Inhibin A, inhibin B and activin A concentrations in follicular fluid from women with polycystic ovary syndrome

Polycystic ovary syndrome (PCOS) is characterized by arrestedfollicle development at the early antral stage. Alterationsin inhibin production by developing follicles could be involvedin PCOS by suppressing follicle stimulating hormone concentrationsduring the follicular phase of the menstrual cycle as well asby increasing thecal androgen production. Inhibin B appearsto be more important than inhibin A during the follicular phase;however, there are no data regarding the follicular fluid concentrationsof inhibin B in PCOS. The purpose of this study was to compareinhibin A, inhibin B and activin A concentrations in the follicularfluid from regularly cycling women and women with PCOS. InhibinA, inhibin B and activin A were measured in the follicular fluidof 4–7 mm follicles from PCOS ovaries and size-matchedfollicles from normally cycling women by specific and sensitivetwo-site enzyme-linked immunosorbent assays. In both controland polycystic ovaries, inhibin B was approximately 10-foldhigher than activin A and more than 100-fold higher than inhibinA. There was no difference in activin A concentrations betweenPCOS and control follicles. In control ovaries, the inhibinB and inhibin A concentrations in dominant follicles were significantlyhigher than in cohort follicles. While inhibin A concentrationswere lower in PCOS follicles than in normal cohort follicles,there was no difference in inhibin B concentrations betweenPCOS follicles and normal cohort follicles. These data are consistentwith the concept that inhibin B is the physiologically mostimportant form of inhibin during the follicular phase of themenstrual cycle and indicate that PCOS is not associated withincreased inhibin B concentrations in follicular fluid.     

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.     

Immunolocalization of Fas and Fas ligand in the ovaries of women with polycystic ovary syndrome: relationship to apoptosis

Both Fas (APO-1, CD95), an apoptosis-inducing receptor, and its ligand, Fas ligand (FasL, CD95L), have been localized to the ovary. Granulosa cell apoptosis occurs in antral follicular atresia. In polycystic ovary syndrome (PCOS), antral follicles accumulate with some atretic features. The ovarian expression of Fas and FasL was examined in PCOS by immunohistochemistry and correlated with immunodetection of apoptotic cells. Fas immunostaining was present in pre-antral follicle oocytes, some primary and secondary pre-antral follicle granulosa cells, and both granulosa and theca of antral follicles. Thecal staining persisted with advancing atresia, while granulosa staining declined. In antral follicles, abundant Fas-positive cells co-localized with scattered nuclei immunopositive for apoptosis. Ovarian vascular myocytes were strongly Fas-immunopositive. FasL immunostaining was present in pre-antral follicles in oocytes and variably in granulosa. In antral follicles, granulosa and thecal FasL staining increased with advancing atresia. Normal control ovaries showed follicular Fas and FasL staining patterns similar to those in PCOS, but vascular staining was less prominent. In one healthy follicle, Fas immunostaining was seen in the oocyte and weakly in mural granulosa and theca interna. The results suggest that in PCOS, an alteration in Fas-mediated apoptosis, does not cause abnormal folliculogenesis, but may promote ovarian vascular remodelling.     

Ultrasound examination of polycystic ovaries: is it worth counting the follicles?

BACKGROUND: This study revisited the ultrasonographic diagnostic criteria of polycystic ovary syndrome (PCOS) and studied the relationship between the major hormonal and metabolic features of PCOS and the follicle number per ovary (FNPO). METHODS: This prospective study included 214 women with PCOS compared with 112 women with normal ovaries. Main clinical, biological and ultrasonographic markers of PCOS were assessed during the early follicular phase. RESULTS: The mean FNPO of follicles 2-5 mm in size was significantly higher in polycystic ovaries than in controls, while it was similar within the 6-9 mm range. Setting the threshold at 12 for the 2-9 mm FNPO offered the best compromise between specificity (99%) and sensitivity (75%). Within the 2-5 mm follicular range, we found significant positive relationships between the FNPO and androgens. The FNPO within the 6-9 mm range was significantly and negatively related to body mass index and fasting insulin serum level. CONCLUSIONS: We propose to modify the definition of polycystic ovaries by adding the presence of > or =12 follicles measuring 2-9 mm in diameter (mean of both ovaries). Also, our findings strengthen the hypothesis that the intra-ovarian hyperandrogenism promotes excessive early follicular growth and that further progression cannot proceed normally because of hyperinsulinism and/or other metabolic influence linked to obesity.     

Selective alterations in insulin receptor substrates-1, -2 and -4 in theca but not granulosa cells from polycystic ovaries

The elevated insulin concentrations that occur in many women with polycystic ovary syndrome (PCOS) can contribute significantly to ovarian hyperandrogenism. The objective of the present study was to compare the content of proximal insulin signalling molecules in theca and granulosa cells between polycystic ovaries and regular cycling controls. Individual follicles (3-7 mm) were obtained from 11 women with PCOS and 10 regularly cycling control women. The theca and granulosa cells were microdissected from each follicle. Total protein was extracted and signalling proteins were measured by western blot analysis. There was no difference in insulin receptor content between PCOS and controls in either theca or granulosa cells. Insulin receptor substrate (IRS)-1 and -2 were increased (P<0.05), but IRS-4 was decreased (P<0.03) in PCOS theca cells. There were no changes in IRS-1, -2 or -4 in granulosa cells. IRS-3 was undetectable in all samples. There were no changes in phosphatidyl inositol-3 kinase catalytic subunits p110alpha or p110beta in either theca or granulosa cells. These data demonstrate cell-specific alterations in IRS protein concentrations in theca cells from polycystic ovaries that are consistent with an exaggerated amplification of the insulin signal and which may play an important role in ovarian hyperandrogenism and thecal hyperplasia.     

Comparison of follicle steroidogenesis from normal and polycystic ovaries in women undergoing IVF: relationship between steroid concentrations, follicle size, oocyte quality and fecundability

Studies of ovarian stimulation for IVF have suggested a relationship between follicle size and pregnancy rates. Furthermore the follicular endocrine environment is correlated with oocyte quality. The aim of this study was first to verify the relationship between follicular steroid content, follicular size, oocyte maturity and fertilization outcome in women with normal ovaries following recombinant human FSH (rhFSH). Secondly this study was extended to women with polycystic ovarian syndrome (PCOS). Fifty-nine patients (31 normal, 28 PCOS) underwent conventional IVF with rhFSH induction. Follicular diameter was classified as small (8-13 mm) or large (>14 mm) and sex steroid content was analysed for each group. Oocyte maturity was studied according to nuclear maturation the day after fertilization. In both ovulation groups, 17 beta-oestradiol and progesterone concentration were significantly higher in large follicles with meiotically competent oocytes compared with those containing meiotically incompetent oocytes. Testosterone levels were increased in PCOS follicles compared with normal patients, with no difference between corresponding sub-groups of follicles with meiotically competent oocytes. The relationship between follicle size and embryo development showed that 14 mm could be a threshold value following rhFSH induction in normal or PCOS women.     

Insulin-like growth factor (IGF)-I and IGF binding protein-3 concentrations in fluid from human stimulated follicles

Insulin-like growth factor-I (IGF-I) and IGF binding protein-3 (IGFBP- 3) play an important role in regulating follicle growth and maturation. We have evaluated whether responsiveness to gonadotrophins during an in- vitro fertilization (IVF) treatment is related to follicular fluid IGF- I and IGFBP-3 concentrations. We also investigated if a difference is present in IGF-I and IGFBP-3 concentrations between patients treated with human menopausal gonadotrophin (HMG) and patients treated with highly purified follicle stimulating hormone (FSH). We have measured IGF-I and IGFBP-3 in follicular fluid from pre-ovulatory follicles in an IVF programme. All 70 patients were stimulated after being down- regulated with a gonadotrophin-releasing hormone (GnRH) analogue. IGF-I concentrations in follicular fluid were significantly inversely correlated with the number of ampoules FSH administered and number of days of FSH administration, and significantly correlated with the number of follicles aspirated. IGFBP-3 concentrations were not correlated with any other parameter measured nor were IGF-I and IGFBP-3 concentrations correlated. IGFBP-3 concentrations were significantly higher in patients receiving highly purified FSH compared with patients receiving HMG (P < 0.005). These results are new evidence that IGF-I concentration in follicular fluid is higher in women who respond better to follicular stimulation, i.e. women who grow many follicles, women who need a shorter duration of stimulation and women who need fewer ampoules FSH before oocyte retrieval.      

Recombinant human follicle stimulating hormone versus human menopausal gonadotrophin induction: effects in mature follicle endocrinology.

To investigate follicular effects of recombinant human follicle stimulating hormone (rhFSH) induction on women with polycystic ovary syndrome (PCOS), steroid content was compared in mature follicles obtained using a long luteinizing hormone-releasing hormone agonist plus rhFSH or human menopausal gonadotrophin (HMG) in PCOS women and controls participating in an in-vitro fertilization programme. Follicular fluids (144 samples) were collected at oocyte retrieval by individual selective aspiration. Oocyte maturity and fecundability were assessed. Plasma and intrafollicular 17beta-oestradiol, progesterone, testosterone concentrations were assayed individually. No significant difference was seen in oocyte maturity and fecundability between PCOS and controls following rhFSH, or between PCOS rhFSH and HMG group. 17beta-oestradiol, testosterone and progesterone concentrations were lower in PCOS follicular fluid following rhFSH than HMG but the difference was not significant. Progesterone concentration, 17beta-oestradiol/progesterone, 17beta-oestradiol/testosterone were significantly different between the two induction groups, for PCOS fertilized oocyte follicles (P = 0.01, P < 0.05 and P < 0.05 respectively). Steroidogenic enzymatic activity seems to be regulated in healthy follicular cells in PCOS as well as in normal patients upon ovarian induction. Following rhFSH, higher PCOS follicular progesterone concentrations leading to a theoretically increased fecundability could suggest that recombinant FSH is a better inducer which needs to be confirmed.     

Current concepts of the roles of follicle stimulating hormone and luteinizing hormone in folliculogenesis

Around 400 follicles sequentially mature and ovulate duringan average women‘s reproductive lifetime. From birth tothe menopause, the other 99.98% of her follicles begin developmentbut never complete it. Instead they default to atresia due toinadequate stimulation by follicle stimulating hormone (FSH).Follicular growth to the stage of antrum formation (0.25 mmdiameter) is independent of gonadotrophic stimulation. Antrumformation and further growth to the stage at which folliclesbecome potentially able to begin pre-ovulatory development (2–5mm diameter) require tonic stimulation by FSH. Before onsetof puberty, blood concentrations of FSH do not rise sufficientlyto sustain development beyond this stage, therefore all antralfollicles become atretic. After puberty, as each menstrual cyclebegins, FSH concentrations rise beyond a critical ‘threshold’and multiple follicles are recruited to begin pre-ovulatorydevelopment. Due to increases in its responsiveness to FSH andluteinizing hormone (LH), one of these follicles becomes selectedto ovulate while the remainder become atretic. At mid-follicularphase, the dominant follicle reaches 10 mm in diameter and increasinglysynthesizes oestradiol. Tonic stimulation by FSH and LH, underpinnedby local paracrine signalling, maintains oestrogen secretionby the dominant follicle, which grows to 20 mm in diameter beforeit ovulates in response to the mid-cycle LH surge. The development-relatedresponse to LH shown by the pre-ovulatory follicle raises thepossibility that exogenous LH might be used as an adjunct totherapy with exogenous FSH in clinical ovulation induction regimenswhere the aim is to induce monovulation.     

Effects of inhibition of vascular endothelial growth factor at time of selection on follicular angiogenesis, expansion, development and atresia in the marmoset

This study determined the effects of inhibiting vascular endothelial growth factor (VEGF) at follicle selection. Marmosets were given an injection of VEGF antagonist, the VEGF Trap on Day 5 of the follicular phase and ovaries were evaluated on Day 10 or 15. Ovaries from controls were assessed on Day 5 (time of selection), Day 10 (peri-ovulatory) and Day 15 (luteal phase). At Day 10, ovaries of four of the five controls contained dominant follicles, while one had ovulated. VEGF Trap-treated ovaries also contained large follicles on Day 10, but VEGF inhibition had suppressed endothelial cell proliferation, leading to reductions in the thecal vascularization and plasma estradiol relative to controls. By Day 15, ovaries of controls contained active corpora lutea whereas ovaries of four of the five treated animals still contained large antral follicles similar in size to pre-ovulatory follicles, and one had small, avascular corpora lutea. However, these follicles had a restricted vasculature, increased incidence of activated caspase-3 staining and morphological features indicating they would become degenerative non-functional cysts. These results show that after follicle selection, VEGF is essential for angiogenesis and the generation of healthy ovulatory follicles and corpora lutea, but fluid accumulation can still occur in selected follicles in the absence of VEGF.     

Follicular fluid concentration of leukaemia inhibitory factor is decreased among women with polycystic ovarian syndrome during assisted reproduction cycles

BACKGROUND: The possibility that a specific cytokine profile could be detected in the ovaries of patients with polycystic ovarian syndrome (PCOS) was investigated. METHOD: Enzyme-linked immunosorbent assay (ELISA) or bioassays were used to assess the concentrations of leukaemia inhibitory factor (LIF), tumour necrosis factor, interleukin 11, gamma interferon, progesterone and oestradiol in follicular fluids from preovulatory follicles collected after ovarian stimulation from 15 PCOS patients, 15 infertile control patients with regular cycles, and 8 oocyte donors. RESULTS: LIF and progesterone concentrations were significantly lower in the follicular fluid of PCOS patients (LIF median: 265 pg/ml) compared with controls (LIF median: 816 pg/ml); LIF and progesterone follicular fluid concentrations were correlated (r = 0.720, P = 0.0001). The LH/FSH ratio was negatively correlated with LIF concentrations (r = - 0.714, P = 0.0075). Although the PCOS and control patients did not differ significantly in age, ovarian reserve or IVF indication, the implantation rate was significantly lower among the women with PCOS (IR = 9 versus 21%, P = < 0.01). CONCLUSION: The specific cytokine profile of the PCOS patients is probably related to the lower implantation rate since follicular fluid LIF appears to function as an embryotrophic agent.     

Comparison of follicular fluid IGF-I, IGF-II, IGFBP-3, IGFBP-4 and PAPP-A concentrations and their ratios between GnRH agonist and GnRH antagonist protocols for controlled ovarian stimulation in IVF-embryo transfer patients

BACKGROUND: Insulin-like growth factors (IGF) and their binding proteins (IGFBP) play a major role in the autocrine and paracrine regulation of folliculogenesis. This is the first study that has compared follicular fluid (FF) IGF-I, IGF-II, IGFBP-3, IGFBP-4 and pregnancy-associated plasma protein (PAPP)-A concentrations, and their ratios, to investigate whether there was any difference in the intrafollicular microenvironment between the GnRH agonist (GnRHa) and antagonist (GnRHant) protocols for controlled ovarian stimulation (COS). METHODS: A total of 68 IVF cycles were included in this study; two groups were studied: GnRHa long protocol group (n = 36) and the flexible GnRHant multiple-dose protocol group (n = 32). FF was obtained from dominant follicles during oocyte retrieval and stored at -70 degrees C until assayed. IGF-I, IGF-II and IGFBP-3 concentrations were measured by radioimmunoassay and IGFBP-4 and PAPP-A by enzyme-linked immunosorbent assay. RESULTS: The duration of COS was significantly longer, and total dose of gonadotrophins used, serum estradiol (E(2)) levels on hCG day and the number of oocytes retrieved were significantly higher in the GnRHa long protocol group. The concentrations of FF IGF-II and IGFBP-4 were significantly higher, and the ratio of IGF-I/IGFBP-4 was significantly lower in the GnRHa long protocol group. Serum E(2) levels per mature follicle were not different between the two groups. CONCLUSIONS: Our data may indicate a difference of intrafollicular microenvironment between cycles using GnRHa long protocols and those using GnRHant protocols. However, the difference in microenvironment does not appear to result in a difference in clinical outcome.     

Physiology: A mathematical model of follicle dynamics in the human ovary

A mathematical model has been developed to describe the ratesof growth and death of follicles in human ovaries between 19and 50 years of age. It was based on the numbers of folliclesat three successive stages of development, which were obtainedby counting follicles in histological sections of ovaries from52 normal women. The model indicated that follicle dynamicswere age dependent, with a transition at 38 years of age whenthe rate of follicle disappearance increased. The rates of folliclegrowth increased at successive stages but did not change withage. The annual egress from stage III (consisting of follicleswith two or more granulosa cell layers) was affected by thedeclining numbers of small follicles, and corresponded to 31,nine and one follicles per day at 29–30, 39–40 and49–50 years of age respectively. The rate of death atstage I (representing small, resting follicles) was the onlyparameter which varied significantly with age: no evidence ofsignificant atresia was found for this stage in ovaries 38 yearsold, but there was significant death above this age. As a consequence,only 40% of follicles leaving stage I reached stage III in olderovaries and just 1500 follicles in toto remained at 50 yearsof age from the 300 000 present at 19 years. This high deathrate of small follicles appears to be responsible for advancingthe timing of ovarian failure, and therefore of menopause, tomidlife in our species.     

Polycystic ovary syndrome: anomalies in progesterone production

The underlying cause of anovulation and miscarriage in polycystic ovary syndrome (PCOS) is unknown. Progesterone may play an important role in oocyte fertilization and embryo implantation. Therefore, in this study we analyse the endocrine function of luteinizing granulosa cells to synthesize progesterone in vivo and in vitro in PCOS and normal patients participating in an in-vitro fertilization programme. Human luteinizing granulosa cells were obtained from 10 patients with normal ovaries (controls) and 10 patients with PCOS by follicular aspiration of individual follicles of each patient and pooled in an attempt to obtain three groups: cells from follicle sizes < or =10,>10< or =15 and > or =16. Serum concentrations of oestradiol and progesterone on the day of human chorionic gonadotrophin (HCG) injection were significantly higher (P < 0.01 and P < 0.05) in PCOS patients than in controls. After HCG stimulation, in-vitro progesterone production was enhanced in granulosa cells of the control group and concentrations increased with follicular size as expected. However, the concentration of progesterone of PCOS patients did not increase with follicular size and there was a significant difference between normal and PCOS groups in follicles >10< or =15 mm (P < 0.05) and > or =16 mm (P < 0.01). Oestradiol production was increased in follicles > or =16 mm in both groups, although this did not reach significance. In summary, it seems that PCOS granulosa cells demonstrate an abnormal capacity to synthesize progesterone in vivo and in vitro. The understanding of granulosa cell function in PCOS may explain the anovulation and miscarriage that occurs in these patients.      

Location and developmental regulation of androgen receptor in primate ovary

Locally produced androgens act via granulosa cell androgen receptors to modulate follicular responsiveness to gonadotrophins and thereby contribute to the paracrine regulation of ovarian function. We used quantitative androgen receptor immunocytochemistry to assess androgen receptor distribution in relation to pre-ovulatory follicular development in the common marmoset (Callithrix jacchus), a New World primate that ovulates two to four follicles in each approximately 28 day ovarian cycle. Ovaries from four adult females in the late follicular phase and from four in the luteal phase were fixed in 4% paraformaldehyde and subjected to an immunocytochemical analysis using a polyclonal androgen receptor antibody with detection by a standard avidin-biotin-peroxidase technique for alkaline phosphatase. Specific androgen receptor immunostaining occurred mainly in granulosa cell nuclei, with little or no specific staining in theca, stroma or oocytes. Granulosa cell androgen receptor immunostaining was most abundant in healthy preantral/early antral follicles, being low or absent from pre-ovulatory follicles and corpora lutea. Differences in granulosa cell androgen receptor immunostaining between immature (0.1- 1.0 mm diameter) and pre-ovulatory (> or = 2.0 mm diameter) follicles were quantified using a videodensitometric analysis of grey-scale values. Readings were taken from the granulosa cell layers of 53 immature follicles and 10 pre-ovulatory follicles in late follicular phase ovaries. The average androgen receptor level in granulosa cells of immature follicles proved to be 4.2-fold higher (P < 0.01) than that in granulosa cells of pre-ovulatory follicles. Because other evidence suggests that paracrine androgen action in granulosa cells converts from stimulation to inhibition as follicles mature, we speculate that a development-related reduction in androgen receptor numbers serves to "protect' granulosa cells against the inhibitory action of androgen, thereby promoting pre-ovulatory follicular dominance in primate ovarian cycles.      

Aromatase mRNA expression in individual follicles from polycystic ovaries

Polycystic ovary syndrome (PCOS) is a common reproductive disorder characterized by arrested follicular development prior to selection of a dominant follicle. Dominant follicles produce large amounts of oestradiol but PCOS follicles do not. With several potential aromatase (P450AROM) inhibitors in follicular fluid, the question arises whether P450AROM is expressed in PCOS granulosa cells, but the activity is inhibited, or whether P450AROM is not expressed in PCOS. The purpose of the present study was to determine whether P450AROM mRNA expression is altered in PCOS and to correlate P450AROM mRNA expression in individual follicles with aromatase stimulatory bioactivity and oestradiol in the follicular microenvironments. P450AROM mRNA was measured in individual follicles from 16 PCOS and 48 regularly cycling control women by quantitative polymerase chain reaction (PCR) and correlated with follicular fluid oestradiol concentrations and aromatase stimulating bioactivity measured by the rat granulosa cells aromatase bioassay. Follicular fluid oestradiol was low in all control follicles <7 mm in diameter. Some follicles > or = 7 mm contained elevated oestradiol values (P < 0.01) and all had an androstenedione:oestradiol ratio of <4. Only in granulosa cells from follicles > or = 7 mm with an androstenedione:oestradiol ratio of <4 were P450AROM mRNA levels increased (P < 0.05). These same follicles also contained increased levels of aromatase stimulating bioactivity whereas follicles <7 mm or with androstenedione:oestradiol ratio of >4 contained little or no bioactivity. All PCOS follicles contained low levels of oestradiol, P450AROM mRNA and aromatase stimulating bioactivity similar to size- matched control follicles. These data indicate that P450AROM mRNA expression and oestradiol production begin in developing follicles when they reach approximately 7 mm in diameter. Oestradiol production is low in PCOS follicles because there is insufficient aromatase stimulating bioactivity to increase P450AROM mRNA expression.      

Regulation of follicle development and novel approaches to ovarian stimulation for IVF

Current ovarian stimulation regimens for IVF are complex and not without risk. Increasing our knowledge of the physiology of follicle development and dominant follicle selection may enable the design of less complex, safer and cheaper ovarian stimulation regimens for IVF. Decremental serum FSH concentrations during the follicular phase of the menstrual cycle are required for single dominant follicle selection. Only the most mature follicle will continue its development due to increased sensitivity for stimulation by FSH. FSH stimulation becomes insufficient for less mature follicles and remaining cohort follicles will therefore go into atresia. The number of days during which FSH is above the threshold for stimulation of follicle development is limited, resulting in a narrow FSH window. More medium sized and large pre-ovulatory follicles and increased oestradiol output can be induced by the administration of small doses of exogenous FSH during the mid- to late follicular phase, preventing the physiological decrease in FSH stimulation. Intervention with decremental serum FSH concentrations in combination with gonadotrophin-releasing hormone (GnRH) antagonists to prevent a premature rise in serum LH may induce ongoing growth of multiple follicles sufficient for IVF. The benefits and risks of these minimal hyperstimulation protocols require further evaluation.     

Ovary and ovulation: Nitric oxide concentrations in the follicular fluid and apoptosis of granulosa cells in human follicles

To study the relationship between follicular atresia, apoptosis,and nitric oxide (NO) generation in follicular development,steroidogenesis, NO levels in follicular fluid and apoptosiswere analysed in the various sized follicles of women receivingovarian stimulation with human meno-pausal gonadotrophin (HMG)—humanchorionic gonado-trophin (HCG) treatments for in-vitro fertilization(IVF)-embryo transfer. The follicles were divided into threegroups by diameter: large follicle, 18 mm; medium follicle,12 and 15 mm; small follicle, 10 mm. Follicular fluid was obtainedfrom 20 women 34 h after HCG administration, and the concentrationsof oestradiol, progesterone and testosterone, and nitrite, nitrate,arginine and citrulline were measured. Granulosa cells obtainedfrom each group of follicular fluid were stained with Hoechstdye, and nuclear morphology was examined by a fluorescence microscopy.Oestradiol and progesterone concentrations in large follicleswere significantly (P < 0.01) higher than those in mediumor small follicles, and testosterone concentrations in smallfollicles were significantly (P < 0.01) higher than thosein large follicles. There were no significant differences inthe concentrations of nitrite, nitrate, arginine and citrullineamong three groups. The percentage of apoptotic cells with nuclearfragmentation was significantly (P < 0.01) higher in smallfollicles than in large follicles. The present results suggestedthat small follicles with poor response to HMG may undergo atresiathrough apoptosis. No significant difference in the follicularNO level between large and small follicles led us to speculateon a different responsiveness to NO in these two types of follicles.     

Dynamics of follicular growth in the human: a model from preliminary results

In the human species, follicle growth is a very long process.The ovulatory follicle originates from a cohort of pre-antralfollicles that have differentiated their theca interna 85 daysearlier under the control of the high peri-ovulatory steroidand gonadotrophin levels. During growth the number of folliclesdecreases by atresia under the influence of factors relatingto the size of the follicles. Their growth rate is regulatedby intra- and extra-ovarian hormonal factors. At the end ofthe luteal phase, 15–20 days before ovulation, the nextovulatory follicle is recruited among a follicle populationof 2–5 mm in diameter. At the beginning of the menstrualcycle it can be distinguished only by its size; a few days laterit starts its maturation and exhibits its dominance on the otherhealthy follicles present in the two ovaries.     

In-vitro ovarian steroidogenesis in women with pelvic congestion

Follicular fluid steroid content and theca and granulosa cell steroidogenesis in pelvic congestion cystic ovaries were compared with steroidogenic function in both normal and polycystic ovaries. Ovaries were obtained at oophorectomy for benign gynaecological conditions, and classified according to gross morphology at dissection. Individual follicles were dissected out, follicular fluid aspirated, and granulosa and theca cells cultured in vitro. Androstenedione, progesterone and oestradiol content of the follicular fluid and overlying culture medium were measured by radioimmunoassay. There was a significant elevation of both basal and LH-stimulated androstenedione production by theca from both polycystic ovaries (n = 10; P < 0.005) and pelvic congestion cystic ovaries (n = 8; P < 0.05 and < 0.01 respectively) as compared with normal ovaries (n = 5). Granulosa cells from pelvic congestion ovaries (n = 7) had a diminished oestradiol response to FSH as compared with those from normal ovaries (n = 8). Follicular fluid from the majority of follicles in the pelvic congestion cystic ovaries had a high androgen:oestrogen ratio consistent with atresia. For the first time, pelvic congestion ovaries characterized by predominantly atretic follicles scattered throughout the stroma in a normal volume ovary are reported. Follicular atresia was reflected by reduced granulosa cell responsiveness to FSH, theca cell hyperplasia and increased basal and LH-stimulated androgen production. These ovaries are functionally distinct from polycystic ovaries, which do not have a higher proportion of atretic follicles than normal ovaries.