Ovary: Follistatin concentrations in follicular fluid of normal and polycystic ovaries

Follistatin (FS) is an activin/inhibin binding protein whichis believed to act in an autocrine/paracrine manner to regulategrowth and differentiation. Although FS has been identifiedin human follicular fluid, it remains unclear how its concentrationchanges during selection and atresia, and what the concentrationsof FS are in follicles of women with polycystic ovary syndrome(PCOS). Towards this goal, we have measured by radioimmunoassaythe concentrations of FS in follicular fluid obtained from dominantand atretic cohort follicles of normal cycling women, preovulatoryfollicles of in-vitro fertilization (IVF) patients, and smallGraafian follicles of patients with PCOS. In all cases, thefollicular fluid concentration of FS was much higher (100-fold)than that reported in serum. The FS concentrations (ng/ml) were203 ± 42 (normal dominant), 185 ± 17 (atreticcohort), 185 ± 5 (IVF), and 250 ± 14 (PCOS). Therewas no statistical difference between these mean values of FS.Further, there were no significant correlations between thefollicular fluid concentrations of FS and the concentrationsof oestradiol, progesterone, or androstenedione. These resultsindicate that human Graafian follicles, regardless of whetherthey are healthy or atretic, normal or PCOS, contain high steady-stateconcentrations of FS in the micro-environment. Collectively,these data fit with the hypothesis that major increases anddecreases in the concentration of FS in the micro-environmentmay not play a key role in the mechanisms of selection, atresia,and PCOS in women. The possibility of regulation of intrinsicactivin and inhibin activity through FS binding is discussed.     

Endocrinology: Vasoactive intestinal polypeptide and peptide histidine methionine. Presence in human follicular fluid and effects on DNA synthesis and steroid secretion in cultured human granulosa/lutein cells

Vasoactive intestinal polypeptide (VIP) and peptide histidinemethionine (PHM) originate from the same precursor molecule,preproVIP. In the present study we examined the concentrationsof VIP and PHM in human follicular fluid and their effects oncultured human granulosa/lutein cells. Follicular fluid andcells were obtained from patients undergoing in-vitro fertilizationfor tubal infertility. The concentrations of VIP and PHM inpre-ovulatory human follicular fluid were measured radioimmunochemically.Granulosa/lutein cells isolated from follicular fluid were culturedunder serum-free conditions with VIP and PHM in varying concentrations(0.1, 10, 1000 nmol/l). [³H]Thymidine incorporation in the cellsand oestradiol as well as progesterone concentrations in theculture medium were measured. The mean (± SEM) concentrationsof VIP and PHM were 6.8 ± 0.1 and 7.7 ± 0.8 pmol/l,respectively. VIP at a concentration of 10 nmol/l caused a significantincrease in (3H) thymidine incorporation, and at 1000 nmol/1a significant increase in oestradiol secretion was observed.VIP had no effect on progesterone secretion. PHM at the concentrationstested did not influence any of the activities. We concludethat VIP and PHM are present in human preovulatory follicularfluid and that VIP stimulates DNA synthesis and oestradiol secretionin cultured human granulosa/lutein cells. This indicates thatVIP and perhaps PHM participate in the local nervous regulationof human ovarian function     

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.     

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.     

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.     

Endocrinology: Effect of follicular fluid on granulosa luteal cells from polycystic ovary

Recent data suggest that follicular fluid may play an importantrole in the endocrine balance of polycystic ovary syndrome,probably by acting on the theca-granulosa cell relationship.The aim of this study was to evaluate the effect of steroid-freefollicular fluid on steroidal response and cell proliferationof human granulosa luteal cells from polycystic (POGC) and normalovary (NC). Granulosa cells (from both POGC and NC) were culturedfor 48 h with or without increasing dilutions of follicularfluid (FF) obtained from polycystic (FFp) and normo-ovulating(FFc) patients. Both follicular fluids were able to elicit aromataseactivity as well as progesterone production and thymidine incorporation.POGC, when incubated with FFp, showed a lower increase of aromataseactivity and progesterone production with respect to NC. Furthermore,the proliferation rate was increased by incubation with eitherfollicular fluid, but the increase was less with FFp comparedto FFc Aromatase/[³H]thymidine (A/T) and progesterone/[³H]thymidine(P/T) ratios could be considered to be representative of thecontribution of the single cell unit to steroidogenesis. Usinghigh concentrations of either follicular fluids, POGC showeda higher A/T ratio compared with NC. Moreover, the same treatmentstrongly decreased P/T ratio in POGC, while it was ineffectivein NC. Our study shows that an abnormal interaction betweenPOGC and their own follicular fluid could be implicated in thepathogenesis of the altered steroidal response in these cells,and that in particular it could affect the proliferation rate.     

Physiology: Human follicular fluid maturity and endothelial cell mitogenesis

Follicular fluid, of varying maturity, (day 5–16 of cycle)was collected from the largest Graafian follicle of each of22 ovulatory patients during laparoscopic procedures. Threesamples were blood-stained and discarded. The mitogenic potentialof each sample was determined using bovine aortic endothelialcells in the CellTiter 96^TM Non-Radioactive Cell Proliferation/CytotoxicityAssay system. Intra- and inter-plate coefficients of variationwere <9%. The follicular fluid samples induced cell doublingtimes which varied from 12–24 h and final cell numberswhich, in the individual wells, ranged from 782–30 900(starting number 2000/well). Follicular fluid total proteincontent was unrelated to the mitogenic potential, (R² = 0%).Serum oestradiol was negatively correlated with the mitogenicpotential (R² = 26%). No correlation was found with day of themenstrual cycle (R² = 4.3%), maximum follicular diameter (R²= 1.8%), or serum concentration of progesterone (R² = 0.7%),luteinizing hormone (LH) (R² = 1.5%) or follicle stimulatinghormone (R² = 0.1%). Five subjects were in ‘early’and six in ‘mid’-follicular phase, six were in ‘early’and two in ‘late’ LH surge. There was no differencein the mitogenic response between these four groups by one-wayanalysis of variance (F = 0.21; P = 0.89). It is concluded thatthe mitogenic potential of human follicular fluid is not relatedto Graafian follicle maturity or, more particularly, to theLH surge.     

Dimeric inhibins and activin A in human follicular fluid and oocyte-cumulus culture medium.

The concentrations of inhibin A, inhibin B and activin A in follicular fluid and oocyte culture medium were analysed to investigate the production of these peptide hormones by ovarian granulosa cells and oocyte-cumulus complexes, as well as their potential as possible biochemical markers for oocyte quality and fertilizing capacity. Follicular fluids were collected from individual follicles during oocyte retrieval for in-vitro fertilization (IVF). Oocyte-cumulus culture media were collected after in-vitro insemination. The concentrations of dimeric inhibin A, inhibin B and activin A were measured using two-site enzyme-linked immunosorbent assays in the follicular fluid and matched oocyte culture medium. Hormone concentrations were compared with oocyte quality and fertilizing capacity. The concentration of inhibin A in follicular fluid increased while that of inhibin B decreased with increasing follicle size. Follicular fluid concentrations of inhibin A inhibin B and activin A were not significantly different in follicles with differing oocyte quality. Oocyte culture medium concentrations of activin A were significantly higher in morphologically good quality oocytes. There was no relationship between the concentrations of the three hormones and oocyte fertilizing capacity. This study confirms that follicular fluid concentrations of inhibin A may prove to be a marker of follicular growth and maturation. Higher concentrations of activin A produced by good quality oocyte-cumulus complexes suggest that activin A may play a role in oocyte maturation.     

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.     

Endocrinology: Growth hormone, oestradiol, progesterone and testosterone concentrations in follicular fluid after ovarian stimulation with various regimes for assisted reproduction

Follicular fluid samples and oocytes were obtained from 75 women(87 cycles), who participated in an assisted conception programme.Determinations of the concentration of oestradiol, progesterone,testosterone and growth hormone were performed in all follicularfluid samples. Patients were stimulated with the following regimes:group A (24 cycles, 94 samples), human menopausal gonadotrophin(HMG) (three ampoules/day) and human chorionic gonadotrophin(HCG); group B (23 cycles, 53 samples), HMG/HCG with prednisolone(7.5 mg/day) after cycle programming with oral contraceptives;group C (40 cycles, 60 samples), buserelin with HMG/HCG. Oestradiolconcentrations (mean ± SEM) were significantly higher(P < 0.05) in group A (320.1 ± 27.3 ng/ ml) and thoseof growth hormone in both groups A and C (3.8 ± 0.2 and3.2 ± 0.15 ng/ml, respectively), as compared to the othergroups, whereas progesterone and testosterone concentrationswere similar in all groups. The mean concentrations of oestradiol,progesterone, testosterone and growth hormone were significantlyhigher (P < 0.01) in follicular fluid with oocytes of intermediatematurity than with mature oocytes (382.5 ng/ml, 7847.5 ng/ml,1704.5 ng/dl and 3.7 ng/ml versus 217.8 ng/ml, 5488.4 ng/ml,1313.6 ng/dl and 2.7 ng/ml, respectively). On the other hand,only oestradiol concentrations were significantly higher infollicular fluid of fertilized compared to non-fertilized oocytes.Concentrations of the other hormones analysed, except growthhormone, were similar in follicular fluid from pregnant andnon-pregnant women after assisted reproduction. Growth hormone,on the other hand, was significantly lower (P < 0.05) infollicular fluid from pregnant compared to non-pregnant women(2.8 versus 3.5 ng/ml). It is concluded that intermediate maturityoocytes and oocytes which will be subsequently fertilized arefound in follicles with higher follicular fluid concentrationsof growth hormone and steroids. Moreover, oocytes leading topregnancy after in-vitro fertilization and embryo transfer arederived from follicles with lower growth hormone concentrationsin follicular fluid.     

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.     

Antioxidant enzymatic defences in human follicular fluid: characterization and age-dependent changes

The aim of this work was to study the antioxidant enzymatic defences in human follicular fluid and investigate their possible changes during reproductive ageing. To this end, we tested the specific activities and protein expression of enzymes involved in reactive oxygen species (ROS) scavenging and in detoxification of ROS byproducts in follicular fluid from young (range 27-32 years, n = 12) and older (range 39-45 years, n = 12) women participating in an IVF programme. Results show that all the tested enzymes [superoxide dismutase (SOD), catalase, glutathione peroxidase, glutathione transferase, glutathione reductase] were significantly expressed in human follicular fluid. However, when the two age groups were compared, we found that follicular fluid from older women exhibited a reduced level of glutathione transferase and catalase activities and a higher level of SOD activity. Immunoblot analysis revealed that ageing was associated with decreased protein expression of GST Pi isoform and did not affect SOD and catalase protein expression. Taken together, these findings indicate that reproductive ageing is accompanied by a change in the antioxidant enzymatic pattern that could impair ROS scavenging efficiency in the follicular environment.     

Concentrations of free oestradiol and progesterone in human preovulatory follicular fluid

This study describes the distribution, based on computer calculations, of the total concentration of oestradiol (E2) and progesterone (P4) between a free and a protein-bound fraction in each of 98 preovulatory follicular fluids (FF). The FFs were obtained from 30 women undergoing in-vitro fertilization--embryo transfer (IVF-ET) treatment. The concentrations of free and total steroid were correlated to oocyte cleavage and establishment of pregnancies. In the FF, 4.3% of E2 was free, 1.5% was bound to sex-hormone-binding-globulin (SHBG), 94.2% to albumin and less than 0.1% was bound to cortisol-binding-protein (CBP). The distribution of P4 in FF was 4.1% free, 5.6% bound to CBP, 90.3% bound to albumin and less than 0.1% was bound to SHBG. These results demonstrate that albumin plays a central role in maintaining the concentration gradient of steroids between the preovulatory FF and the circulation. The concentration of free E2 in fluid from follicles in which the oocyte cleaved was significantly lower in patients who achieved pregnancy (133 +/- 9 nM) (+/- SEM) than in fluid from follicles in which the oocyte cleaved but where the patient did not become pregnant (169 +/- 13 nM: P less than 0.05). Comparing the same two groups, the total concentration of E2 was also significantly lower in FF from patients who became pregnant. By contrast, no such correlation was found for either the free or the total concentrations of P4 in FF.(ABSTRACT TRUNCATED AT 250 WORDS)     

Cortisol and cortisone in human follicular fluid and serum and the outcome of IVF treatment

BACKGROUND: The glucocorticoid status of ovarian follicular fluid has been linked to oocyte quality. The aim of this study was to examine whether the concentrations of cortisone and cortisol and their calculated ratios in the follicular fluid and serum samples are predictive of IVF outcome. METHODS: In the prospective study of 387 patients (420 treatment cycles) undergoing IVF treatment the concentrations of cortisone and cortisol were measured with specific assays, and their calculated ratios in the follicular fluid and serum samples obtained after ovarian stimulation and induced ovulation were determined. RESULTS: In 75 patients, treatment resulted in clinical pregnancy and was associated with significantly lower follicular cortisone (24+/-12 versus 29+/-16 nmol/l, P<0.002) and higher cortisol/cortisone ratio (7.24+/-2.22 versus 6.45+/-2.17 nmol/l, P<0.007). In addition, the ratios of serum cortisone and cortisol to follicular cortisone and cortisol were significantly higher in those women who became pregnant. CONCLUSIONS: We propose that the follicular fluid glucocorticoid concentration resulting from the conditions in the circulation and the course of the intrafollicular cortisol-cortisone interconversion appear to play a role in the outcome of IVF.     

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.     

Aromatase activity of human granulosa cells in vitro: effects of gonadotrophins and follicular fluid.

The aim of this study was to assess whether human dominant follicular fluid has the ability to modulate aromatase activity and/or granulosa cell proliferation. Dominant follicular fluid was obtained by laparoscopy before the luteinizing hormone surge in naturally cycling women while granulosa cells used in the tests were obtained from in-vitro fertilization patients. Aromatase was measured by the tritiated water release assay, following a 48 h incubation with follicular fluid and serum, and expressed for 5x10(4) granulosa cells. The effects of a range of follicular fluid or serum concentrations (2.5, 5, 10 and 20%) were compared. A decrease in aromatase activity was observed when high follicular fluid concentrations (20%) (P < 0.01) were added. Low concentrations (2.5%) of follicular fluid significantly increased cell proliferation (P < 0.01) as compared to basal values (0%). No further stimulation was however observed when concentrations increased up to 20%. Further characterization of these compounds is required to understand how they may modulate maturation of the dominant follicle.     

Melatonin and steroids in human pre-ovulatory follicular fluid: seasonal variations and granulosa cell steroid production

Follicular fluid samples were obtained from the largest pre-ovulatoryfollicle of 120 women undergoing in-vitro fertilization andwere examined for melatonin by enzyme-linked immunosorbent assayand the steroids oestradiol and progesterone by radioimmunoassay.The concentrations (mean ± SE) of melatonin (213.4 ±18.9 pmol/1) and progesterone (20.1 ± 1.1 µmol/l)in follicular fluid during the autumn and winter (dark) monthswere significantly higher than during the spring and summer(light) months, melatonin (138.4 ± 12.5 pmol/1) and progesterone(11.6 ± 0.8 µmol/l). By contrast, oestradiol concentrationswere significantly lower during the dark months than duringthe light months (264.7 ± 44.1 and 661.8 ± 55.1nmol/l respectively). There was a positive correlation betweenfollicular fluid melatonin and progesterone concentrations (r= 0.271, P < 0.05, n = 120) and a negative relationship betweenmelatonin and oestradiol (r = –0.254, P < 0.05, n =120). The effects of melatonin alone and in combination withhuman chorionic gonadotrophin (HCG) or follicle stimulatinghormone (FSH) on steroidogenesis by human granulosa cell culturewere also investigated. Melatonin had minimal effects on oestradiolor progesterone production by granulosa cells. Interestingly,the oestradiol response in culture appeared to be differentaccording to the time of the year when harvested. During thelight period oestradiol production was enhanced. Melatonin alsosynergized with HCG in increasing progesterone production ondays 6 and 7 after treatment during both light and dark periods.FSH stimulated oestradiol production by the cells on day 2 ofculture. Melatonin had no effect on FSH stimulation of oestradiolproduction. The results of this study suggest that melatoninmay be involved in the regulation of steroidogenesis by thehuman ovaries.     

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.     

Ovary and ovulation: Flow cytometric analysis of granulosa cells from follicular fluid after follicular stimulation

Granulosa cells are not easily accessible, unless they are examinedin follicular fluid after oocyte retrieval. These samples areusually contaminated with blood. We have set up a general techniquefor analysis of granulosa cells without physically separatingthem from blood cells. The sample is stained with CD45, whichis a pan-leukocyte marker, and granulosa cells are consecutivelyselected as CD45 negative during flow cytometric analysis. Analysisof forward scatter of the granulosa cells, which is correlatedto cell size, shows a wide size range throughout the whole populationrather than two distinct populations as previously suggested.     

Preliminary characterization of a factor in human follicular fluid that stimulates human spermatozoa motion

Follicular fluid alters the physiology and behaviour of spermatozoaby increasing acrosome reaction, accelerating capacitation,attracting the spermatozoon and enhancing vigorous motion ofthe cell. The objective of this study was to characterize thefactor(s) in human follicular fluid that causes vigorous spermatozoamotion. Follicular fluid and its fractions were tested for stimulationof spermatozoa motion using a standardized assay which employsa computerized digital imaging system. Our results show thatboth follicular fluid and its methanol extract stimulatevigorousspermatozoa motion. To determine the characteristics of theactive factor(s), the methanol extract was subjected to molecularweight fractionation, protease digestion, microcrystalline thinlayerchromatography (TLC) and C18 reverse-phase high-pressure liquidchromatography (HPLC). The spermatozoa motion stimulator inthe methanol extract was dialysable against a low molecularweight membrane (1000 Da), insensitive to boiling and low pH(3.5) and was largely inactivated by proteinase K digestion.The activity was detected near the solvent front on TLC. Usingreverse-phase HPLC monitored at 254 nm (UV), the activity elutedas a single peak of activity at low methanol concentration,indicating that the activity was relatively hydrophilic. Theactivity in the HPLC peak lost most of its motion-stimulatingability after digestion with proteinase K. The motion stimulatorcould be a peptide analogous to the egg-associated peptidescharacterized in echinoderms which stimulate spermatozoa motion,respiration and chemotaxis.