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)     

Amino acid, ammonia and urea concentrations in human pre-ovulatory ovarian follicular fluid

BACKGROUND: This study aimed to determine amino acid (AA), ammonia and urea concentrations in human ovarian follicular fluid and to compare these concentrations with those in the circulation. METHODS: Samples of pre-ovulatory follicular fluid and peripheral venous blood were obtained from 14 IVF patients. High-performance liquid chromatography (HPLC) measurements of 25 AAs were the main outcome measures. RESULTS: There was a significant gradient of most AAs from plasma to follicular fluid, with the exception of glutamate, which demonstrated a three-fold increase in follicular fluid concentration (70.0 +/- 3.80 microM) compared with plasma (23.18 +/- 2.20 microM; P < 0.001). The plasma-to-follicular fluid concentration difference for glutamine (81.83 +/- 9.2 microM) was greatest among all AAs. Among essential AAs, this difference was greatest for the branched-chain AAs, isoleucine, leucine and valine. Ammonia concentrations in follicular fluid and blood were 38.87 +/- 2.23 and 22.11 +/- 1.96 microM, respectively (P < 0.001). Urea concentration in follicular fluid was 3.37 +/- 0.18 mM, a value not significantly different from plasma concentration (3.36 +/- 0.22 mM; P = 0.911). CONCLUSIONS: These plasma-follicular fluid differences may reflect both the utilization of AAs and the transport characteristics of the follicular cells. There is accumulation of glutamate and ammonia in pre-ovulatory follicular fluid. The data for urea are consistent with transport by passive diffusion, with no evidence of an active urea cycle in the cells of the follicle.     

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

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

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.     

Endocrinology: Different follicle stimulating hormone/luteinizing hormone ratios for ovarian stimulation

The aim of the present study was to investigate whether reducingthe amount of luteinizing hormone (LH) in gonadotrophic preparationsimpairs follicular growth in in-vitro fertilization (IVF) cyclesduring suppression of endogenous LH levels. A selected groupof 20 IVF patients was randomly divided into two groups. Onegroup was treated with Org 31338 [follicle stimulating hormone(FSH)/LH 3: 1], the other group with Metrodin® (purifiedFSH), both during pituitary down-regulation with buserelin.A fixed daily dose of 150 IU FSH i.m. was given. Serum concentrationsof FSH, LH, oestradiol and progesterone were determined frequentlyand serial ultrasound examinations were performed. Multiplefollicular growth with concomitant rise of oestradiol levelswas observed in all cycles. The duration of the stimulationphase was shorter in the group treated with Org 31338 than inthe group treated with Metrodin. The number of follicles andoocytes and the fertilization rate was larger and the mean embryoquality was higher in the Org 31338 group, but the differencesdid not reach statistical significance. No significant differenceswere found in hormonal values. In women with normal endocrineprofiles, lowering of the LH activity in gonadotrophic preparationsduring gonadotrophin-releasing hormone agonist treatment resultsin adequate ovarian stimulation. However, a preparation withsome LH needed a shorter stimulation than a purified FSH preparation.Whether the other beneficial effects of Org 31338 also occurin a larger population needs further investigation.     

Endocrinology: Follicular fluid insulin-like growth factor-I and insulin-like growth factor-II concentrations vary as a function of day 3 serum follicle stimulating hormone

We determined follicular fluid concentrations of insulin-likegrowth factor (IGF)-I, IGF-II and inhibin as a function of day3 serum follicle stimulating hormone (FSH) in 16 women undergoingfollicular fluid aspiration in preparation for in-vitro fertilizationand embryo transfer. Follicular fluid concentrations of IGF-Iand IGF-II were significantly less in the ‘low’FSH group as compared to the ‘high’ FSH group. Themean IGF-I concentration was 67.6 ng/ml [confidence intervals(CI) 51.6–92.5] in the ‘low’ FSH group comparedto 87.1 ng/ml (CI 72.8–104.2; P < 0.025) in the ‘high’FSH group. Mean IGF-II concentrations were 354.8 ng/ml (CI 297.8–422.9)in the ‘low’ FSH group compared to 489.8 ng/ml (CI384.6–624.5; P < 0.05) in the ‘high’ FSHgroup. Follicular fluid inhibin concentrations did not differbetween groups. These differences in follicular fluid IGF asa function of day 3 FSH may raise questions regarding the rolegrowth factors play in the physiological processes of the ageingfollicle.     

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.     

Serum levels of free androstenedione, testosterone and oestradiol are lower in the follicular phase of conceptional than of non-conceptional cycles after ovarian stimulation with a gonadotrophin-releasing hormone agonist protocol.

Serum concentrations of total and free androstenedione, testosterone and oestradiol were followed during the follicular phase in women undergoing ovarian stimulation for treatment by in-vitro fertilization and embryo transfer (IVF-ET) and compared to those in natural unstimulated cycles. In addition, 10 conceptional and 18 non-conceptional cycles were compared in an attempt to understand the background for successful IVF cycles. The ultra-short gonadotrophin-releasing hormone agonist protocol was used for ovarian stimulation. Throughout the follicular phase, levels of total and free androstenedione and oestradiol were significantly lower in conceptional than in non-conceptional IVF cycles. In addition, levels of free testosterone during the follicular phase were significantly lower in women who conceived compared to non-conceptional IVF cycles, whereas levels of total testosterone were similar. Levels of both free and total androstenedione increased significantly from the second day of the menstrual cycle until oocyte retrieval in non-conceptional IVF cycles, whereas levels in conceptional IVF cycles and unstimulated cycles showed no increase. On the day of oocyte retrieval levels of free and total androstenedione were significantly higher in non-conceptional IVF cycles than in conceptional IVF cycles and unstimulated cycles, which were similar. This study suggests that appropriate levels of free biologically active androgens and oestradiol are important parameters for successful conception.(ABSTRACT TRUNCATED AT 250 WORDS)     

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.     

Acrosome reactivity in spermatozoa of different morphology in response to stimulation with follicular fluid

The ability of the morphologically abnormal spermatozoon toundergo the processes necessary for fertilization is unknown;one of the essential processes is the acrosome reaction. Inorder to assess this, spermatozoa from 10 known fertile donorswere incubated with either follicular fluid or Earle's mediumcontaining 3 mg/ml bovine serum albumin at 37°C for 6 h.The spermatozoa were then stained with 2% Trypan Blue priorto being fixed in gluteraldehyde and stained for the presenceof the acrosomal cap using the triple stain. Fifty live spermatozoain each of four morphological categories (normal head, largehead, small head, abnormal neck or tail) were examined and thenumber of acrosome-reacted spermatozoa determined. There wasa significant difference between the morphological groups inthe baseline number of acrosome-reacted spermatozoa, determinedby examining sperm samples incubated in Earle's medium; however,the number of spermatozoa undergoing the acrosome reaction inresponse to stimulation with follicular fluid (i.e. the numberof spermatozoa acrosome-reacted in follicular fluid minus thenumber acrosome-reacted in Earle's medium) was similar for allmorphological groups. This suggests that abnormal sperm morphologydid not affect the response of spermatozoa to activation ofthe acrosome reaction by exogenous stimuli.     

Endocrine profile in serum and follicular fluid differs after ovarian stimulation with HP-hMG or recombinant FSH in IVF patients

BACKGROUND: Highly purified menotrophin (HP-hMG) has been associated with fewer oocytes retrieved and a higher proportion of top-quality embryos compared with recombinant FSH (rFSH). METHODS: A randomized, assessor-blind, multinational trial in 731 women undergoing IVF after stimulation with HP-hMG (MENOPUR) (n = 363) or rFSH (GONAL-F) (n = 368) following a long GnRH agonist protocol was conducted. Blood was collected before, during and after stimulation. Fluid was collected from follicles > or =17 mm. RESULTS: Serum androstenedione, total testosterone and free androgen index (FAI) were higher (P < 0.001) with HP-hMG than with rFSH after starting stimulation. At the end of stimulation, serum estradiol was higher (P = 0.031) with HP-hMG, whereas progesterone was higher (P < 0.001) with rFSH, even after adjusting for ovarian response. Serum LH was not different between treatments. Mean mid- and end-follicular hCG levels in the HP-hMG group were 2.5 and 2.9 IU/l, respectively. Follicular fluid levels of FSH, LH, hCG, androstenedione, testosterone, FAI and estradiol and ratios of estradiol:androstenedione, estradiol:total testosterone and estradiol:progesterone were higher (P < 0.001) with HP-hMG, whereas progesterone was higher (P < 0.001) with rFSH. CONCLUSION: Major differences in serum and follicular fluid endocrine profile exist after stimulation with HP-hMG or rFSH. Exogenous LH activity induces a differential endocrine environment influencing oocyte quantity and quality, which may be of relevance for clinical outcome.     

Human chorionic gonadotrophin and steroid concentrations in follicular fluid: the relationship to oocyte maturity and fertilization rates in stimulated and natural in-vitro fertilization cycles

The study investigates the relationship of follicular fluidsteroids and human chorionic gonadotrophin to oocyte maturityand fertilization rates in stimulated and natural cycles. Oestradiol,progesterone, testosterone and human chorionic gonadotrophinwere quantified in 129 samples of follicular fluid and the progesterone:oestradiol ratio calculated. Both stimulated cycles (short andlong luteinizing hormone-releasing hormone/human menopausalgonadotrophin regimens) and natural cycles were compared. Atotal of 60 women were studied, 20 in each group. In the naturalcycles, testosterone was significantly lower in follicles withintermediate oocytes (P = 0.015). Both oestradiol and testosteronewere significantly lower in stimulated cycles compared to naturalcycles (P = 0.032 and P = 0.034 respectively). In the ovarianstimulation cycles, the progesterone: oestradiol ratio was significantlyhigher when oocytes fertilized (P = 0.052). Moreover, in thestimulated cycles, oestradiol and human chorionic gonadotrophinwere significantly lower in the short protocol compared to thelong protocol. The data demonstrate that the hormonal milieuof the follicle is altered in downregulated stimulated cyclesto varying degrees, depending partially on the type of protocolused. Furthermore, the progesterone: oestradiol ratio, ratherthan individual hormone concentrations, may be a useful predictorof the fertilizing capacity of the oocytes.     

Predictive value of serum and follicular fluid leptin concentrations during assisted reproductive cycles in normal women and in women with the polycystic ovarian syndrome

Leptin is an adipocyte-derived hormone which plays a central role in the regulation of body weight and energy homeostasis and in signalling to the brain that adequate energy stores are available for reproduction. Although leptin may affect reproduction by regulating the hypothalamic-pituitary-gonadal axis, recent in-vitro observations indicate that leptin may also have direct intra-ovarian actions. Leptin concentrations were measured in women who succeeded in becoming pregnant within three cycles of in-vitro fertilization (IVF) or gamete intra-fallopian transfer (n = 53), in women who failed to become pregnant within three cycles (n = 50), and in women with polycystic ovarian syndrome (PCOS) (n = 22). It was found that lower follicular fluid leptin concentrations were a marker of assisted reproduction treatment success in normal women. Women with PCOS had higher leptin concentrations than women without such a diagnosis, but this was due to their higher body mass index (BMI). After adjustment for age and BMI, women with PCOS who became pregnant tended to have lower mean follicular fluid leptin concentrations than women with PCOS who did not succeed at becoming pregnant. Further studies exploiting the strengths of the IVF model are needed to assess whether the prognostic role for follicular fluid leptin in human reproduction is independent of other factors, and to elucidate the underlying mechanisms.     

Serum oestradiol and progesterone concentrations during prolonged coasting in 15 women at risk of ovarian hyperstimulation syndrome following ovarian stimulation for assisted reproduction treatment

Serum oestradiol and progesterone concentrations were examined for up to 7 days after withholding gonadotrophins whilst continuing pituitary down-regulation in 15 women at serious risk of severe ovarian hyperstimulation syndrome (OHSS) (serum oestradiol >6000 pg/ml and >15 follicles per ovary). Serum oestradiol concentrations rose on day 1 of coasting in all but two of the 15 women before falling, the decrement being in the order of 40% on each day. This observation permits a rational basis for the estimation of frequency of serum oestradiol measurements and duration of coasting. The trends and rates of fall of serum oestradiol do not seem to predict the occurrence of moderate and severe OHSS, being similar in the six women who developed OHSS compared with nine women without OHSS. The trends in progesterone concentrations were unrelated to any aspects of the clinical outcome.     

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.     

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.     

Physiology: Healthy and atretic follicles: vaginosonographic detection and follicular fluid hormone profiles

The health status of human preovulatory follicles was prospectivelystudied by non-invasive transvaginal ultrasound. Daily ultrasoundscans were performed from the time when the follicle measured15 mm in diameter until formation of corpus luteum or oocyteretrieval. Based on the ultrasound scans two types of follicleswere defined: type A follicles showed a cloud visualized asa cone projecting into the follicular fluid with the base ofthe cone positioned on the follicle wall. A light spot seenat the tip of the cloud in the majority of scans was presumedto be the oocyte-cumulus complex. Type B follicles showed anecho free space without a cloud. Two groups of infertility patientswere studied: Group I received intrauterine insemination; 106patients undergoing a total of 263 cycles (188 spontaneous cyclesand 75 clomiphene citrate cycles). Group II received in-vitrofertilization (IVF); 22 patients undergoing a total of 52 cycles(31 spontaneous cycles and 21 clomiphene cycles). In the firstgroup, the ovulatory potential of the follicle is associatedwith the ultrasound characteristics. From the IVF patients thefollicular fluid was harvested and assessed for the free concentrationof the following steroids: oestradiol, progesterone, testosteroneand androstendione. The endocrine health status of the folliclewas associated with the ultrasound characteristics of the follicle.In patients of group 1, 288 type A follicles ovulated out ofa total of 298 follicles (97%). None of the type B folliclesovulated (0/14). In patients of group II, oocytes were obtainedin 79% of the type A follicles (50/63), whereas 7% of the typeB follicles yielded an oocyte (1/14). The follicular fluid hormoneprofile showed significantly lower free oestradiol, free oestradiol/testosteroneratio and oestradiol/androstenedione ratio and higher free testosteroneand free androstenedione in type B follicles compared to typeA follicles. The hormone status and the high oocyte recoveryrate suggest that type A follicles are healthier than type Bfollicles, which seem to have undergone atretic changes. Itis concluded that non-invasive transvaginal ultrasound witha good degree of accuracy predicts the health status of preovulatoryfollicles. Healthy follicles have a cloud with a light spotat the tip, whereas almost all atretic follicles lack this characteristic.     

Gonadotrophin-releasing hormone agonist and ovarian hyperstimulation syndrome in assisted reproduction

The available literature concerning the association betweengonadotrophin-releasing hormone agonist and ovarian hyperstimulationsyndrome has been reviewed and the different patterns by whichthis agent may contribute to the development of such iatrogeniccomplication has been elicited, and guidelines have been presentedfor prevention of this malady. Gonadotrophin-releasing hormoneagonist acts directly on human granulosa cells, probably inits own dose-dependent manner. The extent of this action isprobably subjected to follicular maturation stage and to thedegree of gonadotrophin pre-treatment. Various agonist effectsin assisted reproduction may be implicated in the developmentof ovarian hyperstimulation syndrome: a higher amount of menotrophin;premature luteinization prevention; ‘flare-up’ effect;and a higher pregnancy rate. Different methods for preventionof ovarian hyperstimulation syndrome may be attempted: (i) allembryo cryopreservation with luteal phase reinitiation of agonist;(ii) avoidance of ovulatory human chorionic gonadotrophin (HCG)and continuation of agonist; (iii) cancellation of ovulatoryHCG, prolongation of agonist and later recommencement of menotrophin;(iv) pre-ovulatory LH surge triggering by agonist instead ofthe conventional HCG. Gonadotrophin-releasing hormone agonistmay affect the steroidogenic ovarian stroma directly and suchinteraction may aggravate the development of ovarian hyperstimulationsyndrome.     

Identification of gonadotrophin surge-inhibiting factor (GnSIF)/attenuin in human follicular fluid

Evidence from several laboratories suggests that the ovariesof many species produce a non-steroidal factor called gonadotrophinsurge-inhibiting or attenuating factor (GnSIF) which may regulatethe response of the pituitary to gonadotrophin-releasing hormone(GnRH) and as such, may modulate the timing and/or amplitudeof the lutein-izing hormone (LH) surge. We have recently isolateda candidate GnSIF from porcine follicular fluid (PFF). PorcineGnSIF is a 69 kDa protein which has undetect-able inhibin andfollistatin immunological and biological activity. The presentstudy was designed to purify and identify GnSIF from human follicularfluid. GnSIF activity was measured as suppression of GnRH-stimulatedLH secretion from rat pituitary cells in primary culture. Humanfollicular fluid (-500 ml) was recovered from patients undergoingin-vitro fertilization (FVF). GnSIF was purified by heparin-sepharose,Q-sepharose, S-sepharose, and hydrophobic interaction chromatographyfollowed by iso-electric focusing. Gel electrophoresis and Westernblot were used to identify human GnSIF and compare it with porcineGnSIF. Using these steps, we obtained a highly-purified preparationof GnSIF that manifests in-vitro bioactivity and chromatographycharacteristics similar to those observed for porcine GnSIFand that hybridizes with a porcine GnSIF antibody. Followingtreatment with human chorionic gonadotrophin/human menopausalgonadotrophin (HMG/HCG), human follicular fluid contained roughly25% of the GnSIF (per mg protein) present in porcine follicularfluid. We conclude that GnSIF is present in human follicularfluid and may participate in the regulation of gonadotrophinsecretion in this species.     

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.