Atrial natriuretic peptide and progesterone in ovarian follicular fluid

Synthesis and secretion of atrial natriuretic peptide (ANP) is not confined to the heart, but also present in other tissues. ANP is known to affect steroidogenesis in the ovary. To assess the possibilities that (a) the human ovary is a source of ANP secretion as well, and (b) ovarian ANP stimulates progesterone secretion, we investigated ANP as well as progesterone concentrations in preovulatory ovarian follicular fluid form women in an in vitro fertilization program. In all women detectable ANP immunoreactivity was found in ovarian follicular fluid (range 3.3-60.1 pg/ml). Follicular fluid concentrations were low in all but 1 women who demonstrated a higher ANP level in follicular fluid than in plasma. Follicular ANP and progesterone concentrations were not significantly correlated. These preliminary results suggest that the ovary could well be a site of ANP secretion. If and how ovarian ANP activity affects progesterone secretion remains as yet unknown.     

Lipidomics analysis of follicular fluid by ESI-MS reveals potential biomarkers for ovarian endometriosis

PurposeThe aim of the present study was to analyze the lipid profile of follicular fluid from patients with endometriosis and endometrioma who underwent in vitro fertilization treatment (IVF).MethodsThe control group (n = 10) was composed of women with tubal factor or minimal male factor infertility who had positive pregnancy outcomes after IVF. The endometriosis group consisted of women with endometriosis diagnosed by videolaparoscopy (n = 10), and from the same patients, the endometriomas fluids were collected, which composed the endometrioma group (n = 10). From the follicular fluid and endometriomas, lipids were extracted by the Bligh and Dyer method, and the samples were analyzed by tandem mass spectrometry.ResultsWe observed phosphatidylglycerol phosphate, phosphatidylcholine, phosphatidylserine, and phosphatidylnositol bisphosphate in the control group. In the endometriosis group, sphingolipids and phosphatidylcholines were more abundant, while in the endometrioma group, sphingolipids and phosphatidylcholines with different m/z from the endometriosis group were found in high abundance.ConclusionThis analysis demonstrated that there is a differential representation of these lipids according to their respective groups. In addition, the lipids found are involved in important mechanisms related to endometriosis progress in the ovary. Thus, the metabolomic approach for the study of lipids may be helpful in potential biomarker discovery.

Electronic supplementary material

The online version of this article (doi:10.1007/s10815-015-0592-1) contains supplementary material, which is available to authorized users.     

Human follicular fluid levels of calcium, copper and zinc

Human follicular fluid was collected under laparoscopic vision from 33 follicles in patients stimulated with clomiphene-human menopausal gonadotrophin in an IVF programme. Thirty-one oocytes were obtained from 26 follicles from which clear follicular fluid was obtained, and 16 oocytes fertilized and cleaved. The follicular fluid was analysed for calcium, copper and zinc using atomic absorption spectroscopy. The mean levels of calcium, copper and zinc were 86.9 +/- 11.6, 1.16 +/- 0.29, and 0.72 +/- 0.12 micrograms/ml, respectively. Their levels were not significantly different in the follicular fluid collected from follicles of different sizes, with or without oocyte, and follicles with oocytes that were fertilized and were unable to be fertilized. Therefore, the concentrations of these elements do not seem to reflect oocyte status or maturity.     

Alteration of human follicular fluid plasminogen activator activity by ovarian hyperstimulation

Recently, it has been shown that granulosa-cell secretion of plasminogen activator (PA) is responsive to luteinizing hormone (LH)/human chorionic gonadotropin (hCG) as well as follicle stimulating hormone (FSH) in the rat and the pig. Accordingly, we asked whether PA activity in follicular fluid from exogenously stimulated human follicles was different from that of normal cycles and whether or not these activities correlated with follicular maturation as determined by follicular fluid steroid concentration. Follicular aspirates were obtained from women who were participating in an in vitro fertilization protocol. Follicular fluid concentrations of estradiol and progesterone were determined by established radioimmunoassay. PA activity, determined using a modified indirect solid-phase radioassay, was significantly less in follicles from patients treated with human menopausal gonadotropin (hMG) plus clomiphene (P<0.05) compared to untreated patients or those receiving hMG or clomiphene alone. Correlations of PA activity and follicular fluid steroid concentrations demonstrated no significant correlation in samples from treated patients. In contrast, untreated spontaneously cycling patients had a significant (r=0.89,P<0.05), positive correlation between follicular fluid estradiol levels. There was no correlation between PA activity and follicular fluid progesterone levels in any of the groups. These results suggest that a subtle balance in granulosa-cell secretion of PA and steroids exists, which appears to be disrupted by follicular hyperstimulation during treatment of patients participating in in vitro fertilization protocols.Supported in part by Clinical Investigator Award HD-00401 and by the University of Southern California Faculty Research and Innovation Award.     

In-vitro culture system for mesenchymal progenitor cells derived from waste human ovarian follicular fluid

To characterize different cell populations in the human ovary, morphological and functional characteristics of cell populations collected during routine IVF procedures were studied. Cells obtained from follicular fluid grew in vitro under minimal medium conditions, without growth factor, including leukaemia-inhibiting factor. Morphological analysis revealed a heterogeneous cell population, with cells displaying a fibroblast-like, epithelial-like and also neuron-like features. Morpho-functional characteristics of fibroblast-like cells were similar to mesenchymal stem cells, and, in particular, were positive for mesenchymal stemness markers, including CD90, CD44, CD105, CD73, but negative for epithelial proteins, such as cytokeratins, CD34 and CD45 antigens. Cell proliferation activity at different times and colony-forming unit capability were evaluated, and multipotency of a subset of granulosa cells was established by in-vitro differentiation studies (e.g. osteogenic, chondrogenic and adipogenic differentiation). This study suggests that cells provided by mesenchymal plasticity can be easily isolated by waste follicular fluid, avoiding scraping of human ovaries, and cultivated in minimal conditions. Successful growth of such progenitor cells on three-dimensional cryogel scaffold provides the basis for future developments in tissue engineering. This culture system may be regarded as an experimental model in which biological behaviour is not influenced by specific growth factors.     

Human preovulatory follicular fluid: the lipids. Are they the trigger for capacitation?

The lipid content of human follicles within 6 h of ovulation was determined. Levels for triglycerides, phospholipids, and total cholesterol were 1/4 to 1/3 of those in serum. An uncommonly high level of free cholesterol was observed, possibly resulting from a high synthetic rate by the granulosa. Follicular lipids appear to be of serum origin since the relative proportions of the various fatty acids are the same. Arachidonic acid was not found to be present at the high levels reported for "large" porcine follicles, possibly due to its active utilization in the synthesis of prostaglandins necessary for the ovulation process. Precise timing relative to ovulation is clearly essential for follicular classification in order to distinguish between before and after the triggering of the ovulation process. A possible role for lipids in the induction of capacitation is proposed.     

基础雄激素及卵泡液雄激素水平与卵巢反应性及妊娠结局关系的探讨

目的:探讨基础雄激素及卵泡液雄激素水平与卵巢反应性及妊娠结局的关系。方法:选取行IVF/ICSI助孕治疗的130例不孕症女性,将研究对象分为卵巢储备功能低下组、卵巢储备功能正常组。ELISA法检测基础雄激素及卵泡液中睾酮浓度。结果:卵巢储备功能低下组中,卵巢低反应组与正常反应组的基础雄激素水平比较,差异有统计学意义(P=0.037);两组卵泡液中雄激素水平比较,差异无统计学意义(P=0.475)。卵巢储备功能正常组中,卵巢低反应组与正常反应组的基础雄激素水平及卵泡液雄激素水平比较,差异均无统计学意义(P=0.426,P=0.594)。20例进行新鲜周期移植的卵巢储备功能低下患者中,未妊娠组与妊娠组基础雄激素水平及卵泡液中雄激素水平比较,差异均无统计学意义(P=0.400,P=0.656)。89例进行新鲜周期移植的卵巢储备功能正常患者中,未妊娠组与妊娠组的基础雄激素水平及卵泡液中雄激素水平比较,差异均无统计学意义(P=0.380,P=0.930)。结论:卵巢储备功能低下患者,基础雄激素水平与卵巢反应性相关,而与妊娠结局无关;卵泡液中雄激素水平与卵巢反应性和妊娠结局无关。卵巢储备功能正常患者,基础雄激素和卵泡液中雄激素均与卵巢反应性及妊娠结局无关。     

Relationship between serum estrogen and level of apolipoprotein E in human ovarian follicular fluid

Apolipoprotein E (apoE) is an important effector of plasma lipoprotein due to its interaction with cell-surface receptors. In addition, it is secreted by rat and human ovarian granulosa cells in tissue culture, and its production is hormonally modulated. Such observations indicate that apoE should be found in human follicular fluid (FF) and that its abundance therein may be subject to hormonal regulation. The authors have collected FF from seven normally cycling women by needle puncture at the time of laparoscopy and from hyperstimulated ovaries of four women undergoing in vitro fertilization (IVF). Apolipoprotein E is present in human FF. The ratio of apoE concentration in FF to that in serum, as determined by western blot analysis, ranges from 0.43 to 4.2 for the normally cycling patients and from 0.16 to 0.77 for the IVF patients. Serum estrogen levels (range, 42 to 477 pg/ml) for the cycling patients are inversely correlated with the FF-to-serum ratio of apoE (r = 0.91). The content of apoE in FF, relative to serum, thus falls dramatically as the follicle approaches ovulation. These data, plus the known ability of apoE to carry cholesterol and other lipids, suggest an important role for apoE in the ovary.     

Ultrasonic and endocrinologic investigation of ovarian follicle and free fluid in cul-de-sac for monitoring follicular development and ovulation

The accuracy of ultrasound as a means of monitoring follicular development and ovulation detection was studied in 31 infertile women. The follicular diameter measured by ultrasound correlated well actual diameter found during laparotomy (r = 0.88), and estradiol-17 beta (E2) and progesterone in follicular fluid aspirated from large follicles (greater than or equal to 16 mm) indicated high levels. Furthermore, in 23 out of the 55 cycles, ultrasound examinations demonstrated free fluid in cul-de-sac at ovulation, and concentrations of E2 and progesterone (P) in peritoneal fluid by the puncture of cul-de-sac dramatically increased after ovulation. We suggested that ultrasound monitoring provided a reliable measure of follicular growth and prediction of ovulation, and a combined use of ultrasound and puncture of cul-de-sac could be developed into a new tool for the study of the mechanism of human ovulation.     

Reactive oxygen species in follicular fluid may serve as biochemical markers to determine ovarian aging and follicular metabolic age

Introduction: The aim of this study was to evaluate the role of oxidative stress in the process of ovarian aging.

Methods: Follicular fluid (FF) from two randomly selected sibling follicles was collected from women undergoing in-vitro fertilization and tested for hydrogen peroxide (H₂O₂) levels.

Results: Group A consists of seven women with whom each of the two sibling separate follicle yielded an oocyte that was later discordantly developed to a low- and top-quality embryo. Group B consists of 13 patients in whom one of the sibling follicle yielded an oocyte while the other did not (empty follicle).

High-quality embryos were derived from follicles with lower H₂O₂ levels compared to follicles from which poor-quality embryos developed (1.004 units?±?0.260 versus 1.145 units?±?0.236, p?2O₂ levels were significantly higher (0.951 units?±?0.233 versus 0.623 units?±?0.309, p?Conclusion: During the process of ovarian ageing, there might be a gradual increase in H₂O₂ level in the follicle. Finally, when the follicle ages and becomes empty of oocyte H₂O₂ levels drops significantly. Therefore, H₂O₂ levels in FF may serve as a possible marker to determine ovarian aging and follicular metabolic age.     

Human chorionic gonadotropin and steroid concentrations in human follicular fluid in relation to follicle size and oocyte maturity in stimulated ovarian cycles

The concentrations in follicular fluid (FF) of beta hCG, progesterone (P), estradiol (E2), androstenedione (A) and testosterone (T) by RIA were determined, in order to study the follicular micro-environment in stimulated cycles. Patients received either clomiphene with hMG plus hCG or hMG plus hCG. With increasing volume of FF (obtained at laparoscopy), P significantly increased (p less than 0.04) and hCG (p less than 0.0004) and T (p less than 0.05) significantly decreased with a borderline decrease in A (p less than 0.07). Progesterone also increased with advancing oocyte maturity. Beta-hCG and P were positively correlated with E2 and negatively correlated with A levels. It is suggested that FF hCG and progesterone levels may be markers of follicular development in stimulated cycles.     

Inhibition of the primate ovarian cycle by a porcine follicular fluid protein(s)

Monkeys received twice daily intramuscular injections of 3 mg of purified porcine follicular fluid protein(s) for the first 14.5 days of the menstrual cycle. Two of five treated monkeys had anovulatory menstrual cycles. Three monkeys had cycles characterized by long follicular phases, low follicular and luteal phase serum estradiol concentrations, and subnormal luteal progesterone production. Serum gonadotropin concentrations were not affected by the follicular fluid protein(s). The data demonstrate in the nonhuman primate that porcine follicular fluid contains a protein(s) that acts at the ovarian level to inhibit gonadotropin action.     

Danazol concentrations in human ovarian follicular fluid and their relationship to simultaneous serum concentrations

Danazol concentrations in follicular fluid and serum were studied in eight women scheduled for laparoscopy because of suspected endometriosis. In order to obtain some variation in follicular maturity, danazol administration was started 2 to 7 days before the expected day of ovulation. A total of nine doses were given, i.e., 200 mg four times daily for 2 days; the last 200-mg dose was given 3 hours before the laparoscopy during which the follicular fluid from the dominant follicle was aspirated. Peripheral venous blood samples were drawn before, during, and after laparoscopy. Danazol concentrations were assayed by means of a high-performance liquid chromatography method. At the time of follicular aspiration, the mean concentration of danazol was estimated at 96 ng/ml in serum and at 71 ng/ml in follicular fluid, i.e., an average of 73% of the simultaneous serum concentration. The data suggest that even short-term therapy with danazol is likely to produce intrafollicular drug concentrations that have a direct inhibitory effect on follicular steroidogenesis.     

Serum and follicular fluid leptin levels in patients undergoing controlled ovarian hyperstimulation for in vitro fertilization cycle

OBJECTIVE: To determine serum and follicular fluid leptin levels in patients undergoing controlled ovarian hyperstimulation (COH) for an in vitro fertilization-embryo transfer (IVF-ET) cycle and their possible correlation to COH variables. Setting: Large university-based IVF unit. Patients: 16 consecutive patients undergoing our routine IVF long gonadotrophin-releasing hormone-analog protocol. INTERVENTIONS AIND MAIN OUTCOME MEASURES: Blood was drawn three times during the COH cycle: 1) day on which adequate suppression was obtained (Day-S); 2) day of or prior to human chorionic gonadotrophin (hCG) administration (Day-hCG); and 3) day of ovum pick-up (Day-OPU). Levels of sex steroids and serum and follicular fluid leptin were compared among the three time points. Serum leptin was measured with a commercial two-site immunoradiometric assay. RESULTS: Results showed significantly higher levels of serum leptin on Day-OPU and Day-hCG than on Day-S, and significantly higher follicular than serum leptin levels on Day-OPU. Though a significant correlation was observed between serum leptin and body mass index (BMI), no correlations were found between serum or follicular fluid leptin and serum sex-steroid levels or IVF treatment variables. CONCLUSION: While serum leptin increases during COH for IVF, there is apparently no correlation of serum and follicular leptin levels with sex-steroid levels or IVF outcome.     

High concentrations of immunoreactive renin, prorenin and enzymatically-active renin in human ovarian follicular fluid

Prorenin (enzymatically inactive) and renin (active) were measured by radioimmunoassay, using monoclonal antibodies reacting either with both prorenin and renin or with renin alone, in pre-ovulatory follicular fluid (FF) from women in an in-vitro fertilization programme who were stimulated with human menopausal/human chorionic gonadotrophin. The concentration of prorenin in FF was 40 times higher than in plasma taken at the time of FF collection; renin in FF was 10 times higher. The plasma concentration of prorenin, but not of renin, in these women was higher than in non-stimulated women in the late follicular phase of the menstrual cycle. The concentration of reninsubstrate and angiotensin-converting enzyme in FF was 60% of that in plasma. Contamination of blood, which may occur at the time of FF collection, was less than 5%. Prorenin in FF was irreversibly converted into renin after adding trypsin or by endogenous serine protease, using procedures that also cause conversion of prorenin in plasma. These results support the hypothesis that the increased plasma level of prorenin in women whose ovulation is stimulated for the collection of oocytes has originated from the ovary and is under gonadotrophic control. This may also be true for the increase of plasma prorenin that has been observed in non-stimulated women during the luteal phase of the cycle and in early pregnancy.     

High concentrations of immunoreactive renin, prorenin and enzymatically-active renin in human ovarian follicular fluid

Summary. Prorenin (enzymatically inactive) and renin (active) were measured by radioimmunoassay, üsing monoclonal antibodies reacting either with both prorenin and renin or with renin alone, in preovulatory follicular fluid (FF) from women in an in-vitro fertilization programme who were stimulated with human menopausal/human chorionic gonadotrophin. The concentration of prorenin in FF was 40 times higher than in plasma taken a! the time of FF collection; renin in FF was 10 times higher. The plasma concentration of prorenin, but not of renin, in these women was higher than in non-stimulated women in the late follicular phase of the menstrual cycle. The concentration of renin-substrate and angiotensin-converting enzyme in FF was 60% of that in plasma. Contamination of blood, which may occur at the time of FF collection, was less than 5%. Prorenin in FF was irreversibly converted into renin after adding trypsin or by endogenous serine protease, using procedures that also cause conversion of prorenin in plasma. These results support the hypothesis that the increased plasma level of prorenin in women whose ovulation is stimulated for the collection of oocytes has originated from the ovary and is under gonadotrophic control. This may also be true for the increase of plasma prorenin that has been observed in non-stimulated women during the luteal phase of the cycle and in early pregnancy.     

Evidence of maternal platelet activation,excessive thrombin generation,and high amniotic fluid tissue factor immunoreactivity and functional activity in patients with fetal death

Objective. Fetal death can lead to disseminated intravascular coagulation or fetal death syndrome. However, currently it is not clear what are the changes in the coagulation system in patients with a fetal death without the fetal death syndrome. This study was undertaken to determine: (1) whether fetal death in the absence of fetal death syndrome is associated with changes in hemostatic markers in maternal plasma and amniotic fluid; and (2) whether maternal hypertension or placental abruption are associated with further changes in the hemostatic profile of these patients.

Methods. A cross-sectional study included the following: (1) determination of changes in markers of coagulation and platelet activation in patients with a normal pregnancy (n = 71) and patients with fetal demise (FD) without disseminated intravascular coagulation (n = 65); (2) determination of the amniotic fluid (AF)–tissue factor concentration and activity, as well as the concentrations of thrombin–antithrombin III (TAT) complexes in patients with a normal pregnancy (n = 25) and those with a FD (n = 36) who underwent amniocentesis. Plasma and AF concentrations of TAT complexes and TF (an index of thrombin generation), as well as maternal plasma concentrations of sCD40L (a marker of platelet activation), tissue factor pathway inhibitor (TFPI) and prothrombin fragments (PF) 1 + 2 (also an indicator of in vivo thrombin generation) were measured by ELISA. TF and TFPI activity were measured using chromogenic assays.

Results. (1) patients with FD without hypertension had a higher median maternal plasma sCD40L concentration than normal pregnant women (P < 0.001); (2) patients with FD had a higher median maternal plasma TAT III complexes than women with a normal pregnancy (P < 0.001); (3) the median AF–TF concentration and activity were higher in the FD group than in the normal pregnancy group (P < 0.001 for both); (4) patients with preeclampsia and FD had a higher median maternal plasma immunoreactive TF concentration than both normotensive patients with FD and women with normal pregnancies (P < 0.001 and P = 0.001, respectively); (5) the median plasma TF activity was higher in patients with preeclampsia and FD than that of women with normal pregnancies (P = 0.003); (6) among patients with a FD, those with placental abruption had a higher median AF–TAT complexes concentration than those without abruption (P = 0.0004).

Conclusions. Our findings indicate that: (1) mothers with a FD have evidence of increased in vivo thrombin generation and platelet activation than women with normal pregnancies; (2) patients with a FD and hypertension had a higher degree of TF activation than those with fetal death but without hypertension; (3) the AF of women with a FD had a higher median TF concentration and activity than that of normal pregnant women. AF can be a potential source for tissue factor and it participates in the development of fetal death syndrome in patients with a retained dead fetus.