Patterns of changes in proteins in the peritoneal fluid of women during the periovulatory phase of the menstrual cycle

During a laparoscopy that was performed between Day -6 and Day +9 of the cycle as related to the day of the LH peak (Day 0), the peritoneal fluid of 100 healthy female volunteers of proven fertility was collected and analysed. Peritoneal fluid volume and concentrations of total protein, albumin, alpha 1-, alpha 2-, beta- and gamma-globulins, IgA, IgG, IgM, haptoglobulin, acid-alpha 1-glycoprotein, alpha 1-antitrypsin, alpha 2-macroglobulin, C3-, C4- and C-reactive protein were determined. The peritoneal fluid volume and the concentrations of most proteins analysed showed an increase during the post-ovulatory phase of the period investigated. The peritoneal fluid:serum ratio of each individual protein showed a significant inverse correlation with its molecular weight. This confirms the assumption that peritoneal fluid is mainly an exudation product, most probably of ovarian origin.     

Changes in uterine protein secretion during luteal and follicular phases and detection of phosphatases during luteal phase of estrous cycle in buffaloes (Bubalus bubalis)

Changes in uterine proteins during different reproductive states and their functional significance though known in other species have not been established in buffaloes. An attempt has been made to unravel the changes in composition of buffalo uterine secretion with growth and regression of corpora-lutea during early, mid and late luteal and follicular phase of estrous cycle using gel filtration and electrophoresis techniques. Also the phosphatases activities in luteal phase uterine secretions have been studied. Gel filtration chromatography analysis revealed a protein peak in void volume of the column, the intensity of which was more in all the luteal phase samples than follicular phase samples. Alkaline phosphatase was also found eluted in the void volume. The other three uterus-specific peaks (Peaks V-VII) were detected below 13.7 kd molecular weight. There were at least five peaks of acid phosphatases activity in chromatogram. Silver staining of SDS-PAGE gel detected as many as 40 protein bands in the uterine fluid of which nine proteins were glycoproteins. Molecular weight (MW) comparison revealed the major protein band at 66 kd which could be serum albumin. Comparison of uterine proteins with serum protein bands revealed a 93.5 kd glycoprotein in buffalo serum that did not appear in uterine fluid and at least 11 uterus-specific protein bands (506, 470, 241, 114, 49, 38, 33, 26, 19.2, 16, and 14.3 kd). The 38 and 19.2 kd bands were luteal-stage specific. Intense periodic acid Schiff's (PAS) stained bands in uterine proteins compared to serum indicated glycosylation process in endometrial epithelial cells. The study suggested that buffalo uterine secretion contained mainly serum and several uterus-specific proteins of which few were luteal phase specific. Further study on characterizing the unique or most abundant proteins and defining their role in uterine functions would help to address the cause of low reproduction rate in buffaloes.     

Functional aspects of peritoneal macrophages in endometriosis of women

Peritoneal fluid was collected in the periovulatory phase of the cycle from 25 women undergoing laparoscopy. Endometriosis was diagnosed in 13 patients (AFS score 1, N = 9; AFS score 2, N = 4) and 12 patients without endometriosis served as controls. In endometriosis patients the total peritoneal fluid cell number and cell concentration was significantly higher than in controls, indicating peritoneal irritation by endometrial implants. Peritoneal fluid macrophages in patients with endometriosis showed significantly increased erythrophagocytosis and lower chemiluminescence than in controls, suggesting an advanced differentiation of the macrophages in endometriosis patients. The macrophages in this stage of differentiation may interfere with gametes and embryos and thus contribute to endometriosis-associated subfertility.     

Suppression of macrophage activation by peritoneal fluid from patients with endometriosis

To investigate the effects of peritoneal fluid from patients with endometriosis on mouse peritoneal macrophages (Mφ), peritoneal fluid from endometriosis patients (n=15) were added to a monolayer of C3H/HeJ mouse peritoneal Mφ. Tumor necrosis factor-producing activity was measured by the L929 assay activated with FK-23 (a preparation of heat-killed Enterococcus faecalis). Tumor necrosis factor-producing activity of C3H/HeJ mouse peritoneal Mφ incubated with peritoneal fluid was suppressed in 14 endometriosis patients. Interestingly, in nine endometriosis patients, tumor necrosis factor-producing activity was much lower than seen with mouse peritoneal Mφ incubated with corticosterone. Peritoneal fluid contains suppressive properties for the activation of peritoneal Mφ, which might allow the implantation of free endometrial cells or the metaplastic phenomena stimulated by retrograde menstruation.     

Proteins secreted from the early ovine conceptus block the action of prostaglandin F2 alpha on large luteal cells.

In this study we evaluated whether the early conceptus secretes a factor that blocks the action of prostaglandin (PG) F2 alpha on cultured ovine large luteal cells. PGF2 alpha inhibited progesterone production by lipoprotein-stimulated large luteal cells and this anti-steroidogenic action was blocked in a dose-dependent manner by conceptus proteins secreted from Day 15 embryos. Purified ovine trophoblast protein-1 (oTP-1) did not exhibit the anti-PGF2 alpha activity, but secreted conceptus proteins devoid of oTP-1 did prevent the anti-steroidogenic effects of PGF2 alpha. This activity does not appear to be a nonspecific effect of protein since neither serum albumin nor thyroglobulin, gamma globulin, insulin, LH, secreted ovine endometrial proteins, or heat-inactivated secreted conceptus proteins had this action. After molecular-sizing chromatography we found a high- and a low-molecular weight fraction with luteal protective activity. Neither of the secreted conceptus protein fractions blocked the binding of 3H-PGF2 alpha to large luteal cells. However, conceptus proteins did block the anti-steroidogenic action of phorbol ester and calcium ionophore on large luteal cells, suggesting that secreted conceptus proteins act after activation of the free calcium/protein kinase C intracellular effector pathways. Thus, the early ovine conceptus secretes a luteal protective protein(s) that may be important for maintaining the corpus luteum during early pregnancy; however, the physiologic significance of this luteal protective protein(s) cannot be stated without further investigation.     

Effect of intraperitoneal furosemide administration on levels of +proteins+ in plasma and signs of their ability to be dialyzed during intermittent peritoneal dialysis]

The study aimed at evaluating an effect of intraperitoneal furosemide on plasma proteins such as albumins, globulins, IgG and IgA and their loss during dialysis. An experiment involved 18 patients with critical renal failure treated with intermittent peritoneal dialyses. Furosemide was administered intraperitoneally with dialysing fluid (40 mg/1) in a total dose of 240 mg. Each patient underwent 2 dialyses of 14 exchanges each. The first dialysis without furosemide served as a control of plasma protein loss during conventional dialysis with a fluid of 369 mOsm/kg at flow rate 2.4 l/hour. Furosemide was given during the second dialysis during three consecutive exchanges. An effect of furosemide on plasma proteins was compared with the results obtained before and after its administration. It was found that furosemide did not change plasma proteins levels and does not increase their loss during exchanges of dialysing fluid containing this drug; during dialysing fluid exchanges without furosemide some indices of IgG and IgA dialysis are significantly decreased due to an increase in ultrafiltration following furosemide cessation. It is important for the increase in intermittent peritoneal dialyses efficiency with the aid of furosemide that its short-term administration does not increase proteins loss during dialysis, if their molecular weight is not exceeding 69,000.     

Inhibitory effects of morphine on some inflammation-related parameters in the goldfish Carassius auratus L

Acute peritonitis induced in the goldfish by intraperitoneal injection of a sterile Thioglycollate solution shows a typical pattern with intraperitoneal exudation of serum proteins followed by influx of leucocytes (mainly heterophils/macrophages) correlated with elevated levels of chemotactic factors in peritoneal fluid and blood plasma. Supplementation of Thioglycollate with morphine (20 mg kg(-1) b.w.) does not affect the leakage of serum proteins into peritoneum. In contrast, it reduces the number of exudate peritoneal leucocytes (among them heterophils/macrophages) to the control level and decreases the level of peritoneal fluid/plasma chemoattractants, both effects being reversed by naltrexone pretreatment. Morphine itself acts as a chemokinetic factor for fish leucocytes as it increases their random movements. Therefore inhibitory effects of morphine on accumulation of exudate cells might be explained by inhibition of the production/release of chemotactic factors and/or reduced sensitivity of leucocytes to chemotactic signals. The effects of morphine on the goldfish peritonitis are in concordance with those described recently in Atlantic salmon and CB6 mice.     

Prostaglandin and leukotriene concentration of the peritoneal fluid of endometriosis and other gynecologic disorders in the secretory phase

In order to investigate whether prostanoids are involved in the pathophysiology of endometriosis, prostaglandin and leukotriene concentrations in peritoneal fluid were measured in adenomyosis, ovarian chocolate cyst and uterine leiomyoma. In the secretory phase, there was no significant difference in 6-keto PGF1 alpha concentration in peritoneal fluid between the adenomyosis group and the leiomyoma group. TXB2 concentration did not significantly differ in the three study groups. Leukotriene C4 level in the adenomyosis group was significantly higher than that of leiomyoma in the secretory phase. Leukotriene B4 could not be detected by our assay system. Our results suggest that leukotriene C4 is possibly involved in the pathophysiology of endometriosis.     

Estimation of the Stokes radii of serum proteins for a study of protein movement from blood to amniotic fluid.

The effective hydrodynamic (Stokes) radii of fifteen serum proteins were estimated by Sephadex G 200 gel filtration and immunochemical methods. The Waldmann-Meyer and Birch equation (Protides Biol. Fluids, Proc. 21st Colloq. (1974) (Peeters, H., ed.), Vol. 21, pp. 653-656, Pergamon Press, Oxford) was used for maximum accuracy. Three replicate column runs were made to assess the precision for the size determinations (coefficients of variation 0.6-2.7(). Quantive two-dimensional immunoelectrophoresis was used to measure eleven proteins in specimens of serum and amniotic fluid collected from twelve normal pregnancies. There was a close inverse linear relationship between the amniotic fluid/serum ratios of the proteins and their Stokes radii. This indicates that the movement of a protein from the blood to amniotic fluid is determined by the size of the protein. The linear correlation of protein amniotic fluid/serum ratio with Stokes radius was better than that with molecular weight. This demonstrates that, as an expression of protein size, Stokes radius should be used in preference to molecular weight when protein filtration systems are being investigated.     

Hyperprolactinemia and luteal insufficiency in infertile patients with mild and minimal endometriosis.

The objective of the present paper was to assess the presence of hormonal alterations in infertile women with stage I or II endometriosis (Group III, n = 20) compared to fertile women without endometriosis (Group I, n = 14) and to fertile women with endometriosis (Group II, n = 7). Serum levels of FSH, LH, estradiol, TSH, and PRL were measured between days 1 and 5 of the early follicular phase; in the luteal phase, three serum samples were collected for progesterone measurement, and endometrial biopsies were performed. Serum estradiol levels were lower (p = 0.035) in infertile patients with endometriosis than in fertile patients without endometriosis. Six infertile patients with endometriosis presented prolactin levels above 20 ng/ml. This was not observed in the other groups. Luteal insufficiency was more frequent in infertile patients with endometriosis (78.9%) than in fertile patients with (42.9%) or without endometriosis (0%). In a multiple logistic regression analysis, only the presence of endometriosis and infertility was significantly associated with luteal insufficiency. The serum levels of LH, FSH, and TSH were not significantly different among the groups. Luteal insufficiency and altered prolactin secretion were associated with endometriosis, and could be important mechanisms causing infertility in this group of patients.     

PGE2 and PGF2α release by human peritoneal macrophages in endometriosis

Objective: To test for differences in the amount and activity of peritoneal macrophages present in the peritoneal fluid of women with, and without endometriosis using prostaglandin release by macrophages in culture as a marker.Patients: Women of reproductive age undergoing laparoscopy for infertility or chronic pelvic pain with postoperative diagnosis of endometriosis and women undergoing laparoscopy for sterilization.Methods: Peritoneal fluid was aspirated during laparoscopy, volume was recorded, macrophages were isolated via a Ficoll Paque gradient and kept in primary culture. PGE₂ and PGF_2α release of the cells were measured before and after stimulation with zymosan.Results: Women with endometriosis had significantly more peritoneal macrophages than controls. Peritoneal macrophages of women with endometriosis released significantly more PGE₂ than those of the control group: 8.4 ± 2.0 versus 1.4 ± 0.4 ng/ml/10⁶cells (mean ± SEM, p=0.0005) and PGF_2α : 10 ± 4.3 (endometriosis) versus 1.8 ± 0.4 (control) ng/ml/10⁶cells (mean ± SEM, p = 0.045).Conclusion: There is a significant increase in the amount of prostaglandins released by peritoneal macrophages from women with endometriosis. These prostaglandins might alter uterine and tubal contractility, thereby affecting fertility.     

Changes in phospholipids, cholesterol and protein content of oviduct fluid of cows during the oestrous cycle

The oviducts of 4 cows were cannulated and oviduct fluid was collected daily from the exteriorized cannulas for a total of 5 oestrous cycles. Daily serum samples were assayed for oestradiol-17 beta and progesterone to monitor the oestrous cycle. Data for each cycle were compared for oviduct fluid collected during the non-luteal phase (serum progesterone less than or equal to 1.5 ng/ml) and the luteal phase (serum progesterone greater than 1.5 ng/ml). During the non-luteal phase oviduct fluid volume was higher and the osmolality was lower than during the luteal phase. Total protein, cholesterol and phospholipid secreted daily was greater during the non-luteal phase. Cholesterol and protein concentrations were generally lower during the non-luteal phase, but phospholipid concentrations were generally higher. About 40% of the phospholipid in oviduct fluid was phosphatidylcholine and lysophosphatidylcholine, while phosphatidylinositol and lysophosphatidylinositol accounted for 20%. The ratio of 1-acyl-phospholipid to diacylphospholipid increased during the non-luteal phase. An increased cholesterol to phospholipid ratio, and a decreased cholesterol to protein ratio in oviduct fluid also were associated with the non-luteal phase. Changes in the lipid composition of oviduct fluid during the oestrous cycle may play a role in the preparation of gametes for fertilization.     

Proteomic approaches in endometriosis research

To date, the quest to develop a noninvasive diagnostic test for endometriosis has mostly concentrated on the levels of cytokines and growth factors that are involved in inflammation, angioneogenesis and tissue remodeling, present in serum, peritoneal fluid, endometrium and endometriotic lesions. As this has not yet translated into the development of such a diagnostic test, proteomic techniques are now being employed to identify proteins that are potential biomarkers for the disease. As proteomics allows the comprehensive analysis of complex fluid and tissue samples with good sensitivity and resolution, it has promise in delivering markers associated with endometriosis. Once identified, the challenge will be in translating these markers into a clinically useful test for endometriosis, as the pathophysiology of this disease is unknown and likely to be complex and multifactorial. Also, with variation between individuals and the influences of steroid hormones during the menstrual cycle, it could be difficult to validate findings relating to a single protein or small groups of proteins differentially expressed in the disease state. Proteomic profiling, using mass spectrometry in combination with sophisticated bioinformatics software to identify protein patterns, may be where a significant clinical diagnostic contribution can be made.     

Proteomic analysis in peritoneal dialysis patients with different peritoneal transport characteristics

Peritoneal membranes can be categorized as high, high average, low average, and low transporters, based on the removal or transport rate of solutes. In this study, we used proteomic analysis to determine the differences in proteins removed by different types of peritoneal membranes. Peritoneal transport characteristics in patients who received peritoneal dialysis therapy were assessed by a peritoneal equilibration test. Two-dimensional differential gel electrophoresis technology followed by quantitative analysis was performed to study the variation in protein expression from peritoneal dialysis effluents (PDE) among different groups. Proteins were identified by MALDI-TOF-MS/MS analyses. Further validation in PDE or serum was performed utilizing ELISA analysis. Proteomics analysis revealed ten protein spots with significant differences in intensity levels among different groups, including vitamin D-binding protein, complement C3, apolipoprotein-A1, complement factor C4A, haptoglobin, alpha-1 antitrypsin, immunoglobulin kappa light chain, alpha-2-microglobulin, retinol-binding protein 4 and transthyretin. The levels of vitamin D-binding protein, complement C3, and apolipoprotein-A1 in PDE derived from different groups were greatly varied (P < 0.05). However, no significant difference was found in the serum levels of these proteins among different groups (P > 0.05 for all groups). This study provides a novel overview of the differences in PDE proteomes of four types of peritoneal membranes. Vitamin D-binding protein, complement C3, and apolipoprotein-A1 showed enhanced expression in PDE of patients with high transporter.     

Identification of two novel cerebrospinal fluid proteins in non specific mental retardation

Cerebrospinal fluid (CSF) from twenty three patients with non specific mental retardation and fourteen age matched normal samples was subjected for qualitative analysis of protein profiles by two-dimensional gel electrophoresis (2-DE) and the proteins were visualised by ultra sensitive silver staining. Two proteins designated as mental retardation associated proteins (MRAP-I and MRAP-II) were identified in six male patients out of twenty three patients CSF samples. MRAP-I had an isoelectric point of 7.4 with a relative molecular weight 16.5 kDa, while MRAP-II had an iso-electric point of 7.2 with a relative molecular weight 16.8 kDa. The two proteins are presumed to be originated from brain, as they could not be traced in the serum of patients, nor due to proteolytic degradation. Despite unknown origin and identity, their presence in the CSF of a specific group of mentally retarded male patients suggest their possible clinical utility and to define protein alterations in mental retardation.     

Soluble Ligands for the NKG2D Receptor Are Released during Endometriosis and Correlate with Disease Severity

Background

Endometriosis is a benign gynaecological disease. Abundant bulk of evidence suggests that patients with endometriosis have an immunity dysfunction that enables ectopic endometrial cells to implant and proliferate. Previous studies show that natural killer cells have a pivotal role in the immune control of endometriosis.

Methods and Findings

This is a prospective laboratory study conducted in a tertiary-care university hospital between January 2011 and April 2013. We investigated non-pregnant, younger than 42-year-old patients (n= 202) during surgery for benign gynaecological conditions. After complete surgical exploration of the abdominopelvic cavity, 121 women with histologically proven endometriosis and 81 endometriosis-free controls women were enrolled. Patients with endometriosis were classified according to a surgical classification in three different types of endometriosis: superficial peritoneal endometriosis (SUP), ovarian endometrioma (OMA) and deep infiltrating endometriosis (DIE). Peritoneal fluid samples were obtained from all study participants during the surgery in order to detect soluble NKG2D ligands (MICA, MICB and ULBP-2). When samples with undetectable peritoneal fluid levels of MICA, MICB and ULBP-2 were excluded, MICA ratio levels were significantly higher in endometriosis patients than in controls (median, 1.1 pg/mg; range, 0.1–143.5 versus median, 0.6 pg/mg; range, 0.1–3.5; p=0.003). In a similar manner peritoneal fluid MICB levels were also increased in endometriosis-affected patients compared with disease-free women (median, 4.6 pg/mg; range, 1.2–4702 versus median, 3.4 pg/mg; range, 0.7–20.1; p=0.001). According to the surgical classification, peritoneal fluid soluble MICA, MICB and ULBP-2 ratio levels were significantly increased in DIE as compared to controls (p=0.015, p=0.003 and p=0.045 respectively). MICA ratio levels also correlated with dysmenorrhea (r=0.232; p=0.029), total rAFS score (r=0.221; p=0.031) and adhesions rAFS score (r=0.221; p=0.031).

Conclusions

We demonstrate a significant increase of peritoneal fluid NKG2D ligands in women with endometriosis especially in those cases presenting DIE. This study suggests that NKG2D ligands shedding is a novel pathway in endometriosis complex pathogenesis that impairs NK cell function.     

The Peritoneum Is Both a Source and Target of TGF-β in Women with Endometriosis

Transforming growth factor-β (TGF-β) is believed to play a major role in the aetiology of peritoneal endometriosis. We aimed to determine if the peritoneum is a source of TGF-β and if peritoneal TGF-β expression, reception or target genes are altered in women with endometriosis. Peritoneal fluid, peritoneal bushings and peritoneal biopsies were collected from women with and without endometriosis. TGF-β1, 2 and 3 protein concentrations were measured in the peritoneal fluid. TGF-β1 was measured in mesothelial cell conditioned media. Control peritoneum and peritoneum prone to endometriosis (within Pouch of Douglas) from women without disease (n = 16) and peritoneum distal and adjacent to endometriosis lesions in women with endometriosis (n = 15) and were analysed for TGF-β expression, reception and signalling by immunohistochemistry, qRT-PCR and a TGF-β signalling PCR array. TGF-β1 was increased in the peritoneal fluid of women with endometriosis compared to those without disease (P<0.05) and peritoneal mesothelial cells secrete TGF-β1 in-vitro. In women with endometriosis, peritoneum from sites adjacent to endometriosis lesions expressed higher levels of TGFB1 mRNA when compared to distal sites (P<0.05). The TGF-β-stimulated Smad 2/3 signalling pathway was active in the peritoneum and there were significant increases (P<0.05) in expression of genes associated with tumorigenesis (MAPK8, CDC6), epithelial-mesenchymal transition (NOTCH1), angiogenesis (ID1, ID3) and neurogenesis (CREB1) in the peritoneum of women with endometriosis. In conclusion, the peritoneum, and in particular, the peritoneal mesothelium, is a source of TGF-β1 and this is enhanced around endometriosis lesions. The expression of TGF-β-regulated genes is altered in the peritoneum of women with endometriosis and this may promote an environment favorable to lesion formation.     

Out-Patient Peritoneal Lavage Cytology In the Detection of Residual Epithelial Ovarian Cancer

Peritoneal lavage fluid cytology was performed in 87 out-patients with histologically proven epithelial ovarian cancer undergoing primary management. A total of 246 peritoneal lavages were attempted, usually with temporary cannulae (n = 229). From these, 184 samples were obtained, of which 156 (85%) were suitable for cytological analysis. The sensitivity of peritoneal lavage fluid cytology in 67 patients with known residual disease was 57% whereas serum CA 125 levels were elevated in 58 (87%). Pre- and post-treatment peritoneal lavage fluid cytology had prognostic value, but this was less than that of serum CA 125 measurements.     

Use of a variety of biological parameters in distinguishing cirrhotic from malignant ascites

Twenty-two different protein measurements were taken in the serum and ascitic fluid of fifty consecutive patients in an attempt to investigate which tests are the most reliable for the differential diagnosis of ascites. Serum and ascitic fluid total proteins (TPR), albumin (ALB), lactate (LAC), ferritin (FER), C3 and C4 complement factors, C-reactive protein (CRP), ceruloplasmin (CER), alpha2-macroglobulin (alpha2MG), haptoglobin (HAP), alpha1-antitrypsin (alpha1AT), alpha1-acid glycoprotein (alpha1AG), transferrin (TRF), immunoglobulins IgG, IgA, IgM and cytokines such as interleukin-1alpha (IL-1alpha), interleukin-1beta (IL-1beta), interleukin-2 (IL-2), interleukin-6 (IL-6), interleukin-8 (IL-8) and tumor necrosis factor-alpha (TNF-alpha) were measured to distinguish between malignant and cirrhotic ascites. Correlations and non-parametric Mann-Whitney tests were used for ascitic fluid:serum ratio comparisons between the two groups. Multivariate analyses were used to determine the most significant biochemical ratio predictors for the differential diagnosis and a recursive partitioning model was constructed. Highly positive correlations (r>0.50) were found between the ratios IgA, IgG, IgM, CER, alpha2 MG, HAP, alpha1AT, alpha1AG and TRF. There was evidence that TPR, ALB, LAC, FER, IgG, CER, alpha2MG, alpha1AT, alpha1AG, TRF and IL-8 ascitic fluid:serum ratios are significnatly higher in patients with malignant neoplasms than in cirrhotics. In the recursive partitioning model the most significant parameters were found to be the ratios of albumin and IL-1alpha. The model fitted allowed for 100% correct classification of ascites. In conclusion, we have shown that a simple and very accurate model based on two ascitic fluid: serum measurements is able to differentiate between malignant and non-malignant ascites.     

中医药防治腹膜纤维化的研究进展

腹膜透析是治疗终末期肾脏病较为有效的方法之一,但是腹膜长期暴露于非生理性的腹膜透析液中（低pH、高浓度葡萄糖以及高渗透压等）会致腹膜纤维化,从而导致腹膜结构和功能的丧失,这是患者放弃腹膜透析的原因之一。现代药理学实验证明,中医药对于腹膜纤维化的防治研究主要集中在腹膜间皮细胞的上皮间质转化方面,通过对细胞因子的作用,保护和／或改善腹膜间皮细胞的功能防治腹膜纤维化。