Immunoreactive endothelin-1, endothelin-2 and big endothelin-1 in follicular fluids of women undergoing ovulation induction for in-vitro fertilization

Endothelin-like immunoreactivity specific for endothelin-1 (ET-1),endothelin-2 (ET-2) or big endothelin-1 (big ET-1) was measured,using commercially available radioimmuno-assay kits, in follicularfluid collected at the time of oocyte aspiration from 36 womenundergoing ovulation induction by human menopausal gonadotrophin(HMG). The relation-ship of ET concentrations to HMG dose, peakserum oestra—-diol concentration, the number and sizeof follicles (by ultrasound), the number of retrieved oocytesand the fertilization rate per retrieved oocyte were studied.Overall, 94% of follicular fluid samples were positive for ET-1,92% were positive for ET-2, and 100% were positive for big ET-1.Mean ET-1, ET-2 and big ET-1 concentrations were 17.23±12.20,32.42± 14.32 and 34.55± 16.34 pg/ml respectively.Endo-thelin-like immunoreactivity in follicular fluid sampleswas found in an order of ET-1<ET-2< big ET-1. There wasa highly significant positive correlation (r=0.8711, P=0.0001,n=32) between follicular ET-1 and ET-2 concentrations. No significantcorrelation of follicular big ET-1 was established either withET-1 or ET-2. However, big ET-1 was found to be negatively correlatedwith number of oocytes (P=0.03) and number of follicles (P=0.04).Control plasma ET-1 and follicular ET-1 were not significantlydifferent. There was no significant correlation between ET concentrationsand any of the other studied parameters. The results demonstratedthat immunoreactive ET-1, ET-2 and big ET-1 exist in human follicularfluid collected at the time of oocytes retrieval for in-vitrofertilization and may be involved in the regulation of reproductivefunction. The clinical significance and physiological role offollicular fluid ET deserve further studies.     

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.     

Purine levels and metabolism in human follicular fluid

Adenosine (ADO) in low micromolar levels and hypoxanthine (HYP) in millimolar levels have been shown to inhibit maturation of cumulus-enclosed oocytes. To determine the effect of ovarian stimulation with gonadotrophins on follicular purine metabolism, we measured ADO, HYP, inosine (INO), adenine (ADE) and cyclic AMP (cAMP) levels in follicular fluid (FF) from natural (n = 7) or human menopausal gonadotrophin/human chorionic gonadotrophin (HMG/HCG)-stimulated (n = 35) cycles. Purines were extracted immediately (natural cycles) or within 30 min of recovery (HMG/HCG cycles) and analysed by high pressure liquid chromatography (HPLC). The concentration of all ADE purines in FF was in the low micromolar range (1-20 microM); cAMP levels were markedly increased (greater than 100 microM) in FF of HMG/HCG-treated patients. While ADO levels were within the range effective for inhibition of oocyte maturation, those of HYP were not. No correlation was found between purine levels in FF and ovum maturation. Purine levels in FF of natural cycles were uniformly lower than those of stimulated cycles. Significant conversion of 5'-AMP into ADO, ADO into INO and INO into HYP occurred within 1 h when FF was incubated at 25 but not at 4 degrees C. These purine levels in human FF confirm our previous findings with bovine FF and suggest a possible role of ADO, but not of HYP, in the inhibition of oocyte maturation in the human.     

Relationship between human oocyte maturity, fertilization and follicular fluid growth factors

Follicular fluid concentrations of growth hormone (GH), insulin-likegrowth factor-I (IGF-I), epidermal growth factor (EGF) and oestradiolwere related to diversities in oocyte maturation and fertilizationamong oocytes obtained for invitro fertilization (IVF). Follicularfluid GH, IGF-I and oestradiol concentrations were significantlycorrelated with increasing follicular size. Follicles with immatureoocytes had concentrations of oestradiol that were significantlylower when compared to follicles with intermediate and matureoocytes. Follicular fluid EGF concentration was similar forall oocyte maturational stages. In follicular fluids with matureoocytes we found IGF-I and GH concentrations were significantlyhigher compared to those of follicular fluid with atretic oocytes.Follicular fluids with Immature and intermediate oocytes hadsimilar concentrations of GH and IGF-I to follicular fluid containingmature oocytes and higher concentrations than follicular fluidwith atretic oocytes. No statistically significant differencewas found between fertilized and unfertilized oocytes. We concludethat maturation of oocytes Is associated with higher concentrationsof GH, IGF-I and oestradiol, but follicular fluid IGF-I andGH concentrations cannot serve as a predictor for IVF.     

Endocrinology: Presence and possible function of activin-like substance in human follicular fluid

We assessed the presence of an activin-like substance in humanfollicular fluid that was obtained from women undergoing in-vitrofertilization using a bioassay for activin A. Activin activitywas not detected in crude follicular fluids; the bioactivityof standard activin A was inhibited by the addition of follicularfluid. After the follistatin (binding protein of activin A)was removed from follicular fluid using a purification procedure,activin activity was detected in the follicular fluids (meanconcentration: 131 ± 40 ng/ml). Activin activity wasinhibited by the addition of follistatin to fluid. The concentrationof activin activity was substantially higher (100-fold) thanthat reported in serum. The concentration negatively and significantlycorrelated with the number of developed follicles in the ovary(r = 0.501, P < 0.01). These results suggest that activinA and its binding protein are present in follicular fluid inlarge amounts and that they may have a role in local ovarianregulation.     

Somatostatin in human follicular fluid

To demonstrate the presence of somatostatin in human pre-ovulatoryfollicular fluid, and to assess the role of this peptide infollicular maturation, a total of 66 follicular fluid sampleswere obtained from 26 patients at the time of oocyte recoveryfor in-vitro fertilization. Follicular fluid concentrationsof somatostatin, oestradiol, progesterone and androstenedionewere measured by immunoassays. Somatostatin concentrations inconcomitantly obtained plasma samples were also analysed. Follicularfluid somatostatin concentrations ranged from undetectable (<1.5pmol/1) to 109.4 pmol/1. The mean ±SE somatostatin concentrationsin follicular fluid (12.8± 1.8 pmol/1) were significantly(P< 0.0001) increased compared to corresponding plasma concentrationsof somatostatin (6.5 ± 0.2 pmol/1). A significant andpositive correlation existed between follicular fluid and plasmasomatostatin concentrations (r = 0.27; P < 0.03). No differencesin either follicular fluid or plasma somatostatin concentrationswere found between different stimulation protocols or diagnosticgroups. Neither did follicular fluid somatostatin concentrationvary with follicular size. Similarly, no differences in somatostatinconcentrations were found between follicular fluids associatedwith fertilized (13.2 ± 2.1 pmol/1) or non-fertilizedoocytes (10.5± 1.6 pmol/1). Follicular fluid concentrationsof somatostatin correlated positively with those of progesterone(r % 0.30; P = < 0.04), but not with those of oestradiolor androstenedione or with the androstenedione/oestradiol ratio.The relationship between follicular fluid somatostatin and progesteroneconcentrations suggests that follicular fluid somatostatin mayhave a physiological role in follicular maturation and the luteinizationprocess.     

Immunosuppressive factor in cow follicular fluid.

The activity of the immunosuppressive fraction and proteinase inhibitor isolated from cow follicular fluid was investigated. The immunosuppressive factor was separated from the accompanying proteinase inhibitor by Sepharose 6B and Sephacryl S-200 column chromatography. In vitro, the fraction significantly reduced mitogen-induced lymphocyte proliferation. In vivo, the fraction inhibited mouse plaque formation. However, the factor had no effect on the development of intact or zona free embryos. The active components have a molecular mass of about 110,000.     

The correlation of interleukin 1 and tumour necrosis factor to oestradiol, progesterone and testosterone levels in periovulatory follicular fluid of in-vitro fertilization patients.

Data has accumulated suggesting reciprocity between cytokines and the reproductive system. The present study was performed in order to evaluate the correlation between interleukin 1 (IL-1) and tumour necrosis factor (TNF) concentrations in follicular fluid and its oestradiol, progesterone and testosterone levels. A total of 39 follicular fluid samples, from eight patients undergoing in-vitro fertilization and embryo transfer were evaluated. All of the patients were treated by a midluteal (long) protocol involving a gonadotrophin releasing hormone agonist (GnRHa) coupled with follicular phase human menopausal gonadotrophin. Mean levels in follicular fluid of IL-1, TNF, oestradiol, progesterone and testosterone were 1.58 +/- 0.42 fmol/0.1 ml, 4.69 +/- 4.18 pg/ml, 28.5 +/- 58.1 ng/ml, 2360.5 +/- 2846.3 ng/ml and 7.22 +/- 7.08 ng/ml respectively. There was a significant (P less than 0.01) positive correlation between IL-1 and progesterone levels. There was no significant correlation between the different lymphokines and oestradiol secretion, oocyte fertilization, embryo quality and pregnancy rates. It is concluded that IL-1 and TNF exist in follicular fluid. It may be hypothesized that IL-1 has a local regulatory action, possibly promoting luteinization.     

Pregnancy: Oxygen promotes contraction by endothelin-1 in human umbilical artery

The influence of oxygen on the contractile response to endothelin-1in the human umbilical artery was investigated in vitro. Segmentsof human umbilical artery were suspended in organ baths to recordthe circular motor activity induced by endothelin-1 at a pO₂of 12 kPascal (kPa) or 45 kPa. Endothelin-1 induced a concentration-dependentcontraction which was significantly larger at 45 kPa O₂ comparedwith the contractile response at 12 kPa O₂.     

Concentrations of monosaccharides and their amino and alcohol derivatives in human preovulatory follicular fluid

The study purpose was to compare sugar and polyol concentrations in preovulatory ovarian follicular fluid (FF) with those in the circulation. Samples of FF and peripheral venous blood were obtained after an overnight fast from 14 women attending an IVF program. High performance liquid chromatography measurements of seven polyols, two aminohexoses and four hexoses were the main outcome measures. Glucose concentrations in FF and plasma were 2781.26 +/- 205.64 and 4431.25 +/- 65.17 microM, respectively (P < 0.001). Mannose concentration in FF was 38.99 +/- 3.33 microM, significantly lower than plasma concentration (55.38 +/- 2.29 microM; P < 0.001). A concentration gradient from plasma to FF was also significant for glycerol (99.41 +/- 8.47 versus 74.32 +/- 6.54 microM; P < 0.002), galactose (31.69 +/- 1.58 versus 26.73 +/- 1.93 microM; P < 0.01) and galactosamine (11.49 +/- 0.69 versus 6.38 +/- 0.59 microM; P < 0.001). The plasma-to-FF concentration difference was greatest for glucose (1649.99 +/- 204.09 microM). There was a significant correlation between plasma and FF concentrations for galactose and glycerol. This study supports a substantial utilization of glucose by the oocyte/granulosa cells complex, and documents a significant concentration gradient from plasma to FF for glycerol, mannose, galactose and galactosamine. These plasma-FF differences may reflect both utilization of these carbohydrates by the cells of the preovulatory ovarian follicle and/or transport characteristics of these cells.     

Maternal serum alpha-fetoprotein and human chorionic gonadotrophin in pregnancies conceived after intracytoplasmic sperm injection and conventional in-vitro fertilization.

Data in the Caucasian population suggest that maternal serum alpha-fetoprotein (AFP) and unconjugated oestriol concentrations are reduced and human chorionic gonadotrophin (HCG) concentrations are elevated in pregnancies conceived after in-vitro fertilization (IVF), leading to a higher than expected Down's syndrome screen-positive rate. There are no previous reports on the serum marker values in pregnancies conceived after intracytoplasmic injection (ICSI). Between 1996 and 1998, we measured maternal serum total HCG and AFP concentrations between 15 and 20 weeks gestation in 42 in-vitro fertilization (IVF) pregnancies and 23 ICSI pregnancies with known normal outcome. The results were compared with that of 2799 naturally occurring singleton pregnancies who were known to have a normal outcome. Median AFP multiple of the median (MOM) in ICSI pregnancies was significantly reduced to 0.76 compared with both that of the controls and that of the IVF pregnancies. For the IVF pregnancies, median HCG MOM was elevated to 1.15, and median AFP MOM was reduced to 0.88 compared with the controls, but these differences were not statistically significant. In both the IVF and ICSI pregnancies the changes might result in a falsely high Down's syndrome risk. In particular, the reduced AFP concentration in ICSI pregnancies was substantial. If this preliminary finding is substantiated by other series, the appropriate adjustment needs to be made to allow for valid interpretation of the screen result and to avoid an unnecessarily high false positive rate.     

Early follicular rise of serum progesterone concentration in response to a flare-up effect of gonadotrophin-releasing hormone agonist impairs follicular recruitment for in-vitro fertilization

A retrospective study of 150 cycles of in-vitro fertilization(IVF) was undertaken to determine the impact of elevated serumprogesterone in the early follicular phase of IVF cycles utilizinggonadotrophin-releasing hormone agonist (GnRHa) initiated inthe follicular phase. A total of 127 patients identified asbeing at risk for poor response to stimulation were treatedwith a flare-up protocol of GnRHa combined with high dose folliclestimulating hormone (FSH). Patients were excluded for severemale factor requiring micromanipulation. Patients were stimulatedwith GnRHa beginning on cycle day 2, and high dose FSH beginningon cycle day 3. Some 85% of the cycles exhibited a rise of serumprogesterone to a peak concentration of > 1.0 ng/ml (range,1.2–4.2 ng/ml) during cycle days 2–6. When comparedto cycles with no demonstrable progesterone rise, cycles witha rise were associated with a significantly decreased ovarianresponse: more ampoules of gonadotrophin were required (mean26.8 versus 22.6, P < 0.05), lower peak oestradiol concentrationwas reached (mean 774 pg/ml versus 1030; P < 0.05), and fewermature oocytes were harvested (mean 4.6 versus 7.5; P < 0.01).Among the different pregnancy outcomes (clinical pregnancy,no pregnancy, ongoing pregnancy, and miscarrige), there wereno significant differences detected in the early follicularprogesterone concentrations as measured by peak progesterone,progesterone area undre the curve (days 2–6), and dayof peak progesterone. The follicular phase initiation of GnRHascan result in significant elevations of serum progesterone inthe early follicular phase, which may impair follicular recruitmentand overall ovarian response.     

Fractured zona oocytes in in-vitro fertilization cycles stimulated with gonadotrophin-releasing hormone analogue and human menopausal gonadotrophin

In order to assess the possible influence of gonadotrophinreleasinghormone analogue and human menopausal gonadotrophin on the occurrenceof fractured zona oocytes (FZOs) in in-vitro fertilization (IVF)treatment cycles, we analysed 267 consecutive cycles in 199patients. In 87 cycles, at least one fractured zona oocyte wasrecovered, and in 180 cycles only intact zona oocytes (IZOs)were recovered. FZOs represented 5.8% of all oocytes retrievedand 14.8% when only cycles with FZOs were considered. Serumoestradiol concentrations were significantly higher at day –3and day –2 (P < 0.02) in cycles yielding at least onefractured zona oocyte compared to IZO cycles (day 0 = retrievalday), and there was a higher incidence of G terminal patternof oestradiol curve (P < 0.01) in cycles with FZOs. The meannumbers of all oocytes retrieved and of mature oocytes weresignificantly higher in FZO than in IZO cycles (P < 0.001).The fertilization rate of mature oocytes was significantly reduced(P < 0.05) in cycles with one or more oocytes with fracturedzonae. There was no significant difference in the number ofembryos transferred, pregnancy and abortion rates in both groups.We conclude that although the occurrence of fractured zona oocytesis a frequent event, it does not affect the overall resultsof our IVF programme. Zona pellucida fragility may be the resultof over-maturation of some oocytes.     

Glucose and lactate in human follicular fluid: concentrations and interrelationships

The concentrations of glucose, lactate, progesterone and oestradiol have been measured in 122 follicular fluids obtained from women attending a natural cycle IVF programme. Some women received low-dose clomiphene. Fluids were recovered 28-35 h after the onset of the spontaneous LH surge. Mean follicular fluid volumes were greater for stimulated (4.46 ml) than spontaneous (3.11 ml) cycles. Mean glucose and lactate concentrations were similar in the two groups, with overall means of 3.29 mM (glucose) and 6.12 mM (lactate). Total glucose and lactate were greater in the fluids from stimulated (14.1 and 29.5 mol, respectively) than from the spontaneous cycles (11.5 and 20.2 mmol, respectively). There were negative correlations between follicular fluid volume and glucose concentration (r = -0.348) and between glucose concentration and lactate concentration (r = -0.367) but no relationship between follicular fluid volume and lactate concentration (r = 0.086). A model is presented to account for these findings in terms of the movement of pyruvate, glucose and lactate from the vascular theca into follicular fluid, and of glycolysis occurring in the granulosa cells.     

The use of human follicular fluid in gamete intra-fallopian transfer

We undertook a prospective study to compare our gamete intra-Fallopian transfer (GIFT) procedure with or without the use of human follicular fluid (FF) as a constituent for the final spermatozoal suspension and as the tubal transfer medium for both eggs and spermatozoa. We routinely perform an intrauterine and intracervical insemination (IUI and ICI) following GIFT, and FF or culture medium was used accordingly as a constituent in this spermatozoal suspension also. When FF was used (26 cycles), clear FF taken from the first egg-bearing follicle was sterilized by micropore filtration, gassed with 5% CO2 in air and warmed to 37 degrees C. This FF was then used to dilute the spermatozoal suspension (50:50, v/v) for both tubal, uterine and cervical inseminations at least 30 min before transfer, and all transferable eggs were placed into this FF before transfer. Alternatively (30 control cycles), eggs and spermatozoa were prepared and transferred in Earle's medium supplemented with 10% pooled fetal cord serum. The FF and control patient groups were relatively homogeneous, with no statistically significant differences in ovarian response, oocyte retrieval or transfer or seminal profiles. The outcome of the GIFT procedures using FF or culture medium showed no significant advantage of the use of FF. The clinical pregnancy rate was similar in both groups: 50% (15/30) control; 46.2% (12/26) FF.     

Ovarian response during IVF-embryo transfer cycles after laparoscopic ovarian cystectomy for endometriotic cysts of >3 cm in diameter.

BACKGROUND: Ovarian response during IVF cycles after laparoscopic ovarian cystectomy for endometriotic cysts >3 cm is controversial. A retrospective study was designed to study this problem. METHODS: At laparoscopy, endometriomas >3 cm were treated by ovarian cystectomy, whilst adhesions and peritoneal endometriosis were treated using conventional techniques. Ovarian stimulation was achieved with clomiphene and gonadotrophins or with gonadotrophins after a desensitization with gonadotrophin-releasing hormone agonists. Three groups of patients were retrospectively selected from an IVF-embryo transfer database: patients who underwent laparoscopic ovarian cystectomy for an endometrioma >3 cm (Group A, n = 41), patients with endometriosis without ovarian endometrioma (Group B, n = 139) and patients with tubal infertility (Group C, n = 59). RESULTS: The groups did not differ in age. In the first IVF cycle, the mean (+/- SD) numbers of oocytes and of embryos were 9.4 +/- 6.2 and 4.7 +/- 3.6 respectively in group A and 11.6 +/- 7.5 and 5.1 +/- 4.9 in group B (not significant). The results did not differ in cycles 2 and 3 or when compared according to age. No difference was found when comparing patients with endometriosis and patients with tubal infertility. CONCLUSION: The number of oocytes and embryos obtained was not significantly decreased by laparoscopic cystectomy, suggesting that in experienced hands this procedure may be a valuable surgical tool for the treatment of large ovarian endometriomas. However, great care must be taken to avoid ovarian damage.     

Extremes of body mass do not adversely affect the outcome of superovulation and in-vitro fertilization

The effect of extremes of body mass on ovulation is well recognized by clinicians. However, the effect of obesity and extreme underweight on the outcome of in-vitro fertilization (IVF) cycles has received relatively little attention. In a retrospective nested case-control study we examined the effect of the extremes of body mass index (BMI) on IVF-embryo transfer outcome at a university-based IVF unit. A total of 333 patients were included in the study; 76 obese patients (BMI > 27.9) with 152 controls, and 35 underweight patients (BMI < 19) with 70 controls. The patients were matched with their controls in age +/- 1 year, day 3 follicle stimulating hormone (FSH) concentration, daily dose of gonadotrophin (+/- 37.25 IU), gonadotrophin preparation and the year of treatment. The following parameters were compared between the study and control groups: duration of administration and dose of gonadotrophin, number of follicles aspirated, number of eggs, fertilization rate, number of embryos, serum oestradiol concentration on human chorionic gonadotrophin (HCG) day (peak oestradiol), clinical pregnancy rate, implantation rate, miscarriage rate, and incidence of ovarian hyperstimulation syndrome. Apart from a significantly lower peak oestradiol concentration (P = 0.009) in the obese patients, they and the underweight patients were not significantly different from their normal controls. The extremes of body mass index do not adversely affect the outcome of IVF-embryo transfer treatment. However, the obese patients had lower peak oestradiol concentrations than their normal controls despite receiving similar gonadotrophin doses.     

Colony stimulating factor-1 concentrations in blood and follicular fluid during the human menstrual cycle and ovarian stimulation: possible role in the ovulatory process.

To evaluate a possible role for colony stimulating factor-1 (CSF-1) in human ovarian function, the peripheral blood CSF-1 concentration throughout the human menstrual cycle and during ovarian stimulation was monitored. Blood was sampled across the menstrual cycle (n = 10) and at specific times during ovarian stimulation. In addition, the CSF-1 concentrations in follicular fluid (FF) during the follicular phase and during the luteinizing hormone (LH) surge of natural cycles, as well as 35-37 h after human chorionic gonadotrophin (HCG) during ovarian stimulation, were determined. There was no significant variation in CSF-1 concentrations during the natural menstrual cycle (median 470, range 212-1364 pg/ml). CSF-1 concentrations in FF (n = 11) were about four-fold higher (P < 0. 0001) than those in plasma of the same patients. CSF-1 concentrations in these FF showed some stage dependent variability, with significantly higher values during the ovulatory phase (median of 2017 pg/ml, range 1131-2236 pg/ml), compared to mid-follicular phase (median 961 pg/ml, range 830-1340 pg/ml; P = 0.02). During ovarian stimulation (n = 20), the plasma concentrations were similar to a time prior to stimulation up to and including 35-37 h after HCG. On day 9 after HCG, the values (median 644, range 357-1352 pg/ml) were significantly higher compared to pre-stimulation (median 422, range 253-1598 pg/ml; P < 0.05) and 35-37 h after HCG (median 458, range 250-658 pg/ml; P < 0.01). FF concentrations (n = 27) of CSF-1 at oocyte retrieval (median 3116, range 1824-5883 pg/ml) were about seven-fold higher than blood concentrations (median 472, range 250-1055 pg/ml; P < 0.0001). These results suggest that the intra-ovarian CSF-1, possibly induced by LH/HCG, plays an important role during ovulation and luteinization.     

Different protein patterns derived from follicular fluid of mature and immature human follicles

The purpose of our study was to compare the protein patternsoriginating from fluids of mature and immature human folliclesin order to gain further insight into their biochemical composition.A total of 10 patients were stimulated for in-vitro fertilization(IVF) using different stimulation protocols. Follicular fluidswere aspirated trans-vaginally and analysed microscopicallyfor the presence of oocytes. Follicular fluids were stored at-18°C. Samples of 500 µl were processed for two-dimensionalgel electrophor-esis. Up to 60 proteins in various groups couldbe detected. Seven protein spots were selected for chemicalanalysis by cutting them out of the gels and subjecting themto internal amino acid sequencing procedures. Our results canbe summarized as follows: (i) major differences were not detectedbetween the protein patterns from the various mature folliclesof a particular patient, nor were significant differences observedin the proteins derived from follicular fluids collected fromthe seven patients with mature follicles; (ii) considerabledifferences were observed in the protein patterns derived fromfluids of immature compared with mature follicles. Fluid fromthe three patients with immature follicles contained many fewerproteins, some of which were expressed at low levels. We concludethat the observed variations in protein composition of folliclesof different developmental age reflect their physiological conditionand serve as biomedical markers for follicular maturity.     

Increasing progesterone secretion in human granulosa-luteal cells induced by human follicular fluid

Increasing evidence suggests that local ovarian agents playa central role in the regulation of follicular maturation andcorpus luteum formation. In previous studies, we have shownthat porcine follicular fluid induces granulosa cell luteinizationin sow, human and rat. In the present study, the effect wasinvestigated of either human follicular fluid (FF) alone, humanfollicle-stimulating hormone (FSH) alone, or both upon progesteronesecretion of human granulosa-luteal cells. Granulosa-lutealcells were cultured in the presence of either FSH (5, 50 and250 ng/ml), lyophilized FF (50 and 250 µg/ml) or both.Secretion of progesterone increased from a minimum of 2.5-foldto a maximum of 23-fold in the presence of FSH alone and, significantlyless (2-fold) in the presence of FF alone, compared to cellscultured in medium alone. The co-administration of FSH and FFwas significantly more effective than either alone, while additionof both FSH (250 ng/ml) and FF (250 µg/ml) gave maximalprogesterone secretion. In granulosa-luteal cells pre-culturedwith both FSH and FF, subsequent exposure to human chorionicgonadotrophin (HCG) alone increased progesterone secretion 1.6-foldto 11-fold, compared to cells pre-cultured with FSH alone. Theeffect of FF from individual follicles was also studied. FFfrom follicles yielding mature cumulus—oocyte complexeswas 4.2-fold more effective, than FF obtained from folliclesyielding immature cumulus—oocyte complexes in enhancingthe FSH stimulation of progesterone secretion. A pre-cultureof granulosa-luteal cells in FSH plus FF from mature follicleswas 1.8-fold more effective in enhancing HCG-induced progesteronesecretion, compared to FF from immature follicles. This studysuggests that progesterone secretion by granulosa-luteal cellsis regulated by gonadotrophins and other factors accumulatingin FF.