adult human BMs.

The efficacy of human placenta as a source of the universal feeder in human and mouse pluripotent stem cell culture.

The use of a mouse embryonic fibroblast (MEF) feeder for culture of embryonic stem cells (ESCs) is a widely accepted method, regardless of the ESCs' origin and type. In this study, we performed the undifferentiated propagation of human ES cell lines (hESCs, H1, and HSF6) and mouse ES cell lines (mESCs, D3, and CE3), which were previously maintained on an MEF feeder, using human placenta-derived fibroblast-like cell (HPC) feeders originated from chorionic villi of women who had undergone therapeutic abortion due to known maternal disease that is aggravated by pregnancy. Moreover, we tried to introduce the HPC feeder for the establishment of inducible pluripotent stem cells (iPSCs) from human placental mesenchymal stem cells (MSCs). On the HPC feeder we were able to propagate ESCs and iPSCs colonies as an undifferentiated state up to the 50th passage and 20th passage, respectively. Maintenance of undifferentiated ESCs was identified by the expression of ALP, SSEA-1, SSEA-4, TRA-81, TRA-60, Oct-4, Nanog, or Rex-1. Also, addition of leukemia inhibitory factor was not required for undifferentiated propagation of mESCs on the HPC feeder. The efficiency and expression of three germ layer markers of embryoid bodies (EBs) from ESCs were satisfactory in both the MEF and HPC group. EBs formed from iPSCs were scant, and differentiation to the three germ layers was identifiable by reverst transcription-polymerase chain reactio (RT-PCR) only in the HPC group. In conclusion, the HPC feeder can efficiently support the undifferentiated propagation of hESCs, mESCs, and iPSCs, suggesting that human placenta may be a useful source of universal feeder cells for hESC, mESC, and iPSC culture.

Relationship between the serum CA-125 level and bone mineral density in healthy pre- and post-menopausal women.

BACKGROUND: Osteoporosis and tumour-associated antigen (TAA) levels are associated with inflammatory processes, but little remains known about TAA levels and bone mineral density (BMD). AIMS: We determined whether or not high-normal TAA levels are associated with a lower BMD in healthy women. METHODS: A total of 3769 healthy women were enrolled from the health screening programme over 5 years. Each participant had undergone a basic health examination. Serum carbohydrate antigen (CA)-125, CA-19-9, carcinoembryonic antigen (CEA) and alpha-fetoprotein levels were evaluated as tumour markers. The correlations between serum TAA levels and BMD were analysed. RESULTS: Carbohydrate antigen 125 and CEA levels were positively associated with a higher BMD in the pre-menopause. In the post-menopause, the CA-125 level was positively associated with BMD. In the pre-menopause, CA-125 (r = 0.102; P < 0.001) and CEA levels (r = 0.134; P < 0.001) had a significant correlation with BMD. In the post-menopause, CA-125 was negatively associated with alkaline phosphatase (r = -0.298; P < 0.001). CONCLUSIONS: There was a significant positive association between CA-125 and BMD in healthy women. Additional basic and clinical studies on the relationship between CA-125 and bone are needed.

Prenatally detected congenital perineal mass using 3D ultrasound which was diagnosed as lipoblastoma combined with anorectal malformation: case report.

We report a case of prenatally diagnosed congenital perineal mass which was combined with anorectal malformation. The mass was successfully treated with posterior sagittal anorectoplasty postnatally. On ultrasound examination at a gestational age of 23 weeks the fetal perineal mass were found on the right side. Any other defects were not visible on ultrasonography during whole gestation. Amniocentesis was performed to evaluate the fetal karyotyping and acetylcholinesterase which were also normal. As the fetus grew up, the mass size was slowly increased more and more. At birth, a female neonate had a perineal mass on the right side as expected. During operation, the anal sphincteric displacement was found near the mass and reconstructed through posterior sagittal incision. This is the first reported case of prenatally diagnosed congenital perineal mass, after birth which was diagnosed as lipoblastoma and even combined with anorectal malformation. This case shows that it can be of clinical importance to be aware of this rare fetal perineal mass in prenatal diagnosis and counseling.