Dose Of Follicle-stimulating Hormone Research Articles

Effects of follicle-stimulating hormone (FSH) and human chorionic gonadotropin in individuals with an inactivating mutation of the FSH receptor

Objective: To study the gonadal steroid responses to FSH and hCG in individuals with the inherited Finnish-type inactivating Ala189Val mutation of the FSH receptor gene. Design: Prospective clinical and descriptive study. Setting: University hospital. Patient(s): Two women and one man homozygous for the Ala189Val mutation of the FSH receptor gene, and ovarian biopsies from four affected and four healthy women, and four normal fetuses. Intervention(s): Individuals were treated with increasing doses of recombinant FSH (300 IU/day start, 900 IU/day final) and/or a single dose of hCG (5000 IU). Ovarian biopsies were used in immunohistochemical analyses for detection of aromatase cytochrome P450 and transcription factor GATA-4. In situ 3′-end labeling analyses were used for detection of apoptosis. Main Outcome Measure(s): Measurements of serum concentrations of follicle-stimulating hormone, leuteinizing hormone, inhibin A and B, estradiol, testosterone (T), androstenedione, and prolactin, immunostaining for ovarian aromatase, GATA-4, and apoptosis. Result(s): Administration of FSH had no effect on production of the steroids. Similarly, human chorionic gonadotropin (hCG) treatment, alone or after FSH administration, failed to raise serum steroid concentrations. Ovarian apoptosis was absent, and the expression of transcription factor GATA-4 and aromatase was negligible in the ovarian biopsies from Ala189Val homozygous individuals. Conclusion(s): The Ala189Val mutation of the FSH receptor gene results in a complete block of FSH action in vivo. Furthermore, the failure of hCG to increase both ovarian estradiol and testosterone secretion emphasizes the possible contribution of FSH in regulating ovarian androgen synthesis, and supports the concept that both gonadotropins are necessary for appropriate ovarian steroidogenesis in humans.

Monitoring during gonadotrophin-releasing hormone antagonist protocols

The results of the four large industry sponsored multicentre clinical trials comparing gonadotrophin-releasing hormone (GnRH) antagonists and agonists in controlled cycles of assisted reproduction are reviewed from the perspective of implications for monitoring ovarian responses. The simplicity and consistency of antagonist-controlled cycles in normal women indicate that patients in assisted reproduction programmes should undergo fewer assessments and will receive shorter treatment cycles. However, the two-stage pattern of follicular recruitment in antagonist-controlled cycles leads to follicles of different size profiles at the end of the follicular phase compared with those obtained in agonist-controlled cycles (one recruitment process only). Increasing the dose of follicle-stimulating hormone (FSH) may increase oocyte yield, but does not obviate the difference between the two types of cycle. This difference has implications for the criteria used to determine timing of administration of the luteinization signal before oocyte retrieval.

Concentrations of insulin-like growth factor (IGF)-I and IGF binding protein-3 in the follicular fluid of women undergoing ovarian hyperstimulation with different gonadotropin preparations

Insulin-like growth factors (IGFs) and their binding proteins (IGFBPs) have an important regulatory role in follicular development and oocyte maturation. The aim of this prospective, randomized, double-blind study was to measure the concentrations of IGF-I and IGFBP-3 in follicular fluids collected from infertile women undergoing ovarian hyperstimulation using three different gonadotropin preparations. Twenty infertile women (mean age 33 years, range 28-40) undergoing in vitro fertilization (IVF) programs were recruited. After a written informed consent, each woman randomly underwent a long-protocol for ovarian hyperstimulation using gonadotropin-releasing hormone (GnRH)-analog and one of the following recombinant- and urinary-gonadotropins--alpha-follitropin, beta-follitropin or urofollitropin. Serum 17 beta-estradiol (E2) levels and follicle growth were assessed during the follicular phase. The concentrations of IGF-I and IGFBP-3 in the follicular fluid of aspirated dominant follicles were measured directly. Women treated with alpha-follitropin needed significantly lower doses of follicle-stimulating hormone (FSH) compared to those receiving beta-follitropin (p < 0.05). No other statistically significant differences were detected between groups. Serum E2 levels increased in the three groups from early to late follicular phase. Follicular fluid IGF-I and IGFBP-3 concentrations did not differ significantly in the three groups of women. A statistically significant relationship was observed between follicular fluid IGF-I and IGFBP-3 levels (r = 0.41, p = 0.001). Oocyte maturation correlated in a positive manner with IGF-I (r = 0.34, p = 0.01) and IGFBP-3 (r = 0.29, p = 0.03). These findings show that both recombinant- and urinary-gonadotropin preparations were equally effective in releasing IGF-I and IGFBP-3 in the follicular fluid of dominant follicles, and confirmed the role of these compounds on oocyte maturation.

A double-blind clinical trial comparing a fixed daily dose of 150 and 250 IU of recombinant follicle-stimulating hormone in women undergoing in vitro fertilization

Objective: To determine the efficacy and efficiency of two fixed doses of recombinant follicle-stimulating hormone (FSH) in controlled ovarian hyperstimulation. Design: Randomized, double-blind clinical trial. Setting: Fifteen IVF clinics in Argentina, Brazil, Chile, Colombia, Mexico, and Venezuela. Patient(s): Women between 30 and 39 years of age undergoing IVF or intracytoplasmic sperm injection (ICSI). Intervention(s): Daily doses of either 150 IU or 250 IU of recombinant FSH (Puregon) until at least two follicles ≥20 mm were seen on ultrasound. Main Outcome Measure(s): Number of cumulus–oocyte complexes retrieved and total dose of recombinant FSH used. Result(s): Two hundred one women received 150 IU and 203 used 250 IU. In the low-dose group 8.9 oocytes were retrieved compared to 10.2 in the high-dose group (not significant). The 150 IU-treated women received a total of 1,589 IU and the total dose used in 250 IU treated women was 2,492 IU. Implantation rates were 10.0% in the 150 IU group and 10.9% in the 250 IU group. The vital pregnancy rates per started cycle in the low-dose and high-dose groups were 17.1% and 16.7%, respectively. Two women, both in the 250 IU group, were hospitalized because of the ovarian hyperstimulation syndrome (OHSS). Conclusion(s): An increase from 150 IU to 250 IU daily dose of recombinant FSH in women between 30 and 39 years of age has only limited value in augmenting ovarian response.

Clinical experience with recombinant follicle-stimulating hormone (FSH) and urinary FSH: a retrospective case- controlled analysis

Objective: To compare the efficacy and efficiency of recombinant FSH (rFSH) and urinary FSH (uFSH). Design: Retrospective case controlled analysis. Setting: An assisted reproduction unit at a university center. Patient(s): 1388 patients undergoing long protocol in vitro fertilization/embryo transfer (IVF-ET) using buserelin acetate from day 2 of the cycle and either rFSH (follitropin beta) (n = 694) or uFSH (n = 694) with equal number of ampules started (rFSH: 50 IU, uFSH: 75 IU). Intervention(s): Patients were included in the two groups of treatment after matching for similarity in age and type of treatment (IVF or intracytoplasmic sperm injection). Main Outcome Measure(s): Total dose of FSH, ovarian response, and IVF outcome. Result(s): Patients who received uFSH experienced a shorter period of stimulation, and a higher number of oocytes were collected. The total FSH used was lower in the rFSH group, and they required a lower FSH dose per oocyte retrieved. The implantation and pregnancy rates were similar between the uFSH and rFSH groups. In both groups implantation and pregnancy rates were higher when intracytoplasmic sperm injection was performed as compared with IVF. Conclusion(s): The implantation and pregnancy rates are similar when either rFSH or uFSH is used (when compared on an ampule-to-ampule basis, rFSH: 50 IU, and uFSH: 75 IU). However, a significantly lower total FSH dose was used in the rFSH group with a lower FSH dose per oocyte collected.

Insulin alters the effects of follicle stimulating hormone on aromatase in bovine granulosa cells in vitro

It is known that follicle-stimulating hormone (FSH) and insulin stimulate estradiol secretion from cultured non-luteinizing granulosa cells. The interaction between these hormones is less well understood. Granulosa cells from small (2–4 mm) bovine follicles were cultured in serum-free medium to determine if cytochrome P450 aromatase activity is regulated by FSH in the presence of different concentrations of insulin. Insulin significantly stimulated aromatase activity in the absence of FSH. There was a significant interaction between insulin and FSH on aromatase activity, such that FSH stimulated activity at low (0.5, 1 and 10 ng/ml) doses of insulin, whereas at higher (100 ng/ml) doses of insulin FSH failed to stimulate aromatase activity. To determine if the lack of a response to FSH with higher doses of insulin is related to gene expression, the effect of FSH on P450 aromatase mRNA levels was measured. An ‘uncoupling’ of mRNA and enzyme activity was observed for cells cultured with 100 ng/ml insulin, as FSH significantly increased P450 aromatase mRNA abundance without affecting estradiol secretion or aromatase activity. We conclude that in the presence of high doses of insulin, FSH decreases aromatase activity, and an uncoupling of P450 aromatase mRNA and aromatase activity occurs. This may have implications for infertility treatments when there is a risk of hyperinsulinemia.

Concentrations of insulin-like growth factor (IGF)-I and IGF binding protein-3 in the follicular fluid of women undergoing ovarian hyperstimulation with different gonadotropin preparations

Insulin-like growth factors (IGFs) and their binding proteins (IGFBPs) have an important regulatory role in follicular development and oocyte maturation. The aim of this prospective ,randomized ,double-blindstudy was to measure the concentrations of IGF-I and IGFBP-3 in follicular fluids collected from infertile women undergoing ovarian hyperstimulation using three different gonadotropin preparations. Twentyinfertile women (mean age 33 years ,range 28-40) undergoing in vitro fertilization (IVF) programs were recruited. After a written informed consent ,each woman randomly underwent a long-protocolfor ovarian hyperstimulation using gonadotropin-releasing hormone (GnRH)-analog and one of the following recombinant- and urinary-gonadotropins - α-follitropin ,β-follitropin oruro-follitropin. Serum 17β-estradiol (E2) levels and follicle growth were assessed during the follicular phase. The concentrations of IGF-I and IGFBP-3 in the follicular fluid ofaspirated dominant follicles were measured directly. Women treated with α-follitropin needed significantly lower doses of follicle-stimulating hormone (FSH) compared to those receiving β-follitropin(p < 0.05). No other statistically significant differences were detected between groups. Serum E2 levels increased in the three groups from early to late follicular phase. Follicularfluid IGF-I and IGFBP-3 concentrations did not differ significantly in the three groups of women. A statistically significant relationship was observed between follicular fluid IGF-I and IGFBP-3 levels(r = 0.41 ,p = 0.001). Oocyte maturation correlated in a positive manner with IGF-I (r = 0.34 ,p = 0.01) and IGFBP-3 (r = 0.29 ,p = 0.03). These findings show thatboth recombinant- and urinary-gonadotropin preparations were equally effective in releasing IGF-I and IGFBP-3 in the follicular fluid of dominant follicles ,and confirmed the role of these compounds onoocyte maturation.

17 Alpha-oestradiol and 17 beta-oestradiol do not affect basal and follicle-stimulating hormone-stimulated inhibin B secretion by highly purified rat Sertoli cells.

The effects of 17 alpha-oestradiol and 17 beta-oestradiol on basal and follicle-stimulating hormone (FSH)-stimulated inhibin B secretion by rat Sertoli cells were studied. Sertoli cells were isolated and cultivated from testes of 18-day-old Wistar rats in the presence and absence of FSH and different doses of oestrogens. On day 4 of culture, secreted inhibin was measured by enzyme-linked immunosorbent assay. Neither 17 alpha-oestradiol nor 17 beta-oestradiol had any effect on the secreted inhibin level in either the presence or absence of FSH. It is concluded that these oestradiols do not play an essential role in regulatory processes involving inhibin or FSH.

Effects of low day 3 luteinizing hormone levels on in vitro fertilization treatment outcome

In a previous study we demonstrated that women with day 3 luteinizing hormone (LH) values < 3 IU/l subjected to controlled ovarian hyperstimulation without pituitary desensitization responded with a lower number of follicles > 15 mm compared to women with a higher basal LH level. The aim of this study was to determine whether in patients with day 3 LH levels < 3 IU/l a further reduction of serum LH concentration by gonadotropin-releasing hormone (GnRH) analog impairs follicular response to follicle stimulating hormone (FSH) and treatment outcome in in vitro fertilization (IVF) cycles.For this purpose we retrospectively studied 249 consecutive women subjected to standard IVF treatment employing pituitary desensitization with buserelin and follicular stimulation with urinary highly purified FSH. The patients were divided into two groups according to their day 3 LH value. The first group (group A) showed day 3 LH levels < 3 IU/l and the second (group B) had day 3 LH levels > 3 IU/l. Group A and B patients did not show statistically significant differences in the ovarian response to FSH, nor in IVF treatment outcome, showing that in FSH treated GnRH analog suppressed cycles, the ovarian responsiveness and IVF outcome do not differ according to basal LH values. However, the high dosage of FSH we employed in group A and B patients could account, at least in part, for this result. Indeed, comparative evaluations with unsuppressed cycles (our previous study) strongly suggest that a reduced ovarian responsiveness to gonadotropins in patients with day 3 LH values < 3 IU/l should be considered in clinical practice.

Prediction of over-response to ovarian stimulation in an intrauterine insemination programme.

Prediction of poor-response is of equal importance to prediction of over-response in intrauterine insemination programmes. The gonadotrophin-releasing hormone agonist (GnRHa) stimulation test (GAST) was assessed as a predictor of over-response to ovarian stimulation in 81 patients. Blood samples were taken on cycle day 2 (before and 24 h after starting the GnRHa). Day 2 and 3 samples were assayed for oestradiol, follicle stimulating hormone (FSH) and luteinizing hormone (LH). Linear and logistic regression analyses were used to assess age, day 2 FSH, day 2 FSH/LH, oestradiol ratio (oestradiol on day 3/oestradiol on day 2) and FSH ratio (FSH on day 3/FSH on day 2) as predictors of the number of follicles (total and > or = 14 mm), oestradiol on HCG day, and clinical pregnancy rate as appropriate. Several parameters were also compared between the patients who produced < or = 3 (> or = 14 mm) follicles (group A) and those who produced >3 (> or = 14 mm) follicles (group B). The mean +/- SEM age of the patients in the study was 32 +/- 0.4 years. The mean total dose of recombinant FSH was 800 +/- 20 IU and the mean duration of stimulation was 7.6 +/- 0.2 days. Nine (11%) and 12 (15%) patients were cancelled for poor and over-response respectively. The oestradiol ratio was significantly positively correlated with oestradiol on HCG day (P < 0.001), and with the number of mature follicles (> or = 14 mm) (P = 0.01). Age, day 2 FSH and FSH ratio were not significantly correlated with oestradiol on HCG day, total follicles and follicles > or = 14 mm. None of the above-mentioned variables was correlated with clinical pregnancy rate. Group A had significantly lower oestradiol ratio (P = 0.007), longer duration of stimulation (P = 0.002), higher total FSH dose (P = 0.001), and lower oestradiol on HCG day (P = 0.001). GAST is therefore useful in predicting the high responders to gonadotrophin stimulation.

In vitro evaluation of aromatase enzyme in granulosa cells using a [ 11C]vorozole binding assay

An in vitro method for measuring aromatase cytochrome P450 enzyme (P450 AROM) in human granulosa cells (GC) has been developed, based on binding of the 11C-labeled aromatase inhibitor vorozole. GC were obtained following superstimulation during in vitro fertilisation. The method revealed a binding affinity ( K d) of 0.4 nM and a maximum binding (B max) at 11 fmol/4000 cells which is equal to 1.6 million binding sites per cell. Linear Scatchard plots indicated a single type of binding site. P450 AROM concentrations measured by [ 11C]vorozole binding correlated positively with aromatisation of [1β- 3H]androst-4-ene-3,17-dione measured as [ 3H]water release, and a positive association was also found with the ovarian in vivo response to follicle-stimulating hormone (FSH) stimulation expressed as 1000 times the ratio of the number of oocytes recovered from a patient and the total dose of recombinant FSH administered. Frozen cells could be used for P450 AROM quantitation, provided the correct freezing procedure was used. Quantitation of P450 AROM, based on binding of [ 11C]vorozole is an accurate and sensitive in vitro method, which might be extended to the measurement of aromatase expression by a noninvasive technique in the intact ovary in vivo using positron emission tomography.

Effect of exogenous FSH on ovulation rate in homozygous carriers or noncarriers of the booroola FecB gene after hypothalamic-pituitary disconnection or after treatment with a GnRH agonist

We have tested the hypothesis “that the ovulation rate in homozygous carriers (BB) and noncarriers (++) of the Booroola FecB gene would not be different if the plasma concentrations of follicle-stimulating hormone (FSH) in the two genotypes were similar.” For this purpose we used two experimental animal models: 1) the hypothalamic-pituitary disconnected (HPD) ovary-intact ewe; and 2) the GnRH agonist (i.e., Deslorelin)-treated ewe. Following HPD or Deslorelin treatment, the animals had low plasma concentrations of gonadotropins and were anovulatory. In both animal models, BB and ++ ewes were treated with exogenous pregnant mares serum gonadotropin (PMSG) and varying doses of FSH to induce preovulatory follicular growth, and human chorionic gonadotropin (hCG) to induce ovulation. HPD or Deslorelin-treated animals administered with pregnant mares serum gonadotropin without FSH followed by human chorionic gonadotropin failed to ovulate. However for both animal models, the proportion of BB and ++ ewes ovulating to various doses of FSH differed such that significantly greater proportions of ++ animals ovulated relative to the BB genotype (P < 0.05). When HPD or Deslorelin-treated BB and ++ ewes were administered identical doses of FSH, the mean ovulation rate and plasma concentrations of FSH in those animals which ovulated was the same in both genotypes. These findings confirm, at least in part, the aforementioned hypothesis. The results also demonstrated that higher ovulation rates were obtained in both genotypes as the FSH dose was increased. Collectively, these findings infer that the higher mean ovulation rate in normal intact BB ewes compared to the ++ genotype is attributable to effects of the FecB gene at the level of ovarian follicular development as well as at the level of pituitary FSH release.

In vitro fertilization outcome according to age and follicle-stimulating hormone levels on cycle day 3.

In a retrospective study, the prognostic factors of in vitro fertilization outcome were studied in women of 39 years of age or older, with an elevated cycle day 3 follicle-stimulating hormone (FSH) level. Ovarian stimulation was achieved with a combination of gonadotropin-releasing hormone agonist/human menopausal gonadotropin in a short protocol. All patients underwent FSH dosage on cycle day 3 prior to stimulation. The pregnancy rate was studied according to age, FSH levels, and stimulation parameters. There was a negative correlation between day 3 FSH levels and the number of ovocytes retrieved. Cycles canceled because of the absence of ovarian response had a significantly higher mean FSH value (18.2 mlU/ml) than cycles ending in ovocyte retrieval (14.6 mlU/ml). Patients with three or more growing follicles during stimulation achieved a significantly higher pregnancy rate per egg retrieval (16%) compared to patients with fewer than three growing follicles (6%). Eighty percent of those pregnancies were obtained during the first two IVF cycles. Even with an elevated FSH level, some patients developed three or more follicles after stimulation. In such cases, the number of embryos available for transfer was the only significant limiting factor to achieving pregnancy. As our results suggest, there is a discrepancy between biological and chronological ovarian age. In patients with an elevated cycle day 3 FSH level and over 40 years of age, alternatives to fertility treatments (ovum donation, adoption, or no treatment) should not be considered as first choices. Indeed, even with elevated FSH levels, a 16% pregnancy rate per egg retrieval may be obtained if three or more growing follicles can be seen during ovarian stimulation. However, in the presence of fewer than three growing follicles during ovarian stimulation, the patient should be informed about the discouraging prognosis of the running cycle.

Histological evaluation of endometrium on the day of oocyte retrieval after gonadotrophin-releasing hormone agonist-follicle stimulating hormone ovulation induction for in-vitro fertilization.

The objective of this study was to evaluate the histopathological characteristics of endometrial biopsies taken on the day of oocyte recovery in in-vitro fertilization (IVF) cycles with a satisfactory response to ovulation induction. A group of 33 patients who went through ovulation induction for IVF, and in whom an endometrial polyp was suspected on transvaginal ultrasonography during the monitoring phase, were studied. Following oocyte recovery, hysteroscopy, polypectomy and endometrial curettage were performed. Dating of endometrial glands and stroma was carried out in the tissue not containing the polyps. The total dose of follicle stimulating hormone (FSH), duration of ovulation induction, peak oestradiol and luteinizing hormone (LH) concentrations, thickness of endometrium and number of oocytes were recorded and compared to the endometrial dating of the specimens. In 15 cycles (45.5%), the endometrium was classified as 'in phase' (group I), 'advanced' by 2-4 days in a further 15 (45.5%, group II), and in the remaining three cycles (9%) it was delayed in maturation (group III). Younger age was correlated with advanced staging of the endometrium (r = -0.42; P = 0.015). Women with 'in phase' and 'advanced' maturation were similar in their response to ovulation induction; however, there was a strong correlation between advanced dating of endometrium and number of oocytes retrieved (r = 0.49; P = 0.04). Endometrial staging on the day of oocyte retrieval varied widely in patients treated by the same gonadotrophin-releasing hormone agonist (GnRHa)/FSH protocol for ovulation induction. This difference was not predictable by parameters monitored through the cycles.

Correlation between the amount of follicle-stimulating hormone administered and plasma and follicular fluid vascular endothelial growth factor concentrations in women undergoing in vitro fertilization

Vascular endothelial growth factor (VEGF) is a powerful mediator for vessel permeability and it is strongly implicated in angiogenesis, stimulating endothelial cell proliferation as well as capillary permeability. We studied 30 women undergoing in vitro fertilization (IVF) programs and evaluated, on the day of oocyte retrieval, VEGF levels in plasma and follicular fluid and related such concentrations to the amount of follicle-stimulating hormone (FSH) administered. Furthermore, the correlation between the number of oocytes retrieved and the VEGF concentrations both in plasma and in follicular fluid were also investigated.Results indicate that follicular fluid VEGF concentrations and the amount of pure FSH administered were directly proportional (p < 0.05). On the day of oocyte retrieval, the VEGF plasma concentrations and the number of oocytes collected were directly proportional (p < 0.05). VEGF plasma levels increased after human chorionic gonadotropin (hCG) administration (30.37± 18.60 pg/ml up to 52.62 ± 43.63 pg/ml).In conclusion, this study demonstrates that the doses of pure FSH administered to women undergoing IVF cycles have a crucial role in hCG-dependent VEGF production.

Differential regulation of leucine-rich primary response gene 1 (LRPR1) mRNA expression in rat testis and ovary.

In immature rat Sertoli cells, leucine-rich primary response gene 1 (LRPR1) represents a follicle stimulating hormone (FSH)-responsive gene; the function of the encoded protein is not yet known. LRPR1 mRNA expression is up-regulated very rapidly and specifically by FSH, both in cultured Sertoli cells and in vivo in regulation in more detail, in testis and ovary of fetal, immature, and adult rats. In addition, we have studied the expression of FSH receptor (FSHR) mRNA in relation to LRPR1 mRNA expression. In rat testis, LRPR1 mRNA and FSHR mRNA followed a similar expression pattern, during postnatal development and also at different stages of the spermatogenic cycle in the adult rat. Furthermore, after short-term challenge of the FSH signal transduction pathway in intact immature rats by injection with a relatively high dose of FSH, an inverse relationship between LRPR1 mRNA (up-regulation) and FSHR mRNA expression (down-regulation) was observed. Similar studies in the ovary provided completely different results. LRPR1 mRNA in the postnatal ovary is present well before expression of FSHR mRNA can be first detected. In addition, incubation of ovaries of immature rats with FSH or dibutyryl cyclic AMP (dbcAMP) did not result in up-regulation of LRPR1 mRNA expression. During fetal development, the LRPR1 mRNA expression pattern involved many more tissues, in contrast to the relatively tissue-specific expression of LRPR1 mRNA in gonads of 21 day old and adult rats. Moreover, LRPR1 mRNA expression could be detected as early as 12.5 days post-coitum, whereas FSHR mRNA is absent at this stage of fetal development. We concluded that the pronounced regulation of LRPR1 by FSH observed in the immature rat testis does not occur in the ovary. Furthermore, in the ovary LRPR1 mRNA expression does not appear to be dependent on FSH action. Finally, the LRPR1 gene product may play a general role during fetal development.

Concerted stimulation of rat granulosa cell deoxyribonucleic acid synthesis by sex steroids and follicle-stimulating hormone.

Although follicle-stimulating hormone (FSH) and estrogens are known to be the main physiological stimuli for the development of the ovarian follicle in mammals, their growth-promoting activity has not been clearly established "in vitro". Furthermore, experimental evidence indicates that FSH and estradiol can independently inhibit granulosa cell proliferation. The present study was aimed at examining the effect of sex steroids in combination with FSH, on DNA synthesis in rat granulosa cells cultured in completely defined medium. Estradiol and FSH, when added separately, produced a significant inhibition of [3H] thymidine incorporation. In contrast, a combination of a low dose of FSH (20 ng/ml) with estradiol (100 ng/ml) produced a shift in the period of maximal DNA synthesis from 96 to 48 h after plating. Dose response studies showed that estradiol effects were produced at physiological intraovarian concentrations (1-100 ng/ml), whereas the effects of FSH were biphasic, with high doses (200 ng/ml) being inhibitory. A similar biphasic dose response curve was observed with increasing concentrations of a cAMP derivative in the presence of maximally effective doses of either an aromatizable steroid (androstenedione), insulin or insulin-like growth factor I. Non-aromatizable androgens (5alpha-dihydrotestosterone, 5alpha-androstane 3alpha-17beta diol and androsterone) showed a potency comparable to that of estradiol. The effect of 5alpha-dihydrotestosterone was completely blocked by a specific antiandrogen (hydroxy-flutamide), indicating that it was mediated by the androgen receptor. The effects of estradiol and androgens were not additive. The interaction between estradiol and FSH was further amplified in the presence of a maximally effective dose of insulin. Data presented herein indicate that both estrogens and androgens are able to elicit a mitogenic response in purified granulosa cells, cultured in a completely defined medium, provided the cells are stimulated by a physiological dose of FSH. These results suggest that, during follicular development, the stimulus for granulosa cell proliferation is given by the concerted action of steroid and peptide hormones acting through different signalling pathways.

Modulation of FSH receptor phosphorylation correlates with hormone-induced coupling to the adenylate cyclase system.

The authors have recently demonstrated that an inhibitor of protein phosphorylation, staurosporine (SSP), can dramatically enhance follicle-stimulating hormone (FSH) stimulated cyclic adenosine monophosphate (cAMP) accumulation in rat granulosa cell line (GFSHR-17) overexpressing about 20-fold FSH receptor than primary granulosa cells. Moreover, incubation with SSP can partially release the cells from FSH-induced desensitization. In this work, it was examined whether coupling of FSH receptor to the adenylate cyclase is correlated with the degree of receptor phosphorylation. Immunoprecipitation of FSH receptor after metabolic labeling of the cells with 32P-orthophosphate revealed that preincubation of the cells with SSP resulted in pronounced reduction in FSH receptor phosphorylation compared to control cells, concomitantly with a dramatic increase in FSH-stimulated cAMP accumulation. In contrast, incubation of the cells with saturating dose of FSH, which leads to uncoupling between the receptor and the adenylate cyclase, resulted in enhanced receptor phosphorylation. Moreover, cells preincubated with FSH could be released from desensitization by further incubation with SSP and a significant reduction in FSH receptor phosphorylation. Immunostaining of the cells with FSH receptor antibody reveal a homogenous distribution of the receptor on the surface of SSP-treated cells. Some aggregation of the receptor was evident in control cells that were not treated with SSP. In contrast, massive clustering and capping of the receptor molecules were observed on the surface of FSH-stimulated cells. The current data suggest that phosphorylation-dephosphorylation of the receptor molecules play an important role in the degree of coupling between the receptor and the adenylate cyclase system. Moreover, desensitization to FSH stimulation that is implicated with high degree of receptor phosphorylation may lead to aggregation of the receptor molecules on the cell surface.

Steroidogenesis in Theca Cells of Chicken Follicles in Response to Ovine Gonadotropins.

The purpose of this study was to assess and compare the steroidogenic response to luteinizing hormone (LH) and follicle-stimulating hormone (FSH) in cells of the theca interna and theca externa isolated from follicles at different stages of maturation. Cells of the theca interna and theca externa isolated from the largest, second largest and third-fifth largest preovulatory follicles of the chicken (Gallus domesticus) were incubated at the concentration of 2×105 cells/ml/tube in Medium 199 containing 10mM Hepes and 0.2% bovine serum albumin with increasing doses (0, 0.1, 1.0, 10 and 100ng/ml/ tube) of ovine LH (oLH) or ovine FSH (oFSH). After the 4hr-incubation, progesterone (P4), testosterone (T), and estradiol-17β (E2) in the medium were measured by RIA. P4 and T were detected in cultured medium of theca interna cells but were undetectable (<25pg/tube) in that of theca externa cells, while E2 was detected in cultured medium of theca externa cells but not (<25pg/tube) in that of theca interna cells. The productions of P4 and T were stimulated in a dose dependent manner by oLH and oFSH. The responses to oLH and oFSH were greater in smaller follicles, although the response to oFSH was less than that to oLH. E2 production was stimulated in a dose-dependent manner by oFSH with greater response in smaller follicles but not by oLH.The present results support a three-cell theory for estrogen production in avian follicles and indicate that, in the chicken, the smaller follicles may play a conspicuous role in gonadotropin-induced steroidogenesis.

The Utility of a Midcycle Follicle-Stimulating Hormone Boost in Addition to Human Chorionic Gonadotropin for Timing of Follicle Aspiration in Unstimulated in vitro Fertilization Cycles

To analyze the effects and potential benefits of a single midcycle dose of follicle-stimulating hormone (FSH) prior to human chorionic gonadotropin (hCG) administration during unstimulated in vitro fertilization (IVF) cycles. Twenty-five cycles from 20 patients receiving 150 IU of FSH 42-44 h and 10,000 IU of hCG 34-36 h prior to follicle aspiration were compared to 110 cycles triggered with hCG alone. Serum E2 levels were significantly lower on the day of hCG treatment in the FSH-treated group (266 vs. 297 pg/ml). On the day after hCG administration, serum E2 was similar in both groups. Maximum follicular diameters the day of and the day after hCG treatment were similar as were the number of oocytes aspirated and embryos transferred, and clinical pregnancy rates in both groups. The number of cycles dropped due to premature luteinizing-hormone (LH) surge was 8% in the FSH group compared to 20% in the group treated with hCG alone. A midcycle FSH boost does not increase pregnancy success of unstimulated IVF cycles but does enhance the increase in serum E2 levels after hCG, thereby potentially allowing earlier hCG administration while incurring decreased cycle cancellation rates due to premature LH surges.