Global increase in O-linked N-acetylglucosamine modification promotes osteoblast differentiation.

The balance between bone formation and bone resorption is maintained by osteoblasts and osteoclasts, and an imbalance in this bone metabolism leads to osteoporosis. Here, we found that osteoblast differentiation in MC3T3-E1 cells is promoted by the inactivation of O-linked ß-N-acetylglucosaminidase (O-GlcNAcase) and suppressed by the inactivation of O-GlcNAc transferase, as indicated by extracellular matrix calcification. The expression of osteogenic genes such as alp, ocn, and bsp during osteoblast differentiation was positively regulated in a O-GlcNAc glycosylation-dependent manner. Because it was confirmed that Ets1 and Runx2 are the two key transcription factors responsible for the expression of these osteogenic genes, their transcriptional activity might therefore be regulated by O-GlcNAc glycosylation. However, osteoclast differentiation of RAW264 cells, as indicated by the expression and activity of tartrate-resistant acid phosphatase, was unaffected by the inactivation of either O-GlcNAcase or O-GlcNAc transferase. Our findings suggest that an approach to manipulate O-GlcNAc glycosylation could be useful for developing the therapeutics for osteoporosis.

Prevalence of hyperglycaemia in singleton versus twin pregnancy.

BACKGROUND: There is conflicting information regarding the risk of gestational diabetes mellitus in twin pregnancies. This study was conducted to determine whether the risk of gestational diabetes mellitus is higher in pregnant Japanese women with twin versus singleton pregnancy. METHODS: The prevalence of gestational diabetes mellitus was studied in two different populations: 144 589 women registered with the Japan Society of Obstetrics and Gynecology (JSOG cohort) over 3 years between 2007 and 2009 in which patient selection bias was unavoidable; and 430 Japanese women who gave birth at a single centre over 5 years between 2008 and 2012 (single-centre cohort), consisting of 86 women with twins and 344 women with singletons matched for maternal age and prepregnancy body mass index. The gestational diabetes mellitus was diagnosed on the basis of the previous criteria in the JSOG cohort. The gestational diabetes mellitus was screened in a stepwise method and diagnosed on the basis of the new criteria in the single-centre cohort. RESULTS: In the single-centre cohort, neither frequency of random glucose level ≥105 mg/dL in the first trimester [9.0% (31/344) vs 5.8% (5/86)], positive result (≥140 mg/dL) on 50 g glucose challenge test in the second trimester [26.5% (90/339) vs 26.7% (23/86)], nor women diagnosed with GDM [8.4% (29/344) vs 9.3% (8/86)] differed between the two groups. The prevalence of hyperglycaemia was higher in singleton than in twin pregnancies in the JSOG cohort (2.6% vs 1.8%, p < 0.001). CONCLUSIONS: The risk of gestational diabetes mellitus may be similar between Japanese women with singleton and twin pregnancies. Difference in the risk of hyperglycaemia in the JSOG cohort may have been due to selection bias.

Autotransplantation or replantation of cryopreserved teeth: a case series and literature review.

BACKGROUND: The aim of this report was to evaluate the outcome of autotransplantation or replantation of cryopreserved teeth clinically and radiographically. Donor teeth were slowly frozen in a controlled-rate freezer using 5% dimethylsulfoxide (DMSO) and 6% hydroxyethyl starch (HES) as protectants. Seven cryopreserved teeth, with duration of storage ranging from 4 to 36 months, were autotransplanted or replanted at Niigata University Medical and Dental Hospital. Endodontic treatment involving root canal debridement followed by interim root canal filling with calcium hydroxide was started 3 weeks after the operation and continued with replacement of the calcium hydroxide filling at 2-week to 3-month intervals. Three transplants showed periodontal regeneration clinically and radiographically, whereas replacement root resorption was observed in the remaining transplants. From the results, it can be concluded that cryopreserved tooth autotransplantation has potential for clinical use; however, the risk of replacement root resorption remains.

Evaluation of the Effect of Thickeners in Enteral Formulas on the Gastric Emptying Rate of Proteins and Carbohydrates Using a Semi-Dynamic Gastric Model.

The emptying rate of specific nutrients in enteral formulas is poorly understood, despite the importance of controlling the emptying rate in tube-fed patients. Because of their viscosity, thickened formulas are widely used to avoid gastric reflux and reduce the burden on caregivers. This study examined how thickeners in enteral formulas affected the gastric emptying rates of proteins and carbohydrates. A semi-dynamic gastric model was used to prepare and digest test enteral formulas that contained either no thickeners or agar (0.2%). The amounts of protein and carbohydrates in each emptied aliquot were determined, and the emptying rate was calculated. We found that agar accelerated protein emptying, and an exploratory experiment with agar (0.5%) suggested the possibility of concentration dependence. Additionally, experiments using gellan gum (0.08%), guar gum (0.2%), or carrageenan (0.08%, 0.2%) suggested that protein emptying could vary depending on the thickener type and that carrageenan might slow it. These results could help with the appropriate selection of thickeners added to liquid foods based on the patient's metabolic profile to manage nutrition, not only for tube-fed patients but also for those with oropharyngeal dysphagia or diabetes.

Effects of caesarean section on serum levels of NT-proBNP.

OBJECTIVE: To determine the effects of delivery by caesarean on serum levels of N-terminal fragment of precursor protein brain-type natriuretic peptide (NT-proBNP). METHODS: Serum NT-proBNP levels were determined longitudinally at 24 and 36 weeks of gestation (GW) and on post-partum day 3 and month 1 (PPD3 and PPM1, respectively) in 78 women with normotensive singleton pregnancies. Thirty-nine women underwent caesarean delivery. Plasma renin activity (PRA) and plasma aldosterone concentration (PAC) were determined on PPD3. Effects of maternal demographic characteristics on NT-proBNP levels were also analysed. RESULTS: NT-proBNP levels (pg/ml) either in pregnancy or on PPM1 did not differ between women with vaginal and caesarean deliveries (44 ± 24 vs 41 ± 30, 24 GW; 37 ± 22 vs 29 ± 22, 36 GW; 43 ± 28 vs 39 ± 24, PPM1, respectively). Levels on PPD3 were significantly higher (94 ± 105 vs 247 ± 186, P < 0.0001) in women with caesarean delivery. Among women with caesarean delivery, a larger rise of NT-proBNP on PPD3 occurred in nulliparous than in multiparous women (319 ± 232 vs 185 ± 107, P = 0.023), while no rise occurred among multiparous women with vaginal delivery (108 ± 115 vs 47 ± 27). NT-proBNP levels on PPD3 were significantly and negatively correlated with PRA, PAC and maternal weight loss after childbirth on PPD3. These 3 variables on PPD3 were significantly lower in women undergoing caesarean than vaginal delivery (0.8 ± 0.4 vs 1.9 ± 1.4 ng/ml/h for PRA; 70 ± 38 vs 136 ± 88 pg/ml for PAC; 2.7 ± 1.2 vs 4.3 ± 1.1 kg for weight loss, each P < 0.0001). CONCLUSIONS: The transient post-partum rise in serum NT-proBNP may reflect transient volume overload after parturition and is remarkable in nulliparous women, especially after caesarean section.

Allergenicity of partially hydrolyzed whey and casein formulas evaluated by ImmunoCAP inhibition assay and basophil activation test.

Background: When exclusive breastfeeding is not possible, partially hydrolyzed formula (PHF) is often used as a starter formula for infants. Some children develop allergic symptoms, including anaphylaxis, after the first intake of cow protein. Therefore, the tolerability of PHF in infants with cow's milk allergy (CMA) is important information. Partially hydrolyzed whey formula (PHWF) is well characterized, but those containing both whey and casein are also available. We evaluated the characteristics of two whey and casein PHFs, PHF1 and PHF2, in vitro and ex vivo, and compared them with a PHWF, PHWF1. Methods: Residual antigenicity of ß-lactoglobulin (ß-LG) and casein in the formulas was measured using ELISA. The molecular weight profile was determined using high-pressure liquid chromatography. IgE reactivity and allergenic activity of the formulas were evaluated by ImmunoCAP inhibition assay and by basophil activation test using blood from patients with CMA, respectively. Results: All the participants (n = 10) had casein-specific IgE. The antigenicity of ß-LG in PHF1 was similar to that in PHWF1, but it was slightly higher than that in PHWF1 for casein. PHF1 had a higher IgE reactivity than PHWF1. However, PHF1 and PHWF1 had a similar ability to activate basophils. PHF2 had lower antigenicity of casein and ß-LG, IgE reactivity and basophil activation than PHWF1. Conclusion: These results suggest that the tolerability of PHF1 and PHF2 in patients with CMA is similar to and higher than that of PHWF1, respectively, and that the degree of IgE binding to PHFs does not necessarily correspond to basophil activation.

Fermentation Supernatant of Elderly Feces with Inulin and Partially Hydrolyzed Guar Gum Maintains the Barrier of Inflammation-Induced Caco-2/HT29-MTX-E12 Co-Cultured Cells.

Intestinal barrier function declines with aging. We evaluated the effect of dietary fibers and indigestible oligosaccharides on intestinal barrier function by altering the microbiota of the elderly. The feces were anaerobically cultured with indigestible dextrin, inulin, partially hydrolyzed guar gum (PHGG), lactulose, raffinose, or alginate, and the fermented supernatant was added to inflammation-induced Caco-2/HT29-MTX-E12 co-cultured cells. Our data showed that inulin- and PHGG-derived supernatants exerted a protective effect on the intestinal barrier. The protective effect was significantly positively correlated with total short-chain fatty acids (SCFAs) and butyric acid production in the supernatant and negatively correlated with the claudin-2 (CLDN2) gene expression in the cultured cells. Furthermore, we showed that the CLDN2 levels are regulated by butyric acid. Thus, inulin and PHGG can change the intestinal environment of the elderly and maintain the intestinal barrier by accelerating the production of SCFAs and modifying the expression levels of barrier function-related genes.

Positive impact of perioperative oral management on the risk of surgical site infections after abdominal surgery: Sixteen universities in Japan.

Surgical site infections (SSI) are associated with increased morbidity and mortality rates. This study aimed to investigate the ability of perioperative oral management (POM) to reduce the risk of SSI in abdominal surgery Real-world data collected from 16 university hospitals in Japan were reviewed. The medical records of consecutive 2782 patients (1750 men and 1032 women) who underwent abdominal surgery under general anesthesia at 16 university hospitals were retrospectively reviewed. Detailed information about SSI was assessed and compared between patients with and without POM in univariate and multivariate analyses. SSI were observed in 275 patients (incidence rate:9.9%), and POM was administered to 778 patients (28.0%). Univariate analyses revealed that diabetes mellitus, Eastern Cooperative Oncology Group performance status, American Society of Anesthesiologists classification, surgical site, preoperative Prognostic Nutritional Index score, POM, extent of surgery, operation time, and intraoperative blood loss were significantly associated with postoperative SSI (Chi-square or Mann-Whitney U test, P < .01). Multivariate analysis revealed that POM had significant preventive effects against postoperative SSI (estimate: -0.245, standard error: 0.080, P < .01). Surgical site, American Society of Anesthesiologists classification, and operation time were also significant and independent clinical predictors of SSI. The analysis of real-world data from 16 university hospitals revealed that, regardless of the content and degree of the problem, the addition of POM has significant beneficial effects in reducing the risk of SSI in patients who undergo abdominal surgery. Medical records from each hospital and data from the Health Care Payment Fund were collected and analyzed retrospectively.

Keratinocytes of tissue-engineered human oral mucosa promote re-epithelialization after intraoral grafting in athymic mice.

PURPOSE: The objective of this study was to investigate the role of grafted oral keratinocytes in a transplanted ex vivo-produced oral mucosa equivalent (EVPOME) in the regeneration and/or healing process of the oral mucosa at the recipient site. MATERIALS AND METHODS: The EVPOME was developed in a serum-free defined culture system without a feeder layer. EVPOME is composed of a stratified layer of human oral keratinocytes that are seeded onto a human cadaveric dermis, AlloDerm (LifeCell, Branchburg, NJ). Intraorally grafted EVPOMEs in athymic mice (BALB/c) were excised, contiguous with the surrounding oral mucosa, on days 5, 7, 14, and 21 after grafting. Serial sections were stained with hematoxylin-eosin and immunohistochemically analyzed for cytokeratin 17 (CK17) expression to distinguish the human-cultured EVPOME epithelial keratinocytes from murine oral keratinocytes. RESULTS: All EVPOME epithelial cells showed intense immunoreactivity for CK17, whereas mouse buccal mucosal epithelial cells did not show CK17 immunoreactivity. The grafted EVPOME maintained a stratified epithelial layer for up to 5 days after grafting. By day 7 after grafting, a portion of the EVPOME epithelial layer peeled away from the AlloDerm, and a thin, CK17-immunonegative epithelial layer extended from the adjacent thick epithelial layer of the mouse and contacted the CK17-immunopositive EVPOME epithelium. From days 14 to 21 after grafting, the stratification of the CK17-immunonegative continuous mouse epithelium increased compared with earlier time points and showed a similar appearance to the epithelium of the adjacent mouse mucosa. In contrast, no epithelial coverage of the AlloDerm that was grafted without keratinocytes was observed for up to 21 days after grafting. The grafted AlloDerm without cells resulted in tissue necrosis that was accompanied by a dramatic infiltration of inflammatory cells by day 14. CONCLUSIONS: These findings suggest that grafting of EVPOME with viable oral keratinocytes onto an intraoral mucosal wound plays an active role in promotion of re-epithelialization of the oral wound during the subsequent healing process.

Characteristics of insulin secretion patterns in Japanese women with overt diabetes and gestational diabetes defined according to the International Association of Diabetes and Pregnancy Study Groups criteria.

AIM: To characterize patterns of insulin secretion in women with overt diabetes and gestational diabetes (GDM) defined by the International Association of Diabetes and Pregnancy Study Groups (IADPSG) criteria. MATERIAL AND METHODS: A total of 228 Japanese women were examined retrospectively. All 228 women had a positive 50-g glucose challenge test (GCT) result at 25.2±1.2weeks of gestation and underwent a 75-g glucose tolerance test (GTT) at 27.4±1.8weeks of gestation. The immunoreactive insulin levels were determined during the GTT in four groups of pregnant women: five with overt diabetes, 20 with GDM according to both the previous Japan Society of Gynecology and Obstetrics (JSOG) and current IADPSG criteria (traditional GDM group), 43 with GDM according to only the IADPSG criteria (new GDM group), and 160 with non- GDM, but with a positive GCT result. RESULTS: Attenuated and slow rise in plasma insulin in concert with prolonged hyperglycemia were characteristic in women with overt diabetes, compared with women with GDM in whom excessive insulin secretion in the presence of hyperglycemia was characteristic. The new GDM group did not differ significantly from the traditional GDM group with respect to scores of such indices as the insulinogenic index, the homeostasis model assessment for insulin resistance, and the quantitative insulin sensitivity check index. CONCLUSION: Women with overt diabetes have both an impaired capacity for insulin secretion and elevated insulin resistance, while women with GDM exhibit a maintained insulin secretory capacity with an elevated insulin resistance.

Orofacial pain related to traumatic neuroma in a patient with multiple TMJ operations.

The diagnosis of orofacial pain associated with temporomandibular disorders after repeated temporomandibular joint (TMJ) surgeries can be quite difficult. This case report describes a 52-year-old woman who had previously undergone five TMJ surgeries and developed divergent pain caused by a trigger point in the left preauricular area. Computed tomography and magnetic resonance imaging could not be used to identify a lesion because of metallic artifacts from a TMJ prosthesis. However, sonography indicated the location of the suspected lesion. Moreover, a neurological examination performed with local anesthesia was clinically effective in ruling out other diagnoses of orofacial pain. Ultimately, a histopathological examination of a biopsy specimen from the painful site confirmed the lesion to be a traumatic neuroma. This case report suggests the value of including traumatic neuroma in the differential diagnosis of patients with a history of previous TMJ surgery who present with orofacial pain in the region of the TMJ.

Antithrombin activity, platelet count, hemoglobin concentration and hematocrit value determined immediately before vaginal delivery among healthy women.

AIMS: To determine the normal reference values for antithrombin (AT) activity, platelet count (Plt), hemoglobin concentration (Hb), and hematocrit value (Ht) immediately before vaginal delivery among healthy pregnant women with singleton pregnancies and to determine association of these blood parameters with fetal growth. METHODS: A complete blood count was performed and the AT activity was examined in 300 consecutive women admitted to hospital at > or = gestational week 36 for labor pains and/or the rupture of fetal membranes. All the women were normotensive and had singleton pregnancies, and none of the women had proteinuria, a weekly weight gain > or = 0.5 kg, or other specific complications upon admission. All the women attempted a vaginal delivery. RESULTS: The medians (5th-95th percentile) were 90% (71-110%) for AT activity, 234x10(9)/L (150-337x10(9)/L) for Plt, 11.0 g/dL (9.5-12.8 g/dL) for Hb, and 34.0% (30.4-38.6%) for Ht. Women with an Hb value of > or = the median (11.0 g/dL) gave birth to significantly smaller infants than their counterparts. CONCLUSIONS: A considerable number of healthy women exhibit a reduced AT activity and/or platelet count immediately before delivery. Hemoconcentration evidenced by a raised Hb value adversely effects on infant growth. Our data may be helpful when considering the normal ranges of these blood parameters for healthy parturient women.

Whey protein hydrolysate mitigates both inflammation and endotoxin tolerance in THP-1 human monocytic leukemia cells.

INTRODUCTION: It is important to control both inflammation and immunosuppression after severe insults, such as sepsis, trauma, and surgery. Endotoxin tolerance is one of the immunosuppressive conditions and it has been known that endotoxin tolerance relates to poorer clinical outcomes in patients with severe insults. This study investigated whether whey protein hydrolysate (WPH) mitigates inflammation and endotoxin tolerance in THP-1 human monocytic leukemia cells. METHODS: Endotoxin tolerance can be experimentally reproduced by two consecutive stimulations with lipopolysaccharide (LPS). THP-1 cells were incubated with LPS and WPH (first stimulation). After collecting the culture supernatant to evaluate the effect on inflammation, the cells were washed and restimulated by 100 ng/ml LPS (second stimulation). The culture supernatant was again collected to evaluate the effect on endotoxin tolerance. Concentrations of LPS and WPH in the first stimulation were adjusted to evaluate their dose dependency. Cytokine levels in the supernatant were determined by enzyme-linked immunosorbent assay. Statistical analysis was performed using the student's t-test or Dunnett's test. RESULTS: Five mg/ml WPH significantly decreased interleukin (IL)-6 (p = .006) and IL-10 (p < .001) levels after the first LPS stimulation (1000 ng/ml). WPH significantly increased tumor necrosis factor-alpha (p < .001) and IL-10 (p = .014) levels after the second LPS stimulation. The suppressive effect of WPH on inflammation and endotoxin tolerance was dependent on the concentrations of LPS and WPH. The effective dose of WPH for endotoxin tolerance was lower than its effective dose for inflammation. CONCLUSION: WPH mitigated both inflammation and endotoxin tolerance. Therefore, WPH might be a candidate for valuable food ingredients to control both inflammation and immunosuppression after severe insults.

Large presacral Tarlov cysts in pregnancy.

This is the first report of large presacral Tarlov cysts (cerebrospinal fluid-filled perineural cysts) diagnosed during pregnancy in which a cesarean delivery mode was selected to avoid the risk associated with vaginal delivery.

Do uterotrophic drugs increase the risk of fatal hemorrhagic brain stroke?

OBJECTIVE: To evaluate whether uterotrophic agents increase the risk of fatal hemorrhagic brain stroke. METHODS: Between 1991 and 1992, there were 230 maternal deaths among 2,420,000 pregnant women in Japan and the causes of these deaths was investigated in 1994. Using information provided in this report, we identified 35 women who died from or were assumed to die from hemorrhagic brain stroke. We assumed that 93% of women would have tried vaginal delivery. The risk of fatal hemorrhagic brain stroke after uterotrophic agent use was calculated according to the assumption that 5.0-40% of women received uterotrophic agents. RESULTS: Use of uterotrophic agents for induction/augmentation of labor was confirmed in five (14.3%) of the 35 women who died from hemorrhagic brain stroke. The incidence of fatal brain stroke after the use of uterotrophic agents was only significantly higher than that for spontaneous hemorrhagic brain stroke if these agents were administered in ≤ 6.0% of women. CONCLUSIONS: Because more than 6.0% of women received uterotrophic agents, these agents are unlikely to increase the risk of fatal hemorrhagic brain stroke.

Risk factors for postpartum hypertension in women with twin pregnancies.

OBJECTIVE: Which physical findings and blood parameters predict postpartum hypertension remain to be studied in women with twin pregnancies. METHODS: The antenatal systolic and diastolic blood pressures (SBP and DBP, respectively), and 16 laboratory variables were investigated in 150 normotensive women who gave birth to twins. RESULTS: When the median values of the 18 continuous variables were used as cut-off values, an SBP>120 mm Hg (relative risk [95% confidence interval], 2.81 [1.94-4.08]), a DBP>70 mm Hg (2.42 [1.68-3.49]), an aspartate aminotransferase level>18 U/L (2.22 [1.55-3.19]), and a uric acid level>5.3 mg/dL (1.68 [1.20-2.36]) were independent risk factors for postpartum hypertension. CONCLUSIONS: Antenatal blood pressure measurements and a laboratory work-up may be useful clinically for predicting postpartum hypertension in women with twin pregnancies.

Relationships between fetal growth and maternal body mass indices, plasma glucose level, and plasma insulin level in Japanese women with mildly impaired glucose tolerance.

AIM: To identify variables which are strongly associated with fetal growth. MATERIAL AND METHODS: The study subjects were 203 Japanese women who had a plasma glucose level ≥140 mg/dL (7.8 mmol/L) on a 50-g glucose challenge test but were normoglycemic when challenged with a 75-g oral glucose tolerance test (GTT) according to the criteria previously used in Japan. All the subjects subsequently gave birth to singleton infants. The correlations between the standard deviation for birthweight (birthweight SD) and 15 other variables, including the maternal body mass index (BMI), plasma levels of glucose and insulin on the GTT, and various indices calculated using data from the GTT, were then examined. Univariate and multivariate regression analyses were performed. RESULTS: Univariate analyses revealed significant correlations between the birthweight SD and the fasting plasma glucose level (P = 0.0063), the pre-pregnancy BMI (P = 0.0001), and the BMI at delivery (P < 0.0001). Only the BMI at delivery remained as a significant factor that was independently correlated with the birthweight SD after a multivariate regression analysis. CONCLUSION: The suppression of maternal weight gain, rather than the suppression of the plasma glucose level, may be effective for avoiding infants with overgrowth among Japanese women with mildly impaired glucose tolerance.

Pregnancy-induced antithrombin deficiency.

OBJECTIVE: This retrospective study was performed to characterize the laboratory features and water metabolism of women with pregnancy-induced antithrombin deficiency (PIATD). METHODS: Among 1493 women who gave birth to a singleton infant at our institution, 114 women who developed PIATD and/or pregnancy-induced hypertension (PIH) were reviewed with respect to perinatal changes in laboratory variables (hematocrit value, fibrinogen, fibrinogen degradation product, D-dimer, uric acid, aspartate aminotransferase, lactate dehydrogenase) and body weight. PIATD was defined as a gradual decline in antithrombin (AT) activity to

Evidence of the escape of antithrombin from the blood into the interstitial space in pregnant women.

OBJECTIVE: we investigated whether ascites samples obtained from pregnant women during cesarean sections contained antithrombin because it is unknown whether antithrombin escapes from the blood and passes into the interstitial space during pregnancy. METHODS: the concentration and activity levels of antithrombin were determined in six ascites samples obtained from six consecutive women who exhibited generalized edema, ascites, and a gradual decline in antithrombin activity. RESULTS: all six ascites samples contained antithrombin (mean ± SD, 4.9 ± 2.2 mg/dL; range, 2.7-8.8 mg/dL) and exhibited an antithrombin activity level of 15.5 ± 6.0% (range, 10-24%). CONCLUSIONS: antithrombin escapes from the blood into the interstitial space in pregnant women. This phenomenon partially explains the gradual decline in antithrombin activity observed in these six pregnant women with generalized edema and large volumes of ascites.

Marked gestational edema as a clinical sign of life-threatening condition.

A 35-year-old Japanese nulliparous woman exhibited rapid weight gain (6 kg/7 days), reduced antithrombin activity and platelet count at 37 weeks of gestation without hypertension or proteinuria, and underwent cesarean section. Postnatally, pulmonary edema developed for 7 days, with transient hypertension and proteinuria, and bodyweight loss (14.6 kg) by 14 days postpartum. Platelet count and antithrombin activity normalized promptly postpartum. Despite a life-threatening clinical condition due to enhanced vascular permeability, neither hypertension nor proteinuria appeared antenatally. Determining antithrombin activity and platelet count may be useful for distinguishing between women with pathological edema and physiological edema.