Ovary and ovulation: Nitric oxide concentrations in the follicular fluid and apoptosis of granulosa cells in human follicles

To study the relationship between follicular atresia, apoptosis,and nitric oxide (NO) generation in follicular development,steroidogenesis, NO levels in follicular fluid and apoptosiswere analysed in the various sized follicles of women receivingovarian stimulation with human meno-pausal gonadotrophin (HMG)—humanchorionic gonado-trophin (HCG) treatments for in-vitro fertilization(IVF)-embryo transfer. The follicles were divided into threegroups by diameter: large follicle, 18 mm; medium follicle,12 and 15 mm; small follicle, 10 mm. Follicular fluid was obtainedfrom 20 women 34 h after HCG administration, and the concentrationsof oestradiol, progesterone and testosterone, and nitrite, nitrate,arginine and citrulline were measured. Granulosa cells obtainedfrom each group of follicular fluid were stained with Hoechstdye, and nuclear morphology was examined by a fluorescence microscopy.Oestradiol and progesterone concentrations in large follicleswere significantly (P < 0.01) higher than those in mediumor small follicles, and testosterone concentrations in smallfollicles were significantly (P < 0.01) higher than thosein large follicles. There were no significant differences inthe concentrations of nitrite, nitrate, arginine and citrullineamong three groups. The percentage of apoptotic cells with nuclearfragmentation was significantly (P < 0.01) higher in smallfollicles than in large follicles. The present results suggestedthat small follicles with poor response to HMG may undergo atresiathrough apoptosis. No significant difference in the follicularNO level between large and small follicles led us to speculateon a different responsiveness to NO in these two types of follicles.     

Follicular fluid concentrations of adrenomedullin, vascular endothelial growth factor and nitric oxide in IVF cycles: relationship to ovarian response

Marked granulosa cell proliferation along with important changes in the vascular bed of the ovary characterize IVF cycles associated with multiple follicular growth and maturation. The present report investigated follicular fluid (FF) and circulating concentrations of adrenomedullin, vascular endothelial growth factor (VEGF) and nitric oxide (NO) in 70 IVF patients (14 of whom became pregnant); these three vasoactive substances may be implicated in extensive ovarian tissue remodelling. Serum and FF concentrations of oestradiol and progesterone were also measured in the 70 IVF cycles studied. Follicular fluid concentrations of VEGF and adrenomedullin but not nitrite/nitrate (the two stable oxidation products of NO metabolism) were significantly higher (P < 0.0001) than the corresponding circulating concentrations. Follicular fluid concentrations of oestradiol and progesterone were not correlated with those of adrenomedullin, VEGF or nitrite/nitrate. No relationship existed between circulating concentrations of adrenomedullin, VEGF or nitrite/nitrate on the day of oocyte aspiration and parameters of ovarian response to gonadotrophin stimulation. In contrast, FF adrenomedullin concentration showed a direct relationship with day 3 FSH serum concentration (r = 0.53, P < 0.01) and the number of ampoules of gonadotrophin administered (r = 0.36, P < 0.005), but an inverse correlation with the total number of oocytes retrieved (r = -0.29, P < 0.01) and the number of mature oocytes (r = -0.25, P < 0. 05). A positive correlation was found for FF VEGF concentration and chronological age (r = 0.29, P < 0.05) and ampoules of gonadotrophins administered (r = 0.30, P < 0.05). There was no relationship between nitrite/nitrate FF concentrations and parameters of ovarian response. Neither serum concentrations nor FF concentrations of adrenomedullin, VEGF or nitrite/nitrate were correlated with IVF outcome. This study suggested for the first time that increased FF concentrations of adrenomedullin can be a marker of decreased ovarian response in IVF. Our results also provide further evidence favouring an association between FF VEGF and patient's age, while on the basis of our findings NO measurements are not a useful marker of ovarian response.     

Nitric oxide (NO) and interleukin-1beta (IL-1beta) in follicular fluid and their correlation with fertilization and embryo cleavage

PROBLEM: Using an IVF model, the goal of the study was to investigate the relationship between follicular fluid (ff) NO and IL-1beta levels, as well as their correlation with fertilization of mature oocytes and embryo cleavage rates. METHOD OF STUDY: Follicular fluid was collected from 17 patients at the time of transvaginal oocyte retrieval following controlled ovarian stimulation. Oocytes harvested from these follicles were followed through fertilization and embryo cleavage. The NO metabolites nitrate/nitrite (NO3/NO2) were measured using the Griess reaction as an indirect assessment of NO activity. IL-1beta was measured using a high sensitivity ELISA system (Amersham, UK). The Student's t-test was utilized for unpaired data with the means considered significantly different when P < or = 0.05. RESULTS: Follicular fluid NO3/NO2 levels were significantly lower in follicles containing mature oocytes that fertilized (n = 30; 9.7 +/- 1.0 microM), versus those that did not fertilize (n = 23; 15.4 +/- 2.4 microM; P < 0.05). Follicles that contained oocytes that fertilized and went on to divide beyond the 6 cell stage had significantly lower ff levels of NO3/NO2 (n = 18; 7.5 +/- 0.9 microM), as compared to ff that contained oocytes that did not fertilize or failed to develop beyond the 5 cell stage (n = 35; 14.6 +/- 1.7 microM; P < 0.01). No correlation was found between ff NO3/NO2 levels (n = 28; 13.8 +/- 2.0 microM) and ff IL-1beta levels (n = 28; 0.5 +/- 0.08 pg/mL). An analysis of ff IL-1beta levels in relation to fertilization and embryo cleavage rates revealed no correlation. CONCLUSIONS: Lower ff NO3/NO2 levels at the time of oocyte retrieval are associated with adequate fertilization and embryo cleavage rates. In our IVF model, no correlation was found between ff IL-1beta levels and ff NO3/NO2, fertilization, or embryo cleavage rates.     

Adjuvant L-arginine treatment in controlled ovarian hyperstimulation: a double-blind, randomized study

BACKGROUND: Enhanced vascularization appears to be important for follicular selection and maturation in both spontaneous and stimulated IVF cycles. Nitric oxide, formed in vivo from L-arginine, may play a key role in follicular maturation and ovulation. METHODS: To evaluate the role of L-arginine supplementation in controlled ovarian hyperstimulation, 37 IVF patients were divided into two groups according to ovarian stimulation protocols: group I, GnRH agonist plus pure (p)FSH plus oral L-arginine (n = 18); and group II, GnRH agonist plus pFSH plus placebo (n = 19). Hormonal, ultrasonographic and Doppler evaluations were performed, and plasma and follicular fluid nitrite/nitrate concentrations were monitored. RESULTS: Thirty-two patients completed the study. In group I (n = 16), plasma L-arginine concentrations increased from (basal) 87 +/- 12 micromol to 279 +/- 31 micromol (P = 0.002) on the day of beta-HCG administration. In this group, pFSH treatment was shorter (P = 0.039) than in group II (n = 16). The number of the follicles > or =17mm was lower (P = 0.038) in group I than group II. The "good quality" embryos were fewer in number (P = 0.034) and pregnancy rate, both per patient (P = 0.024) and per embryo transfer (P = 0.019), was lower in group I. In the L-arginine group, an increased follicular fluid concentration of nitrite/nitrate was observed. On day 8 of the cycle, elevated plasma estradiol levels were associated with decreased blood flow resistances of perifollicular arteries. Follicular fluid concentrations of nitrite/nitrate were inversely correlated with embryo quality (r = -0.613; P = 0.005) and perifollicular artery pulsatility index (r = -0.609; P = 0.021). CONCLUSIONS: L-Arginine supplementation may be detrimental to embryo quality and pregnancy rate during controlled ovarian hyperstimulation cycles.     

Differential induction of nitric oxide synthase in various organs of the mouse during endotoxaemia: role of TNF-alpha and IL-1-beta.

BALB/c mice injected intraperitoneally with bacterial lipopolysaccharide (LPS) developed lethal septic shock. This was accompanied by significantly elevated concentrations of nitrite and nitrate in the plasma and expression of high levels of nitric oxide (NO) synthase activity in the lungs, heart, spleen and peritoneal macrophages. Mice pretreated with anti-tumour necrosis factor-alpha (TNF-alpha) monoclonal antibody or anti-interleukin-1 beta (IL-1 beta) polyclonal antibody were protected, in a dose-dependent manner, from endotoxin-induced mortality. This effect was accompanied by a significant reduction in plasma levels of nitrite and nitrate. Antibody treatment also reduced the level of NO synthase activity in peritoneal macrophages, spleen and heart but had no effect on enzyme expression in the lung. These results demonstrate that TNF-alpha and IL-1 beta play an important role in the induction of NO following administration of LPS and in the development of endotoxin-induced shock. In addition, NO synthase activity is differentially expressed in various organs and this may not always require TNF-alpha and IL-1 beta.     

Concentrations of immunoreactive interleukin-1 and interleukin-2 in human preovulatory follicular fluid.

The presence of immunoreactive interleukin-1 (IL-1) and interleukin-2 (IL-2) in human follicular fluid obtained at the time of oocyte collection for in-vitro fertilization was ascertained by radioimmunoassay. In group I (20 fluids from 20 patients), the concentrations of IL-1 were 0.9 +/- 0.06 and 1.9 +/- 0.04 (mean +/- SEM) fmol/l in follicular fluid and plasma respectively. A positive correlation existed between IL-1 levels in follicular fluid and plasma (r = 0.56, P less than 0.01). Concentrations of IL-2 were 3.5 +/- 0.2 and 6.1 +/- 0.3 fmol/l in follicular fluid and plasma respectively. A positive correlation of IL-2 levels was also found between follicular fluid and plasma (r = 0.65, P less than 0.01). There was no association between IL-1, IL-2 and steroid levels, regardless of whether they were compared in follicular fluid or plasma. Group II was composed of a series of fluids (two to seven samples for each of seven patients) in which the follicular concentrations of IL-1 and IL-2 did not show a positive correlation with the volume of follicular fluid or the concentrations of follicular fluid steroids. It is concluded that human preovulatory follicular fluid contains immunoreactive IL-1 and IL-2. The role of IL-1 and IL-2 in ovarian physiology remains to be determined.     

Inhibition of microglial inflammatory responses by norepinephrine: effects on nitric oxide and interleukin-1beta production

BACKGROUND: Under pathological conditions, microglia produce proinflammatory mediators which contribute to neurologic damage, and whose levels can be modulated by endogenous factors including neurotransmitters such as norepinephrine (NE). We investigated the ability of NE to suppress microglial activation, in particular its effects on induction and activity of the inducible form of nitric oxide synthase (NOS2) and the possible role that IL-1beta plays in that response. METHODS: Rat cortical microglia were stimulated with bacterial lipopolysaccharide (LPS) to induce NOS2 expression (assessed by nitrite and nitrate accumulation, NO production, and NOS2 mRNA levels) and IL-1beta release (assessed by ELISA). Effects of NE were examined by co-incubating cells with different concentrations of NE, adrenergic receptor agonists and antagonists, cAMP analogs, and protein kinase (PK) A and adenylate cyclase (AC) inhibitors. Effects on the NFkappaB:IkappaB pathway were examined by using selective a NFkappaB inhibitor and measuring IkappaBalpha protein levels by western blots. A role for IL-1beta in NOS2 induction was tested by examining effects of caspase-1 inhibitors and using caspase-1 deficient cells. RESULTS: LPS caused a time-dependent increase in NOS2 mRNA levels and NO production; which was blocked by a selective NFkappaB inhibitor. NE dose-dependently reduced NOS2 expression and NO generation, via activation of beta2-adrenergic receptors (beta2-ARs), and reduced loss of inhibitory IkBalpha protein. NE effects were replicated by dibutyryl-cyclic AMP. However, co-incubation with either PKA or AC inhibitors did not reverse suppressive effects of NE, but instead reduced nitrite production. A role for IL-1beta was suggested since NE potently blocked microglial IL-1beta production. However, incubation with a caspase-1 inhibitor, which reduced IL-1beta levels, had no effect on NO production; incubation with IL-receptor antagonist had biphasic effects on nitrite production; and NE inhibited nitrite production in caspase-1 deficient microglia. CONCLUSIONS: NE reduces microglial NOS2 expression and IL-1beta production, however IL-1beta does not play a critical role in NOS2 induction nor in mediating NE suppressive effects. Changes in magnitude or kinetics of cAMP may modulate NOS2 induction as well as suppression by NE. These results suggest that dysregulation of the central cathecolaminergic system may contribute to detrimental inflammatory responses and brain damage in neurological disease or trauma.     

Nitric oxide and pro-inflammatory cytokines correlate with pain intensity in chronic pain patients

Objective: Inflammatory cytokines as well as nitric oxide (NO) play a key role in the pathogenesis of persistent and exaggerated pain states. To document this, we investigated whether a range of cytokines and NO were detectable in the plasma of chronic pain patients and whether cytokine and NO levels correlated with pain severity. Methods: Plasma samples of 94 chronic pain patients and 6 healthy volunteers were obtained. Average pain intensity during the last 24h was assessed on a 11-point numeric rating scale and patients were distributed to three groups: light, moderate and severe pain. The concentrations of TNF-α, GM-CSF, interleukin (IL)-1β, IL-6, IL-8, interferon (IFN)-γ, IL-2, IL-4, IL-5, IL-10 and nitrate/nitrite were determined. Results: Patients with light pain demonstrated significantly increased levels of IL-6 compared to controls. In the severe pain group IL-6 and nitrate/nitrite were significantly increased. Serum concentrations of IL-1β, TNF-α, IL-2 and IL-4 were increased but as we adjusted the level of significance at p = 0.0045, most cytokine plasma levels failed to reach statistical significance. Conclusions: Pro-inflammatory cytokines (IL-1β, IL-2, IL-6, IFN-γ, TNF-α) in the plasma correlate with increasing pain intensity. Chronic pain patients show a significant increase in plasma levels of NO in comparison to healthy controls. Received 30 May 2006; returned for revision 17 July 2006; accepted by G. Geisslinger 7 August 2006     

Seminal plasma nitrite/nitrate and intratesticular Doppler flow in fertile and infertile subjects

The objective of the present study was prospectively to evaluate the role of nitric oxide (NO) in modulating intratesticular blood flow and sperm function. A total of 56 males, undergoing assisted reproduction, were divided into three groups according to semen analysis: (i) normozoospermic (n = 16); (ii) oligozoospermic (n = 21); and (iii) azoospermic (n = 19). All the subjects were submitted to hormone analysis [luteinizing hormone, follicle stimulating hormone (FSH), growth hormone, testosterone, androstenedione, insulin], and to ultrasonographic (testicular volume) and Doppler (transmediastinal artery) evaluations. Plasma and seminal plasma nitrite/nitrate concentrations, and plasma insulin-like growth factor-I were assayed. All 56 patients completed the study. In normozoospermic patients, significantly greater testicular volume, lower transmediastinal resistances, and higher seminal plasma nitrite/nitrate concentrations were observed in comparison with both oligo- and azoospermic subjects. Testicular volume was inversely correlated with plasma FSH (r = -0.589; P = 0.005) and pulsatility index of transmediastinal artery (r = -0.402; P = 0.049). Furthermore, the seminal plasma nitrite/nitrate concentrations were inversely correlated with pulsatility index of transmediastinal artery (r = -0.511; P = 0.015). It was concluded that NO is involved in vascular modulation of testicular vessels and ultimately in sperm output.     

Aromatase mRNA expression in individual follicles from polycystic ovaries

Polycystic ovary syndrome (PCOS) is a common reproductive disorder characterized by arrested follicular development prior to selection of a dominant follicle. Dominant follicles produce large amounts of oestradiol but PCOS follicles do not. With several potential aromatase (P450AROM) inhibitors in follicular fluid, the question arises whether P450AROM is expressed in PCOS granulosa cells, but the activity is inhibited, or whether P450AROM is not expressed in PCOS. The purpose of the present study was to determine whether P450AROM mRNA expression is altered in PCOS and to correlate P450AROM mRNA expression in individual follicles with aromatase stimulatory bioactivity and oestradiol in the follicular microenvironments. P450AROM mRNA was measured in individual follicles from 16 PCOS and 48 regularly cycling control women by quantitative polymerase chain reaction (PCR) and correlated with follicular fluid oestradiol concentrations and aromatase stimulating bioactivity measured by the rat granulosa cells aromatase bioassay. Follicular fluid oestradiol was low in all control follicles <7 mm in diameter. Some follicles > or = 7 mm contained elevated oestradiol values (P < 0.01) and all had an androstenedione:oestradiol ratio of <4. Only in granulosa cells from follicles > or = 7 mm with an androstenedione:oestradiol ratio of <4 were P450AROM mRNA levels increased (P < 0.05). These same follicles also contained increased levels of aromatase stimulating bioactivity whereas follicles <7 mm or with androstenedione:oestradiol ratio of >4 contained little or no bioactivity. All PCOS follicles contained low levels of oestradiol, P450AROM mRNA and aromatase stimulating bioactivity similar to size- matched control follicles. These data indicate that P450AROM mRNA expression and oestradiol production begin in developing follicles when they reach approximately 7 mm in diameter. Oestradiol production is low in PCOS follicles because there is insufficient aromatase stimulating bioactivity to increase P450AROM mRNA expression.      

Polycystic ovary syndrome: anomalies in progesterone production

The underlying cause of anovulation and miscarriage in polycystic ovary syndrome (PCOS) is unknown. Progesterone may play an important role in oocyte fertilization and embryo implantation. Therefore, in this study we analyse the endocrine function of luteinizing granulosa cells to synthesize progesterone in vivo and in vitro in PCOS and normal patients participating in an in-vitro fertilization programme. Human luteinizing granulosa cells were obtained from 10 patients with normal ovaries (controls) and 10 patients with PCOS by follicular aspiration of individual follicles of each patient and pooled in an attempt to obtain three groups: cells from follicle sizes < or =10,>10< or =15 and > or =16. Serum concentrations of oestradiol and progesterone on the day of human chorionic gonadotrophin (HCG) injection were significantly higher (P < 0.01 and P < 0.05) in PCOS patients than in controls. After HCG stimulation, in-vitro progesterone production was enhanced in granulosa cells of the control group and concentrations increased with follicular size as expected. However, the concentration of progesterone of PCOS patients did not increase with follicular size and there was a significant difference between normal and PCOS groups in follicles >10< or =15 mm (P < 0.05) and > or =16 mm (P < 0.01). Oestradiol production was increased in follicles > or =16 mm in both groups, although this did not reach significance. In summary, it seems that PCOS granulosa cells demonstrate an abnormal capacity to synthesize progesterone in vivo and in vitro. The understanding of granulosa cell function in PCOS may explain the anovulation and miscarriage that occurs in these patients.      

Inhibin A, inhibin B and activin A in the follicular fluid of regularly cycling women

Recent measurements of circulating inhibin A and inhibin B concentrations indicate that inhibin B may play an important role in the selection of dominant follicles. The concentrations of inhibin A, inhibin B and activin A were measured in the follicular fluids of 61 individual follicles (4.8-20 mm in diameter) from 47 regularly cycling women using specific two-site enzyme-linked immunosorbent assays. The microenvironment of each follicle was characterized by measuring follicular fluid androstenedione and oestradiol concentrations. The mean activin A concentrations were < 8 ng/ml for follicles of all sizes (4-17 mm). Inhibin A concentrations were < 1 ng/ml in follicles < 6 mm, and progressively increased to concentrations > 50 ng/ml in follicles > or = 13 mm. Follicles with androstenedione/oestradiol ratios < or = 4 had higher concentrations of inhibin A than follicles with androstenedione/oestradiol ratios > 4. Inhibin B concentrations were higher than inhibin A concentrations in all follicles, increasing from 19.2 +/- 8.3 ng/ml in 4 mm follicles to 409 +/- 9.6 ng/ml in 13 mm follicles and then declining to 275 +/- 47 ng/ml in 17 mm follicles. These results support the hypothesis that inhibin B may play a more important paracrine role in developing follicles and a greater regulatory role with respect to follicle stimulating hormone (FSH) secretion than inhibin A.      

Complete and partial luteinized unruptured follicle syndrome after ovarian stimulation with clomiphene citrate/human menopausal gonadotrophin/human chorionic gonadotrophin

A total of 31 clomiphene citrate/human menopausal gonadotrophin(HMG)/human chorionic gonadotrophin (HCG)-stimulated cyclesin 28 patients were investigated to determine the fate of eachof the matured follicles. A standard stimulation regimen wasadhered to, and ultrasound as well as hormonal monitoring wasperformed. All follicles were measured by vaginal ultrasoundat –12, +35 and +45 h relative to HCG administration andat 7 days after HCG administration. Of the 220 follicles, 107(48.6%) ruptured. The number of ruptured follicles per cyclewas correlated with the mid-luteal progesterone concentration(r = 0.63, P = 0.0005). The probability of follicular rupturewas related to follicular diameter at 12 h before HCG administration;6% of follicles <12 mm in diameter ruptured compared with87% of follicles 18–19 mm. A complete luteinized unrupturedfollicle (LUF) syndrome was observed in six cycles (20%). Inthese cycles, follicular growth and oestradiol, progesterone,luteinizing hormone (LH) and follicle stimulating hormone (FSH)concentrations at 12 h before HCG administration were similarto those in cycles with follicular rupture. However, mid-lutealprogesterone concentrations were lower in complete LUF cycles(46.97 ± 8.95 nmol/1 versus 108.74 ± 12.27 nmol/1;P = 0.02). These data demonstrate that in stimulated cyclesmany follicles, usually the smaller ones, fail to rupture, evenafter HCG administration. Complete LUF syndrome, despite a strongexogenous ovulatory signal, and the absence of any differencein peri-ovulatory hormonal parameters, indicates that the defectcausing LUF resides in the follicle itself and/or hormonal changesduring the follicular phase.     

The influence of ovarian follicular activity on late proliferative phase serum IGFBP-1 in down-regulated assisted conception cycles

Serum insulin-like growth factor binding protein-1 (IGFBP-1)concentrations were measured at the end of the proliferativephase in infertility patients undergoing normal menstrual cyclefrozen embryo transfer, exogenous hormone-supported frozen embryotransfer and in-vitro fertilization (IVF) treatment cycles.These patients were divided into five groups according to theirovarian follicular activity. The exogenous hormone-supportedfrozen embryo transfer group, who had no ovarian follicles,and the IVF groups (number of follicles ranging from 4–38)showed statistically higher serum IGFBP-1 concentrations whencompared to the normal menstrual cycle group (P0.01). Therewas no significant difference in the serum IGFBP-1 concentrationsbetween the exogenous hormone support frozen embryo transfergroup and the poor or normal response IVF groups (number offollicles ranging from 4 to 16). An IVF group that displayedan excessive response to our standard human menopausal gonadotrophinstimulation (>>20 mature follicles or oestradiol >>10000 pmol/1) showed a significantly higher serum IGFBP-1 concentrationwhen compared with the other groups (P = 0.001). This subgroupwas subsequently given a modified (follicle-stimulating hormone)stimulation regime which resulted in a significant reductionin serum IGFBP-1 concentrations (P << 0.05). There wasno correlation between serum oestradiol and IGFBP-1 overallor within the patient groups. We conclude that serum IGFBP-1concentrations in our down-regulated assisted conception cyclesdid not increase in line with ovarian follicular activity, unlessan excessive response was displayed.     

The correlation between interleukin 2 and soluble interleukin 2 receptors to oestradiol, progesterone and testosterone levels in periovulatory follicles of in-vitro fertilization patients.

The present study was performed to evaluate the correlation between follicular fluid levels of interleukin 2 (IL-2) and IL-2 soluble receptor (sIL-2R), oestradiol, progesterone and testosterone levels, oocyte fertilization, embryo quality and pregnancy rates. Twenty-eight patients with a pure tubal factor and undergoing in-vitro fertilization and embryo transfer were randomly chosen and treated with gonadotrophin releasing hormone agonist (GnRHa) in the midluteal phase (long protocol) coupled with follicular phase administration of human menopausal gonadotrophin. Transvaginal follicular aspiration was performed 36 h after human chorionic gonadotrophin administration, followed 48 h later by embryo transfer. One hundred and twenty-three follicular fluids were sampled. The mean follicular fluid levels (+/- SD) were 2.30 +/- 0.80 fmol for IL-2, 458.2 +/- 236.0 units/ml for sIL-2R, 28.5 +/- 58.1 ng/ml for oestradiol, 2360.5 +/- 2846 ng/ml for progesterone and 7.22 +/- 7.08 ng/ml for testosterone. There was a significant (P less than 0.01) correlation between IL-2 and testosterone levels. No correlation was found between the lymphokines and serum oestradiol, follicular fluid progesterone, oocyte fertilization, embryo quality and pregnancy. It may be concluded that significant concentrations of IL-2 and sIL-2R exist in follicular fluid. Wide variations in follicular IL-2 and sIL-2R concentrations of different follicles were found in the same patients.     

Upregulation of interleukin-8 by hypoxia in human ovaries

PROBLEM: To evaluate the effect of hypoxia on interleukin (IL)-8 expression in human ovarian follicles. METHOD OF STUDY: Follicular fluid (FF) from each follicle was separately collected from women undergoing in vitro fertilization and embryo transfer. Concentrations of oxygen, progesterone, estradiol, IL-1alpha/beta, IL-8, and tumor necrosis factor (TNF)-alpha in FF were measured. Isolated granulosa-lutein cells (GLC) from obtained FF were cultured under normoxic or hypoxic conditions, and concentrations of IL-8 in culture media were measured. RESULTS: Simple regression analysis demonstrated a significant negative correlation between the concentrations of IL-8 and oxygen in FF (r = 0.50, P < 0.0001). However, none of the concentrations of progesterone, estradiol, IL-1beta, and TNF-alpha in FF showed a significant correlation with IL-8 concentrations. Hypoxia stimulated the secretion of IL-8 by cultured GLC over twofolds compared with a normoxic control (P < 0.05). CONCLUSIONS: These findings suggest that IL-8, like other angiogenic factors, is upregulated under hypoxic condition, which argues that hypoxia in the ovarian follicles comes into play in ovarian functions by inducing a range of proangiogenic and chemoattractive substances.     

High concentrations of inhibin A and inhibin B in ovarian serous cystadenoma: relationship with oestradiol and nitric oxide metabolites

Inhibin production has been demonstrated in malignant epithelial ovarian tumours, but secretion of inhibins by benign cystadenoma has not yet been reported. The present study evaluated the concentrations of inhibin A and inhibin B and the relationship with oestradiol and nitric oxide metabolites in fluid collected from benign ovarian serous cystadenomas (n = 15). In addition, follicular fluid samples (n = 14) from women with regular ovulatory cycles undergoing ovarian stimulation for IVF were studied as a reference group. High concentrations of inhibin A (median = 89.3 ng/ml) and inhibin B (median = 116.1 ng/ml) were found in the cystic fluid of ovarian serous cystadenomas. These inhibin concentrations were even higher than in follicular fluid of stimulated follicles (inhibins A and B = 41.2 and 46.8 ng/ml respectively; P: < 0.001), whereas oestradiol was approximately 18-fold lower in cystic fluid than in follicular fluid (median = 34 versus 622 pg/ml, P: < 0.001). In ovarian cysts, the concentrations of inhibin A and oestradiol were inversely correlated (r = -0.678, P: = 0.008). Cystic fluid samples containing the highest concentrations of NO(2)(-)/NO(3)(-) (45-60 micromol/l) had lower inhibin A and higher oestradiol concentrations than those samples containing lower concentrations (10-25 micromol/l) of NO(2)(-)/NO(3)(-). It is concluded that high amounts of dimeric inhibins are present in ovarian serous cystadenoma. The source of inhibins and the determinants of the inverse association of inhibin A with oestradiol and nitric oxide remain to be determined.     

The clinical role of interleukin-6 and interleukin-6 soluble receptor in human follicular fluids

In order to investigate the role of interleukin-6 (IL-6) and interleukin-6 soluble receptor (sR) in human ovulation, we evaluated the concentrations in human follicular fluid and analyzed the correlation of IL-6 and IL-6 sR with oocyte maturation. The oocytes were obtained from the follicular fluid of 45 women undergoing in vitro fertilization and embryo transfer. The concentrations of IL-6 and IL-6 sR in follicular fluid were measured by ELISA. In addition, granulosa cells obtained from the follicular fluid were cultured and treated with forskolin and 12-o-tetradecanoylphorbol 13-acetate for 24–48 h. The concentration of IL-6 was significantly higher in the follicular fluid than in the serum (P<0.01). In contrast, the concentration of IL-6 sR was significantly lower in the follicular fluid than in the serum (P<0.001). The concentrations of IL-6 and IL-6 sR were significantly higher in the follicular fluid containing mature oocytes than in fluid containing immature oocytes (P<0.05). The production of IL-6 was markedly increased over the basal level after 24 h of treatment with forskolin(P<0.001) and 48 h of treatment (P<0.01) with cultured granulosa cells. Our data suggest that IL-6 and IL-6 sR may play an important role in follicular growth and development in human preovulatory processes. It is possible that IL-6 in particular may be regulated by cAMP. IL-6 and IL-6 sR might also be valuable biochemical markers in the evaluation of oocyte maturation. Received: 6 July 2002 / Accepted: 18 December 2002 Correspondence to Y. Kawano     

Cytokine regulation on the synthesis of nitric oxide in vivo by chronically infected human polymorphonuclear leucocytes.

To determine if nitric oxide (NO) is produced by chronically infected human polymorphonuclear leucocytes (PMNs) in vivo, inflamed exudates (periapical exudates: PE) collected from periapical periodontitis patients were examined. Cell-free supernatants and cells were separated by centrifugation. Significant levels of nitrite concentrations were observed in the supernatants. The production of inducible NO synthase (iNOS) in highly purified PMNs derived from PEs was then immunocytochemically determined using rabbit anti-human iNOS antiserum. In vitro, human peripheral blood PMNs (PB-PMNs) isolated from patients were cultured with a combination of Esherichia coli-lipopolysaccharide (LPS), recombinant human interferon-gamma (rhIFN-gamma) and/or interleukin-1 beta (rhIL-1 beta). The stimulated PB-PMNs showed steady-state levels of nitrite. The stimulation of LPS, rhIFN-gamma and rhIL-1 beta showed more NO induction than that of LPS with either IFN-gamma or IL-1 beta, suggesting the synergistic effects of cytokines. Cryostat sections of surgically removed periapical tissues were also immunohistochemically examined for iNOS, IFN-gamma and IL-1 beta. Two-colour immunohistochemistry revealed the interaction of iNOS-producing PMNs and IFN-gamma- or IL-1 beta-producing mononuclear cells. On the basis of these data, we concluded that with the stimulation of inflammatory cytokines derived from mononuclear cells, PMNs can spontaneously produce NO at the site of chronic infection. The present studies are consistent with a hypothesis suggesting that PMNs could be regulated and delicately balanced to produce NO by mononuclear cell-derived cytokines in vivo. NO-producing cells may play a pivotal role in chronic inflammation.     

In-vitro modulation of plasminogen activator activity, prostaglandin E and nitric oxide production by interleukin-1 in pregnant mare serum gonadotrophin-primed theca-interstitial cells

To examine the participation of the theca-interstitial (TI) compartment in cytokine modulation of ovarian function, the effects of interleukin- 1beta (IL-1) on plasminogen activator (PA) activity and on prostaglandin E (PGE) and nitric oxide (NO) production were examined in cultures of pregnant mare serum gonadotrophin (PMSG)-primed rat TI cells. Exposure to IL-1 (10 ng/ml) resulted in a 25% reduction (P < 0.001) in PA activity, concurrent with a 4.6-fold increase in the ability of the corresponding conditioned media to inhibit exogenous urokinase activity. IL-1 also produced a 4.7-fold increase in PGE content and a 2.8-fold increase in NO generation. These effects of IL-1 were abolished by the IL-1 receptor antagonist, suggesting specific IL- 1 receptor-mediated effects. Transforming growth factor (TGF)-beta1 (10 ng/ml) significantly attenuated the IL-1-stimulated PGE production and NO generation but did not affect the ability of IL-1 to suppress PA activity and stimulate urokinase inhibitor production. The NO synthase inhibitor N-nitro-L-arginine attenuated the IL-1-induced NO generation but had no effect on PA activity or PGE production. Thus, NO is not an obligatory mediator of IL-1 effects on plasminogen activation and PGE generation in rat ovary. The present observations attest to a pleiotropic response of PMSG-primed TI cells to IL-1, and suggest a paracrine/autocrine function for the TI compartment in ovulation and corpus luteum formation.