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.     

Follistatin and activin A serum concentrations in obese and non-obese patients with polycystic ovary syndrome.

BACKGROUND: Activin promotes ovarian follicular development, inhibits androgen production and increases FSH and insulin secretion. Follistatin, an activin-binding protein, neutralizes activin bioactivity. Therefore, a decrease in the ratio of activin/follistatin might encourage characteristic features of polycystic ovary syndrome (PCOS). We investigated whether women with PCOS showed disordered follistatin and/or activin serum concentrations. METHODS: The study group included 24 obese and 20 non-obese (body mass index vertical line and <27 kg/m2 respectively) clomiphene-failure PCOS patients. The control group included 16 obese and 46 non-obese patients with normal ovulatory cycles. Blood samples were obtained from the patients on day 3-5 of a progesterone-induced or spontaneous cycle and were assayed for LH, FSH, testosterone, 17-hydroxy-progesterone, androstenedione, follistatin, activin A, fasting glucose and insulin. RESULTS: Follistatin concentrations were comparable between obese and non-obese PCOS patients (mean +/- SE; 1171 +/- 103 and 1045 +/- 159 pg/ml respectively) and significantly higher than their respective controls (628 +/- 61 and 592 +/- 49 pg/ml, P < 0.0001 and P < 0.02 respectively). Activin A concentrations were comparable between the four groups (590 +/- 35, 513 +/- 74, 661 +/- 87 and 595 +/- 43 pg/ml in obese and non-obese PCOS and controls respectively). Stepwise regression analyses for relationships between follistatin or activin A levels and all other variables indicated that follistatin was significantly and independently positively affected by PCOS (P < 0.0001), age (P < 0.02), androstenedione (P < 0.03) and weight (P < 0.05). Activin A was significantly and independently negatively affected by PCOS (P < 0.003) and FSH (P < 0.03), and positively affected by weight (P < 0.009) and androstenedione (P < 0.02). CONCLUSIONS: Serum follistatin is increased in PCOS patients, regardless of obesity. PCOS is the most significant variable that relates to high follistatin and low activin A serum concentration. A high follistatin/activin ratio could well contribute to the pathophysiology of PCOS.     

Follistatin and activin A in extra-embryonic coelomic and amniotic fluids and maternal serum in early pregnancy

Follistatin is a specific binding protein which controls bioavailability of activins and inhibins which have an important role in fetal development. In the first trimester of pregnancy bioactive dimeric inhibins are found at high concentrations in the extra- embryonic coelomic fluid, but the distribution of follistatin and activins is not known. We have used recently developed immunoassays for follistatin, activin A and activin AB to determine their presence in the intrauterine compartments during early pregnancy. Follistatin was present in highest concentrations in the extra-embryonic coelomic fluid (11.72 +/- 1.70 ng/ml; median +/- SEM), with less in maternal serum (6.35 +/- 4.58) and lowest amounts in amniotic fluid (0.97 +/- 0.52). Follistatin concentrations in extra-embryonic coelomic fluid were highly correlated with both dimeric inhibin isoforms. Activin A was present in only barely detectable amounts in some samples of extra- embryonic coelomic fluid (41% of samples) and maternal serum (26%) and was undetectable in all amniotic fluid samples. Activin AB was undetectable in all compartments. The presence of follistatin in the amniotic and extra-embryonic coelomic fluids may regulate the availability of bioactive activins and inhibins which are released into the intrauterine compartments during the development of the fetus and placenta in early pregnancy.      

Identification of Activin A and Follistatin in Human Milk

Activin A is a dimeric protein member of the transforming growth factor- &#103 (TGF- &#103 ) family: it is synthesized by a variety of organs and follistatin is an activin-binding protein. A sensitive and specific assays for bioactive dimeric activin A and follistatin have recently allowed to measure these proteins in blood and other biological fluids, giving a new insights into their possible physiological role. Since human breast is able to produce activin A, the aim of the present study was to evaluate whether it and follistatin are measurable in breast milk of women during lactation. Concentrations of activin A and follistatin were measured in milk samples collected at 3, 5 and 30 days after delivery by using specific and sensitive two-site ELISAs. For the first time the presence of immunoreactive activin A and follistatin in human milk has been shown; no significant different concentration between the third and the fifth day after delivery was found. Furthermore, no difference of activin A and follistatin concentration between the whole and the skim milk or between spontaneous delivery and cesarean section was found. Milk activin A and follistatin concentrations after 1 month of lactation were significantly decreased (P <0.01). Activin A and follistatin are present in human milk in high concentrations in the first week of lactation, while decrease after a month suggesting a possible role as growth factors in human milk.     

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.     

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.     

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.     

Serum activin A and follistatin concentrations during human pregnancy: a cross-sectional and longitudinal study

Activin A, a dimer of the betaA-subunit of inhibin, has been shown to have multiple biological activities and sites of production. Follistatin is a high-affinity binding protein for activin, which neutralizes its activity. This study provides the first data, using a cross-sectional design, on the measurement of both these proteins in the maternal circulation of a large cohort of women (6-39 weeks of gestation, n = 2-20 women/time point) during normal pregnancies, and confirms that similar patterns are seen in nine women studied longitudinally during pregnancy. The concentrations of total activin A were measured using a specific two-site enzyme-linked immunosorbent assay (ELISA), and a new radioimmunoassay for measuring total follistatin in serum utilizing dissociating reagents to eliminate the interference of activin is described. At 38-39 weeks gestation, both activin A and follistatin concentrations rose to a peak (4.59 +/- 0.54 ng/ml and 72.7 +/- 3.31 ng/ml, respectively). The activin A and follistatin concentrations were highly correlated both in the cross-sectional study (P <0.0001) and in individual women in the longitudinal study (P <0.05-0.0001). Concentrations of follistatin showed a greater increase in the second trimester of pregnancy relative to activin A concentrations. The parallel increase in the secretion of these two proteins throughout pregnancy probably reflects feto-placental secretion.     

Identification of activin A and follistatin in human milk

Activin A is a dimeric protein member of the transforming growth factor-beta (TGF-beta) family: it is synthesized by a variety of organs and follistatin is an activin-binding protein. A sensitive and specific assays for bioactive dimeric activin A and follistatin have recently allowed to measure these proteins in blood and other biological fluids, giving a new insights into their possible physiological role. Since human breast is able to produce activin A, the aim of the present study was to evaluate whether it and follistatin are measurable in breast milk of women during lactation. Concentrations of activin A and follistatin were measured in milk samples collected at 3, 5 and 30 days after delivery by using specific and sensitive two-site ELISAs. For the first time the presence of immunoreactive activin A and follistatin in human milk has been shown; no significant different concentration between the third and the fifth day after delivery was found. Furthermore, no difference of activin A and follistatin concentration between the whole and the skim milk or between spontaneous delivery and cesarean section was found. Milk activin A and follistatin concentrations after 1 month of lactation were significantly decreased (P < 0.01). Activin A and follistatin are present in human milk in high concentrations in the first week of lactation, while decrease after a month suggesting a possible role as growth factors in human milk.     

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.     

Infertility: Ovarian synchrony factor: a new ultrasound parameter in the prognosis of follicular rupture

The present study was carried out to present a new ultra-soundparameter used in stimulated cycles and called the ovarian synchronyfactor(OSF), which reflects the response of the total follicularcohort. It is calculated from the formula: OSF = (total no.of follicles 16 mm)/(total no. of follicles 10mm). OSF wasdetermined on the day of human chorionic gonadotrophin (HCG)administration (indicated when at least one follicle was 16mm)by measuring the widest follicular diameters with an ATL ultrasoundapparatus model Ultramark 4, with a 5.0 MHz vaginal transducer,in a total of 221 cycles stimulated for non-invasive assistedreproduction techniques (i.e. no oocyte retrieval). A new ultrasoundexamination was performed 56–60 h after HCG administrationto determine the possible presence of follicular rupture indicatedby the disappearance of the follicular image and/or a >5mmdecrease in the widest follicular diameter. The mean OSF inthe group of patients with rupture of at least one follicle(195 cycles) was mean ±SD, 0.57 ±0.25, as opposedto a mean ±SD of 0.39 ± 0.25 for the group withoutfollicular rupture (26 cycles). The Mann–Whitney testshowed that the OSF of the group with follicular rupture wassignificantly higher than that detected in the group withoutfollicular rupture (P <0.01). This information suggests thatovarian stimulation protocols should produce a synchronous follicularresponse (an OSF as close as possible to 1), i.e. that the follicularlots developing in both ovaries should not vary widely in size.This follicular homogeneity should facilitate the follicularrupture process.     

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.     

Follistatin and activin A production by the male reproductive tract

Follistatin is a binding protein for the activin and inhibin family of hormones, regulating their biological activity. In the male reproductive tract, the interaction of these factors is likely to be involved in the regulation of the proliferation of several cell types. We have investigated the presence of follistatin and activin A in seminal plasma using specific immunoassays and have localized follistatin and activin/inhibin subunits in the adult human testis, prostate and seminal vesicle to establish their likely sources. High concentrations of immunoreactive follistatin were present in seminal plasma in normal men (mean 97.9 ng/ml; 1.43 ng/ml in peripheral plasma) and were similar in men with oligo/azoospermia and following vasectomy. Follistatin immunoreactivity was localized to both Leydig and Sertoli cells of the testis, and to epithelial cells of the prostate gland and seminal vesicle, which are likely to be the predominant sources of the hormone in seminal plasma. Activin A was also present in seminal plasma in normal men but was undetectable following vasectomy, thus deriving from the testis. Consistent with this finding, the betaA-subunit was immunolocalized in Sertoli and Leydig cells but was not present in seminal vesicle or prostate gland. The functional significance of the high concentrations of follistatin secreted into seminal plasma by the prostate gland and/or seminal vesicle is uncertain, but they may regulate the biological activity of testis-derived activin A and inhibin B.      

Expression of activin receptors, follistatin and betaglycan by human endometrial stromal cells; consistent with a role for activins during decidualization

Decidualization of the human endometrium is critical for implantation, but the mechanisms involved are largely unknown. Activin subunits are expressed in endometrium during decidualization. From its known actions in cell differentiation and tissue remodelling, we hypothesized that activin A is involved in the paracrine regulation of decidualization. We examined the expression of activin receptors (ActRs) by semi-quantitative and real-time RT-PCR. mRNA for all ActR subtypes (Ia, Ib, IIa and IIb) was detected in endometrium, with maximal expression in the early secretory phase and in early pregnancy. ActR protein was localized exclusively to stromal and endothelial cells. This expression pattern was confirmed by in-situ hybridization. Activin bioavailability is locally regulated by its binding protein, follistatin, and also by the antagonist, inhibin. Inhibin competition for ActRII binding is enhanced by the binding protein, betaglycan. Follistatin and betaglycan were also detected in the endometrium, localized to stromal and epithelial cells. This co-expression of activin subunits, receptors and binding proteins indicates that stromal cells are capable of responding to activin, and that there is tight local regulation of activin action within the endometrium. As activin production is up-regulated in decidual cells, this provides further evidence for an involvement of activins during stromal cell decidualization.     

Gonadotrophin surge-attenuating factor bioactivity is present in follicular fluid from naturally cycling women

Rat pituitary monolayer bioassays were used to compare gonadotrophinsurge-attenuating factor (GnSAF) bioactivity in follicular fluidfrom 12 follicles in 10 spontaneously cycling women with thatin pooled follicular fluid from women undergoing ovulation induction.Expressed as ED₅₀s (µl follicular fluid/well producing50% of maximal effect), GnSAF bioactivity was detectable inall spontaneous follicular fluid samples (1.4–33.3 µl/well)and in follicular fluid from women undergoing ovulation induction(6.8 µl/well). This GnSAF bioactivity was unaffected bypre-incubation with an inhibin antibody. When the data weregrouped according to whether the recovered oocytes fertilizedin vitro or not, the fertilized group contained significantlygreater GnSAF bioactivity than the unfertilized group (5.3 ±1.1 and 14.1 ± 2.6 µl/well respectively, P <0.05). While both inhibin bioactivity (9.7 ± 1.4 and28.9 ± 12.1 µl/well) and immunoreactivity (36.8± 2.2 and 21.0 ± 3.0 and ng/ml) were also greater(P < 0.01) in the fertilized compared with the unfertilizedgroups respectively, there were no other significant differencesbetween the two groups. We conclude that GnSAF is found in follicularfluid from spontaneously cycling women, supporting in-vivo evidencefor the involvement of GnSAF in feedback control of the ovary-pituitaryaxis.     

High levels of immunoreactive endothelin-1 in human follicular fluids

Follicular fluids were obtained from 180 follicles of 15 womenundergoing follicular aspiration for in-vitro fertilization.Follicular development was induced by a combination of buserelinacetate and human menopausal gonadotrophin. Endothelin-1 (ET-1)concentrations in human follicular fluids were determined byspecific radioimmunoassay. ET-1-like immunoreactivity (ET-1-LI)ranged from 338 to 928 pg/ml. ET-1-LI concentrations in follicularfluids obtained from immature (< 15 mm) follicles were significantlyhigher than those from mature (15–25 mm) and post-mature(25 mm) follicles. No correlation was found between the concentrationof ET-1-LI, on the one hand, and that of oestradiol, progesterone,testosterone, prolactin, luteinizing hormone, insulin-like growthfactor-I, prostaglandin E₂ or platelet activating factor onthe other, in follicular fluids. However, a significant positivecorrelation was observed between ET-1-LI concentration and folliclestimulating hormone and IGF-II concentrations, respectively.These data suggest that the high concentration of ET-1 foundin follicular fluids may play some physiological role in folliculardevelopment.     

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.     

Effect of peritoneal fluid supplemented with exogenous progesterone on sperm motility in vitro

Since progesterone has been claimed to induce acrosomal reactionand hyperactivated motility of human spermatozoa, the presentstudy was undertaken to determine if its presence at concentrationssimilar to those of peri-ovulatory follicular fluid could influencethe effect of peritoneal fluid on spermmotility in vitro. Tothis end, 11 sperm samples were incubated at 37°C with fiveperitoneal fluids with/without exogenous progesterone, and spermmotility was assessed using a computer-assisted analyser attime (t) = 0, 2.5, 5 and 24 h. Overall there was no observableconstant trend for enhancement or inhibition of sperm motility.Progesterone generally induced a negative effect on those spermsamples with high velocities in the native peritoneal fluidsand a positive effect on those sperm samples demonstrating lowmotility in the native peritoneal fluids. The incorporationof progesterone into the incubation medium seemed to resultin a ‘tuning’ of sperm velocity to around 30–50µm/s. However, a given sperm sample reacted differentlywhen incubated with various peritoneal fluids and, reciprocally,different semen samples incubated with the same peritoneal fluidshowed very variable motility patterns. The greater variabilityof the effects exerted by progesterone on sperm motility couldarise from the fact that each sperm sample may contain subpopulationsof gametes with different sensitivity to progesterone.     

Follistatin is a candidate endogenous negative regulator of activin A in experimental allergic asthma

BACKGROUND: Activin A is a member of the transforming growth factor-beta superfamily which is directly implicated in airway structural change and inflammation in asthma. In vitro, the biological effects of activin A are neutralized by the soluble binding protein follistatin. OBJECTIVE: To determine the potential of endogenous follistatin to suppress activin A in vivo by analysing their relative tissue and kinetic compartmentalization during the effector phase of subchronic Th2-driven mucosal inflammation in a murine model of allergic asthma. METHODS: Eosinophilic mucosal inflammation was elicited by triggering Th2 recall responses by antigen challenge in ovalbumin-sensitized BALB/c mice. The kinetics and distribution of activin A and follistatin protein were assessed in lung tissue and bronchoalveolar lavage fluid and measured in relation to airway eosinophilia, goblet cell metaplasia and Th2 cytokine production in mediastinal lymph nodes. RESULTS: Follistatin was released concurrently with activin A suggesting it acts as an endogenous regulator: peak BAL concentrations coincided with maximal airway eosinophilia, and frequency of IL-4, IL-5 and IL-13 producing cells in mediastinal lymph nodes but induction lagged behind the onset of inflammation. Follistatin and activin A immunoreactivity were lost in airway epithelial cells in parallel with goblet cell metaplasia. Exogenous follistatin inhibited the allergen-specific Th2 immune response in mediastinal lymph nodes and mucus production in the lung. CONCLUSION: Follistatin is preformed in the normal lung and released in concert with activin A suggesting it serves as an endogenous regulator. Disturbance of the fine balance between activin A and its endogenous inhibitor follistatin may be a determinant of the severity of allergic inflammation or tissue phenotypic shift in asthma.     

Hormonal characteristics of follicular fluid from women receiving either GnRH agonist or hCG for ovulation induction

BACKGROUND: A recent prospective randomized study from our groupcompared GnRH agonist (0.5 mg buserelin) and hCG (10 000 IU)for triggering of ovulation following a flexible antagonistprotocol. The agonist group showed a poor reproductive outcomedespite luteal phase support with progesterone and estradiol(E₂). In the present prospective observational study, the healthstatus of follicles from the above study was monitored by analysingthe hormonal content of frozen/thawed follicular fluid samples.The aim was to test whether the poor reproductive outcome couldbe related to a defective pre-ovulatory follicular maturationresulting in oocytes with a compromised developmental competence.METHODS: Hormone concentrations were measured in two individualfollicular fluid samples from each of 32 women receiving buserelinand 37 receiving hCG, thus representing a subset of the folliclesretrieved. RESULTS: Follicular fluid levels of LH in the agonistgroup as compared with the hCG group was 11.1 ± 0.5 versus3.6 ± 0.3 IU/l (mean ± SEM; P < 0.001); FSH,6.3 ± 0.6 versus 3.3 ± 0.2 IU/l (P < 0.001);hCG, not determined versus 139±8 IU/l; E₂, 1.9 ±0.2 versus 1.8 ± 0.2 µmol/l (P > 0.10); progesterone,70 ± 4 versus 93 ± 6 µmol/l (P < 0.001);inhibin-A, 36.9 ± 3.1 versus 37.1 ± 2.5 ng/ml(P > 0.10) and inhibin-B, 35.6 ± 2.8 versus 40.1 ±3.1 ng/ml (P > 0.10). Thus, pronounced hormonal differencesexist in follicular fluid, and the collective concentrationof all three gonadotropins and the follicular fluid concentrationof progesterone were much higher in the group of women receivinghCG for ovulation induction. CONCLUSION: The study suggeststhat GnRH agonist results in proper pre-ovulatory follicularmaturation, but the ovulatory signal – probably in synergywith the resulting pituitary down-regulation – is toolow to support appropriate corpus luteum (CL) function.