Effect of prednisolone on serum and follicular fluid androgen concentrations in women with polycystic ovary syndrome undergoing in-vitro fertilization.

Increased androgen concentrations are thought to be detrimental to oocyte quality and reproductive potential. Adjuvant treatment with glucocorticoids has been tried to suppress androgens in women undergoing infertility treatment. In the present study 20 infertile women with polycystic ovary syndrome were prospectively randomized in a placebo-controlled study to receive either placebo or prednisolone 10 mg at night, during standard in-vitro fertilization (IVF) treatment. Serum samples for assays of gonadotrophins, steroids and sex hormone-binding globulin (SHBG) were collected before treatment, at down-regulation, and at oocyte retrieval. Up to five follicles in each ovary were analysed separately regarding follicular fluid and oocytes, the rest according to the clinic's routines. In the placebo group, serum dehydroepiandrosterone (DHEA) and dehydroepiandrosterone-sulphate (DHEA-S) did not change between down-regulation and oocyte retrieval, whereas adjuvant prednisolone resulted in a significant decrease. In follicular fluid, adjuvant prednisolone resulted in significantly lower concentrations of DHEA-S as compared to placebo, no other significant differences were found. No significant differences were found in embryo characteristics or pregnancy rates between the groups.     

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.     

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)     

Do analogues of gonadotrophin releasing hormone influence follicular fluid steroid levels, oocyte maturity and fertilization rates?

One-hundred-and-twelve samples of follicular fluid from 32 patients undergoing in-vitro fertilization and embryo transfer were analysed in this study. The follicular fluids were analysed for any relationships between oestradiol, progesterone and 17 alpha-hydroxyprogesterone levels, the progesterone/oestradiol and 17 alpha-hydroxyprogesterone/oestradiol ratios and oocyte maturity and fertilization rates. In Group A, consisting of women who used analogues of gonadotrophin-releasing hormone during ovarian stimulation with human menopausal gonadotrophin, the progesterone/oestradiol ratio rose in parallel with the fertilization rate (P less than 0.05). Group B comprised patients treated with human menopausal gonadotrophin alone. No significant relationship was found between the other parameters, oocyte maturation and fertilization rates in either group.     

Andrology: Acrosome reaction inducing activity in follicular fluid correlates with progesterone concentration but not with oocyte maturity or fertilizability

Follicular fluid is a potent mediator of sperm acrosome reaction(AR) in vitro. The aim of this study was to investigate whetherindividual follicular fluids vary quantitatively in their abilityto stimulate an AR, and whether such variability relates tofertilizability of the corresponding egg, its maturational leveland/or progesterone content. Individual follicular fluids wereobtained from 24 women undergoing in-vitro fertilization andassayed for their ability to induce an AR in normal human spermatozoa.After incubation in capacitation medium for 18 h, spermatozoawere challenged with the individual follicular fluids for 30min. AR was detected by immunofluorescence, using fluorescein-labelledPisum sativum lectin. We found that individual follicular fluidsvaried markedly in their ability to induce AR. Acrosome reactioncorrelated linearly with progesterone concentration (Spearman'sr = 0.735, P = 0.01) at constant protein level, but no correlationwas found between AR and protein concentration at constant progesteronelevel. Progesterone concentrations were not only higher (ANOVA,P = 0.002) in fluids from mature oocytes compared to those fromless mature or post-mature eggs but also in fluids from fertilizedcompared to unfertilized eggs (ANOVA, P = 0.015, n = 13 patientswith both fertilized and unfertilized eggs). In contrast, AR-inducingability of individual follicular fluids did not differ for fertilizedand unfertilized eggs. While AR-inducing ability appeared toincrease with maturational stage of the egg, this trend wasnot statistically significant, probably due to small samplesize. Our data suggest that progesterone rather than proteinis the principal mediator of acrosome reaction induced by follicularfluid in vitro. Though progesterone concentration correlateswith both the ability of the fluid to induce an AR in normalspermatozoa and with fertilization of the corresponding oocyteby husband's spermatozoa, the lack of direct correlation betweenAR-inducing ability and fertilization implies that other aspectsof gamete function are also important in determining fertilizationsuccess.     

The role of follicular fluid in inducing the acrosome reaction of human spermatozoa incubated in vitro

Motile human sperm populations were prepared from liquefiedsemen (5 donors x 3 replicates) using Percoll gradients at 30–60 min post-ejaculation and preincubated in a complex ‘syntheitictubal fluid’ culture medium (STF) at 37° C under 5%CO₂ in air for 6h. Aliquots of these suspensions were then incubatedfor a further 2 h in STF containing 0, 5, 25, 50, 75 and 100%(v/v) pooled human follicular fluid (FF). Another aliquot wastreated with 10 µm A23187 in STF for 20min and then incubatedin fresh STF medium for a futher 2h to induce maximal acrosomeloss. Acrosome reactions were assessed using both the triple-staintechnique and fluorescent peanut agglutinin lectin-labelling.Sperm motility and movement characteristics were assessed fromvideorecordings using digital image analysis (CellSoft). Exposureto FF caused only relatively small proportions of the preincubatedspermatozoa to undergo acrosome reactions. The size of theseresponsive sub-populations was smaller than that capable ofresponding to a Ca²⁺ influx generated by A23187. Increased FFconcentrations induced a progressive loss of motility and trendsfor changes in movement characteristics that may have been relatedto reduced intracellular Ca²⁺. This interpretation of theseobervations is that while FF may act to stimulate or promotethe human sperm acrosome reaction it does not appear to be aspecific inducer of it. Consequently, a precise role for FFat the relatively low concentrations that would be expectedto be present in the tubal ampulla in the physiological regulationof human fertilization remains unproven     

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.     

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.     

Fertilization and early embryology: Human follicular fluid inhibits the binding of human spermatozoa to zona pellucidain vitro

The effect of human follicular fluid on human zona pellucidabinding of spermatozoa was investigated using the hemizona bindingassay (HZA). This effect was compared to that of progesterone,a known component of human follicular fluid. Exposure of spermatozoato 25% pooled human follicular fluid for 1 h significantly reducedthe number of spermatozoa bound to zona pellucida when comparedto those without human follicular fluid treatment (149.1 ±30.7 versus 177.1 ± 33.8, P 0.01). The same phenomenonwas observed after 3 h of treatment The corresponding numbersof bound spermatozoa were 140.4 ± 19.1 and 200.2 ±23.4 (P 0.0001). Progesterone (1.0µg/ml) stimulated thezona pellucida-binding capacity of spermatozoa significantlyunder the same conditions (P 0.01). The numbers of bound spermatozoaafter 1 and 3 h progesterone treatment were 235.5 ± 44.7(control, 168.1 ± 32.9) and 204.3 ± 27.4 (control,162.3 ± 20.1) respectively. HZA comparing the effectsof human follicular fluid and progesterone at concentrationsequivalent to those found in human follicular fluid using matchinghemizonae confirmed the inhibitory effect of human follicularfluid on sperm binding to zona pellucida (80.4 ± 28.4versus 149.8 ± 35.2, P 0.05). This inhibitory effectwas also found in another eight individual human follicularfluid samples. Both human follicular fluid and progesteronedid not affect the motility and viability of the treated spermatozoawhen compared to the controls with the same incubation period.Although more spermatozoa underwent the acrosome reaction after1 and 3 h of human follicular fluid treatment than in the control,the extent was comparable to those after progesterone treatmentThese results suggested that human follicular fluid inhibitedthe zona pellucida-binding capacity of spermatozoa in vitro.This inhibitory effect of human follicular fluid was not mediatedby progesterone, and did not result from the effects of humanfollicular fluid on sperm motility, viability and acrosome reaction.     

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.     

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: 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.     

Comparison of plasma and follicular fluid hormone profiles following stimulation with HMG, with or without LHRH agonists, for in-vitro fertilization.

Plasma and follicular fluid (FF) hormone assays for follicle stimulating hormone (FSH), luteinizing hormone (LH), prolactin (PRL), oestradiol (E2), progesterone (P), delta-4-androstenedione (A4) and testosterone (T) were performed on the day of oocyte retrieval in two groups of normo-ovulatory women enrolled in an in-vitro fertilization (IVF) programme: 24 were treated using the decapeptyl agonists DTRP6, of luteinizing hormone-releasing hormone (LHRH) in the long protocol associated with human menopausal gonadotrophin (HMG) (49 FF) and 14 were stimulated with HMG alone (33 FF). In both FF and plasma the mean concentration of P was greater, and the E2/P ratios as well as the LH levels were lower in the agonist-treated group. In this group the follicular concentration of P was greater and the E2/P ratio lower when pregnancy occurred following IVF. The hormonal modifications may be due to greater functional maturity of the granulosa cells.     

Concentrations of carotenoids,retinol and alpha-tocopherol in plasma and follicular fluid of women undergoing IVF

BACKGROUND: Carotenoids are transferred into follicular fluid where they might serve as antioxidants and/or as precursors of retinoids which might modulate follicular or oocyte functions. METHODS AND RESULTS: In 77 women undergoing IVF differences between plasma and follicular fluid in the levels of carotenoids, retinol and alpha-tocopherol were evaluated especially with regard to fertilization success. Concentration of total carotenoids, retinol and alpha-tocopherol determined by HPLC in follicular fluid and plasma were 0.06 +/- 0.02 versus 0.56 +/- 0.23 micro mol/l, 1.26 +/- 0.52 versus 1.66 +/- 0.37 micro mol/l and 4.89 +/- 2.13 versus 21.0 +/- 5.7 micro mol/l (mean +/- SD) respectively (P < 0.001 for all). Differences between plasma and follicular fluid were greater for beta-carotene and lycopene (<20% of plasma concentration) than for lutein and zeaxanthin (>40%). Intrafollicular retinol and retinol-binding protein (RBP) levels represented 58 +/- 23% and 60 +/- 19% of plasma level. Similar molar ratios of retinol/RBP were observed. While no differences in the mean values of all components investigated were observed in plasma and follicular fluid between women with and without reproductive success, the variability in the concentration was much greater in follicular fluid obtained from women without reproductive success. CONCLUSIONS: It remains to be elucidated, if this is indicative of a disturbed sieving effect of the blood-follicle barrier with possibly negative consequences for oocyte maturation.     

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.     

An unexpected guest in follicular fluid

Parasitic infection as the only or concomitant cause of infertilityin Caucasian women is rare. A parasitic infection may also presentitself quite unexpectedly as a coincidental finding as shownwith this case report Moving microfilariae of Mansonella perstanswere found in the aspirated follic-ular fluid of a patient whounderwent in-vitro fertilization (TVF) with embryo transferbecause of tubal pathology due to Chlamydia trachomatis. Thepatient also appeared to have a Schistosoma infection. To ourknowledge, the presence of parasites in follicular fluid hasnever been reported before. We expect that infertility physiciansmay be confronted with parasitic infections more often, notonly in patients originating from tropical countries but alsoin Western women as a result of a tendency to travel more frequentlyto exotic and (sub)tropical countries.     

Reproductive implication of D-aspartic acid in human pre-ovulatory follicular fluid

BACKGROUND: In the present study, we report that D-aspartic acid (D-Asp) occurs in human ovarian follicular fluid and that a relationship may exist between the concentration of this amino acid and oocyte quality. METHODS: Samples of pre-ovulatory follicle fluid were obtained from 20 patients undergoing an IVF programme. The concentration of D-Asp was measured by using specific high-performance liquid chromatography (HPLC) combined with a d-aspartate oxidase. RESULTS: D-Asp occurs in human follicular fluid at a mean concentration of 14.98 +/- 4.51 nmol/ml. A significant difference in the content of this amino acid in the follicular fluid in relation to patient's age exists. In younger women aged 22-34 years (group A), D-Asp was found at a concentration of 19.11 +/- 1.91 nmol/ml, whereas in patients aged 35-40 years (group B), it decreased to 10.86 +/- 1.22 nmol/ml (P < 0.01). In addition, this amino acid was linked to oocyte quality; a relationship exists between D-Asp follicular concentration and the percentage of good quality metaphase II oocytes (P < 0.01), as well as the fertilization rate. CONCLUSIONS: In human follicular fluid, D-Asp is present at a relatively higher concentration in younger women than in older patients and there appears to be a relationship between the concentration of d-Asp and fertility outcome parameters. These findings suggest that follicular D-Asp concentration may be considered as an alternative or additional biochemical marker for oocyte quality in patients undergoing IVF programmes.     

Immunology: Assessment of the relevance of zona pellucida antibodies in follicular fluid of in-vitro fertilization (IVF) patients

The presence of anti-zona pellucida antibodies in the follicularfluid of 11 women who underwent in-vitro fertilization (IVF)and embryo transfer was analysed. Only infertile couples withtubal or unexplained pathologies were included in our study,which was aimed at investigating the relationship between anti-zonapellucida antibodies in follicular fluid and failed fertilization.Whether or not these antibodies were present in some or allfollicles in the same patient was also investigated. Out of55 follicular fluids analysed, 36.3% were positive to the testand no fertilization was observed in oocytes from these follicles,while 63.6% were negative, and the oocyte fertilization rateassociated with these was 51.4%. The presence of anti-zona pellucidaantibodies was positively correlated with the degree of fertilizationfailure (P <0.001 x² test).     

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.