Detection of immunoreactive interleukin-11 in human follicular fluid: correlations with ovarian steroid, insulin-like growth factor I levels, and follicular maturity

Objective: To prove the presence of interleukin-11 (IL-11) in the follicular fluid (FF), to determine its source and the correlation between IL-11 and fertilization outcome, follicular size, number of follicles per patient, steroids, and insulin-like growth factor-1 (IGF-I) levels.

Design: Interleukin-11 levels were measured in FFs, aspirated during oocyte pickup for IVF.

Setting: Academic hospital and research environment.

Patient(s): Follicular fluid and serum were obtained with informed consent from 44 patients undergoing IVF-ET. Granulosa cells were isolated from 17 patients.

Main Outcome Measure(s): We hypothesized that IL-11 might play a role in follicular development, as do other related cytokines present in FF. Interleukin-11 was measured with ELISA.

Result(s): Interleukin-11 was absent in the serum but present in FF and in conditioned medium from granulosa cells. Atretic follicles had higher concentrations of IL-11. No correlation was found between IL-11 and fertilization outcome, follicular size, steroid, IGF-I, and total protein concentrations.

Conclusion(s): We conclude that IL-11 is present in FF. The role of IL-11 in follicular development should be the object of further investigations.     

Cortisol levels in human follicular fluid

This study shows that cortisol levels in follicular fluids in stimulated cycles were correlated with oocyte maturity and in vitro fertilizability. The levels were significantly higher than the concentrations found in spontaneous cycles. Our findings suggest that the presence of cortisol in follicular fluid may play a role in follicular development and oocyte maturation.     

Vascular endothelial growth factor, interleukin-6 and interleukin-2 in serum and follicular fluid of patients with ovarian hyperstimulation syndrome

BACKGROUND: The pathogenesis of ovarian hyperstimulation syndrome (OHSS) is not completely understood. OBJECTIVE: To investigate the presence of VEGF, IL-6 and IL-2, in serum and follicular fluid, in patients developing severe OHSS. STUDY DESIGN: We enrolled 101 women undergoing in vitro fertilization. Eight patients developing severe OHSS were compared with 43 high risk patients and 50 controls. We analyzed VEGF and IL-6 in serum collected before hCG administration, and in both serum and follicular fluid on the day of oocyte retrieval. RESULTS: OHSS patients presented follicular fluid IL-6 levels higher than both the patients at risk and controls (P<0.05). On the day of the oocyte retrieval the patients developing OHSS showed serum and follicular VEGF values higher than the ones of the patients at risk (P<0.05). Serum and follicular fluid IL-2 levels showed no differences between the examined groups. IL-2, IL-6 and VEGF values were not correlated with each other. CONCLUSIONS: Angiogenesis and inflammation processes are both present in severe OHSS.     

Immunosuppressive properties of human follicular fluid

Human preovulatory follicular fluids (FF) obtained in the course of stimulated cycles were analyzed for their possible immunologic functions. Different concentrations of FF (20%, 2%, 1%) inhibited the mitogenic response of normal human lymphocytes to concanavalin A (Con A). Lymphocytes were assessed for immunosuppressor activity after preincubation with FF. Lymphocyte mitogenic response to Con A was only suppressed by cells preincubated with FF at concentrations of 2% and 1% for at least 48 hours. No evidence of suppressor cell induction was seen following incubation of lymphocytes with 20% FF, nor was any significant relationship between FF immunosuppressor activity and the outcome of in vitro fertilization observed. We conclude that some factor(s) in FF may be capable of directly inhibiting lymphocyte response and inducing immunosuppressor cell activity in vitro.     

Studies on inhibin in human follicular fluid

In order to investigate whether inhibin (FSH-suppressing activity) is present in human follicular fluid (hFF) and whether inhibin in hFF could be correlated with the FSH level in peripheral serum, the effect of hFF on FSH secretion was studied using monolayer culture of rat anterior pituitary cells. Dextran-coated charcoal (DCC)-treated hFF exerted an inhibitory effect on pituitary FSH secretion but not on LH. The inhibitory effect of hFF upon basal FSH secretion was different from those of steroids such as estradiol, progesterone, testosterone and androstenedione. hFF inhibited the LHRH-stimulated secretion of both FSH and LH. Since hFF and porcine follicular fluid (pFF) produced a parallel dose-dependent decrease of basal FSH secretion, the same suppressing activity may be present in both hFF and pFF. Inhibin activity increased gradually during the follicular phase, but decreased in the luteal phase. Inhibin activity in hFF except during preovulatory surge showed a significant inverse correlation with the FSH level in peripheral serum. These results might indicate that inhibin activity in hFF increased according to follicular maturation and that inhibin (non-steroidal substance) may be one of the important regulators of FSH secretion in the human pituitary.     

Glutathione S-transferases in human ovarian follicular fluid

Objective: To study the levels of glutathione S-transferase Alpha 1-1 and glutathione S-transferase Pi 1-1 in human preovulatory ovarian follicular fluid (FF) and pooled granulosa and cumulus cells.Design: The relation of glutathione S-transferase Alpha 1-1 and glutathione S-transferase Pi 1-1 with P and 17β-E₂ concentrations were studied.Setting: The Department of Obstetrics and Gynecology, the Department of Gastroenterology, and the Laboratory of Endocrinology and Reproduction of the University Hospital Nijmegen in Nijmegen, the Netherlands.Patient(s): Infertile women participating in an IVF program.Result(s): Detectable amounts of glutathione S-transferase Alpha 1-1 and glutathione S-transferase Pi 1-1 were found in ovarian FF and pooled cumulus and granulosa cells. Concentrations of glutathione S-transferase Alpha 1-1 were always much higher than those of glutathione S-transferase Pi 1-1. Both ovarian FF concentrations of glutathione S-transferase Alpha 1-1 and glutathione S-transferase Pi 1-1 did not correlate with ovarian FF concentrations of 17 β-E₂ and P.Conclusion(s): The high FF concentrations of glutathione S-transferase Pi 1-1 and especially of glutathione S-transferase Alpha 1-1 suggest that these enzymes may play an important role in the detoxification processes in the follicles. The lack of correlation between follicular P and 17β-E₂ and glutathione S-transferase Alpha 1-1 and glutathione S-transferase Pi 1-1 indicates that both enzymes presumably are not present as a result of the high steroid levels.     

Platelet-activating factor-acether is a component of human follicular fluid.

OBJECTIVE: Platelet-activating factor-acether (PAF-acether) presence was investigated in 27 human follicular fluids (FFs). DESIGN: Aggregation of washed rabbit platelets was used to measure PAF-acether. Data were compared using Student's t-test. SETTING: Follicular fluids came from the in vitro fertilization program at Antoine Béclère Hospital, and PAF-acether was assayed at Institut National de la Santé et de la Recherche Médicale, Unités 187 and 200, Clamart, France. PATIENTS, PARTICIPANTS: The study concerned five infertile women 29 to 39 years of age. INTERVENTIONS: Ovaries were stimulated with human menopausal gonadotropin under gonadotropin-releasing hormone agonist (GnRH-a) action. MAIN OUTCOME MEASURES: The height of platelet aggregation was compared between FFs and synthetic PAF-acether. RESULTS: Mean FF concentration of PAF-acether was 1,367 to 3,467 pg/mL among women. Values were higher for patients in a long than in a short GnRH-a protocol (P less than 0.05). However, PAF-acether concentration was not related to fertilization rate. CONCLUSIONS: Platelet-activating factor-acether is possibly involved in oocyte release from the follicle, a process occulted by follicular puncture.     

Detection of T8 (suppressor/cytotoxic) lymphocytes in human ovarian follicular fluid

Clear ovarian follicular fluid and the follicular flush (bloody) of aspirated follicles from 18 women undergoing oocyte retrieval for in vitro fertilization were tested for the presence of T-lymphocyte subpopulations with the use of a panel of monoclonal antibodies in indirect immunofluorescence assay. A sample of peripheral venous blood from each patient was run as a control. There was no difference in the percentage of cells positive for the various markers between bloody follicular fluid (i.e., ovarian blood) and peripheral venous blood. T8-positive (suppressor/cytotoxic) lymphocytes were recovered from all clear follicular fluids; ten of the patients exhibited a dramatically decreased T4/T8 ratio (peripheral blood, 2:1; clear follicular fluid, 1:25; P less than 0.001). These lymphocytes may be involved in the suppression of autoimmune responses directed against ovarian antigens.     

Pregnancy-related chemotactic activity of human follicular fluid

We measured chemotactic activity in 238 follicular fluids (FF) aspirated from 45 women who had undergone ovarian stimulation with a combination of clomiphene citrate and human menopausal gonadotropin for oocyte retrieval, in vitro fertilization, and embryo transfer. Fifteen of the treatment cycles resulted in pregnancy. The mean chemotactic activity, measured as the distance in microns granulocytic leukocytes migrated through a 3.0-micron membrane, was significantly higher in FF from conceptual cycles, compared with nonconceptual cycles. Serum chemotactic activity was significantly lower in conceptual cycles, compared with nonconceptual cycles. A chemotactic gradient appears to exist between the peripheral circulation and the ovarian follicle. The gradient favors the follicle in conceptual cycles, as indicated by the chemotactic quotient (the ratio of chemotactic activity of FF to serum). In conceptual cycles the chemotactic quotient was 1.7 +/- 0.17, compared with 0.7 +/- 0.03 for nonconceptual cycles. The presence of leukocyte chemotactic factor in FF appears to discriminate prospectively with a 90% degree of confidence between conceptual and nonconceptual in vitro fertilization and embryo transfer cycles.     

The detection of blood contamination in human follicular fluid

Purpose: The aim of this study was to compare the reliability of the methods conventionally used to identify low levels of blood contamination in human follicular fluid (hFF) as applicable in the clinical environment. Methods: Follicular fluid (n=339) and plasma samples (n=20) were collected from patients (n=138) attending the Centre for Fertility Studies, HF Verwoerd Hospital, University of Pretoria, South Africa. hFF blood contamination was assessed by means of (a) visual inspection, (b) hematocrit (Hct), (c) spectrophotometric analysis, (d) spectrophotometric hemoglobin kit, and (e) Combur-9-test urine sticks. Results: (1) Neither hematocrit nor spectrophotometry provided reliable detection at low levels of blood contamination. (2) Visual inspection presented with a better discriminatory ability than either Hct or spectrophotometry. (3) Combur-9-test sticks identified up to 50% of blood-contaminated fluids. (4) Spectrophotometrically determined hemoglobin levels presented with weak discriminatory abilities for detecting blood-contaminated fluids. Conclusions: Visual inspection as performed in this study provides a fast and relatively reliable method for the determination of blood-contaminated hFFs. In a laboratory environment, however, it would be recommended that a combination of visual inspection, Hct, and spectrophotometric evaluation be employed for the selection of blood-free fluids.     

Prolactin size heterogeneity in human follicular fluid: a preliminary study.

Follicular fluid from mature preovulatory follicles was examined for the presence of prolactin (PRL) heterogeneity. Two pools of follicular fluid aspirates were used: one from follicles in which the ova were successfully fertilized in vitro and the other from follicles in which the ova were not successfully fertilized. Follicular fluid aspirates were concentrated by ultrafiltration and subjected to Sephadex G-100 gel chromatography. Fractions were assayed for immunoactive PRL by radioimmunoassay (RIA-PRL) and for bioactive PRL by the Nb2 lymphoma cell bioassay (BA-PRL). The major portion of RIA-PRL activity appeared as a low-molecular-weight component and accounted for 88% of the total PRL activity in the fertilized group and 87% in the unfertilized group. A high-molecular-weight component was also evident (12% and 13% for the two groups, respectively). The high-molecular-weight component in both groups was not active as PRL in the bioassay. These results demonstrate that while two immunoactive, heterogeneous forms of PRL exist in human follicular fluid when measured by RIA, it is only the low-molecular-weight form of PRL that is biologically active as established by Nb2 lymphoma cell bioassay.     

Angiotensin-I-converting enzyme and its correlation with human follicular fluid steroids.

Angiotensin-I-converting enzyme (ACE) is a peptidyl-dipeptide hydrolase which splits off the dipeptide His-Leu from the decapeptide angiotensin I and thus converts it to angiotensin II. We determined ACE activity in human preovulatory follicular fluid to further establish the intraovarian activity of the renin angiotensin system. Follicular fluids (n = 18) were obtained from eight patients undergoing in vitro fertilization (IVF) and embryo transfer (ET). ACE activity in follicular fluid and serum was determined by fluorescent spectrophotometry. The median follicular fluid ACE activity was 1.12 (range: 0.19-1.56) nmol/min/ml. This value was significantly lower than ACE activity in serum, 1.50 (range: 1.22-1.57) nmol/min/ml (P less than 0.001). In contrast to this 3:4 ratio between follicular fluid and serum ACE when expressed per ml fluid, the values were very similar when expressed per mg of protein: 0.025 vs. 0.023 nmol/min/mg in follicular fluid and serum, respectively. Correlations were sought between follicular fluid ACE activity and both serum and follicular fluid E2 and P4. A highly significant correlation (P less than 0.0005, r = 0.73) was found between ACE activity in follicular fluid and follicular fluid P4. The presence of significant ACE activity in human follicular fluid further supports the local-ovarian activity of the renin angiotensin cascade.     

Type IV collagenolytic activity in human preovulatory follicular fluid

During normal ovulation, the two basement membrane layers of the ovarian follicle are degraded locally. The main component of basement membranes is type IV collagen, which is specifically cleaved by type IV collagenase. According to our results, type IV collagenolytic activity is present within follicular fluid and increases toward ovulation, decreasing rapidly as the follicles rupture. These results suggest that importance of type IV collagenolytic activity in the ovulatory process.     

Human follicular fluid stimulates hyperactivated motility in human sperm

Since human follicular fluid (FF) is known to enhance the acrosome reaction and capacitation, we investigated whether hyperactivated motility is stimulated by FF. Follicular fluid-treated sperm exhibited a threefold increase in hyperactivation compared with the controls. The use of fetal cord serum in the medium, instead of bovine serum albumin, supported the same high levels of hyperactivation, although the peak occurred at 3 hours rather than 5 hours of capacitation. When sperm were treated with a steroid-rich fraction of the FF, hyperactivation was stimulated to the same degree as with whole FF. In contrast, no stimulation occurred when sperm were treated with a FF fraction stripped of steroids. The FF enhancement of hyperactivation in vitro could augment the fertilizing capacity of subfertile sperm samples, providing also a glimpse of possible in vivo events as sperm traverse the FF-laden cumulus oophorus.