Development of human primordial follicles to antral stages in SCID/hpg mice stimulated with follicle stimulating hormone

In contrast to the many detailed studies of Graafian follicles, the biology of small follicles in the human ovary is poorly understood and the trigger for follicular growth initiation remains unknown. No practical model exists to study preantral follicle growth in the human because of their slow growth rate and lack of an effective culture system. We therefore tested ovarian xenografts as a new strategy to study the early stages of ovarian follicular growth in vivo. Mice homozygous for severe combined immunodeficiency (SCID) and hypogonadism (hpg) received human ovarian xenografts under their kidney capsules. Follicle growth was assessed by morphology and proliferating cell nuclear antigen (PCNA) immunostaining. The grafts were recovered after 11 (short-term) and 17 weeks (long-term), and serially sectioned. During the last 6 weeks of long-term grafting, mice were randomized to receive either placebo or 1 IU of purified follicle stimulating hormone (FSH) s.c. on alternating days. After 11 weeks of grafting, the most advanced follicles had a maximum of two granulosa cell layers. In the absence of FSH administration, follicles did not progress beyond the two-layer stage even after 17 weeks of grafting, and the oestradiol levels remained undetectable. In the FSH-treated long-term grafts, follicles had grown to antral stages and resulted in oestradiol levels as high as 2070 pmol/l. Growth initiation indices did not differ between control and FSH-treated grafts. This study demonstrates that follicles can survive and grow in human ovarian tissue grafted under the renal capsules of immunodeficient mice for at least 17 weeks, and indicate that xenograft models are potentially useful for studying human follicle development. Using this physiological model, we showed that FSH is required for follicle growth beyond the two-layer stage, although growth initiation is independent of gonadotrophin stimulation.      

Differential responses of granulosa cells from small and large follicles to follicle stimulating hormone (FSH) during the menstrual cycle and acyclicity: effects of tumour necrosis factor-alpha

This study determined effects of follicle stimulating hormone (FSH) alone and in combination with tumour necrosis factor (TNF), on granulosa cells from small (5-10 mm diameter) and large (>10-25 mm) follicles during follicular and luteal phases of the cycle and during periods of acyclicity. Granulosa cells were collected from ovaries of premenopausal women undergoing oophorectomy. The cells were cultured with human FSH (2 ng/ml) and testosterone (1 microM) in the presence or absence of human TNF-alpha (20 ng/ml). Media were removed at 48 and 96 h after culture and progesterone, oestradiol and cAMP in media were measured by radioimmunoassays. FSH stimulated the accumulation of oestradiol from granulosa cells of small follicles during the follicular and luteal phases but not during acyclicity; and TNF reduced oestradiol accumulation in the presence of FSH. Interestingly, in granulosa cells from small follicles, progesterone and cAMP secretion increased in response to FSH and neither was affected by TNF. Thus, TNF specifically inhibited the conversion of testosterone to oestradiol in granulosa cells from small follicles. FSH stimulated oestradiol production by granulosa cells of large follicles obtained only during the follicular phase of the cycle and TNF inhibited the FSH-induced oestradiol secretion. Granulosa cells obtained from large follicles during the luteal phase and during acyclicity did not accumulate oestradiol in response to FSH. However, FSH increased progesterone and cAMP secretion by granulosa cells obtained from large follicles during the follicular and luteal phases. During the luteal phase alone, TNF in combination with FSH increased progesterone accumulation above that of FSH alone. FSH did not increase progesterone, oestradiol or cAMP secretion by granulosa cells obtained from large follicles during acyclicity. Thus, FSH increases progesterone, oestradiol and cAMP secretion by granulosa cells of small follicles during the follicular and luteal phases and TNF appears to inhibit FSH-induced oestradiol secretion specifically in those cells. In large follicles, FSH- stimulated granulosa cell secretion of oestradiol is limited to the follicular phase and this effect can be inhibited by TNF. In addition, when granulosa cells of large follicles do not increase oestradiol secretion in response to FSH, TNF stimulates progesterone secretion.      

Recombinant follicle stimulating hormone stimulation in poor responders with normal basal concentrations of follicle stimulating hormone and oestradiol: improved reproductive outcome.

A total of 30 young infertile patients who exhibited a poor response in two previous consecutive cycles, despite having normal basal follicle stimulating hormone (FSH) and oestradiol concentrations, were invited to participate in a prospective randomized study comparing the clinical efficacy of recombinant (rFSH) and urinary (uFSH) follicle stimulating hormone. An evaluation of the total dose used (3800 IU versus 4600 IU, P < 0.05) and duration of treatment (10.2 days versus 13.2 days, P < 0.05) showed a significantly shorter treatment period as well as a significantly lower total dose of FSH required to induce ovulation successfully in the group of patients treated with rFSH. Significantly more oocytes (7.2 versus 5. 6, P < 0.05) as well as mature oocytes (5.9 versus 3.2, P < 0.01) were retrieved after rFSH treatment. In addition, significantly more good quality embryos were obtained (3.4 versus 1.8, P < 0.05) in the group of patients treated with rFSH and, as a result, higher pregnancy (33 versus 7%, P < 0.01) and implantation (16 versus 3%, P < 0.01) rates were achieved in these patients. It is concluded that rFSH is more effective than uFSH in inducing multifollicular development and achieving pregnancy in young low responders.     

Role of the prostate in the regulation of pituitary secretion of follicle stimulating hormone

The secretion of Follicle Stimulating Hormone by the pituitary gland is not regulated entirely by the negative feedback from gonadal steroids. There is ample evidence that the secretion of FSH is regulated by a non-steroidal hormone of gonadal origin termed "inhibin". Using a radioimmunoassay procedure, immunoreactive inhibin-like material was quantified in tissue and biological fluids to study its role in reproductive pathophysiology. High levels of immunoreactive inhibin were observed in human prostate as compared to testis. Biologically active as well as immunoreactive inhibin-like material (ILM) was obtained from human prostate, and was similar to that obtained from human seminal plasma under identical conditions. In patients with benign prostatic hypertrophy, high inhibin levels were observed which reduced after prostatectomy. The presence of immunoreactive inhibin in urine as well as semen of vasectomized and gonadectomized subjects indicates that prostate also secretes inhibin-like material. The specific receptors for inhibin as well as biosynthesis of inhibin in human prostate have been reported. Elevation of serum FSH levels following castration of adult male rats was further enhanced in animals where castration as well as prostatectomy was carried out. All these results demonstrate that apart from the testis, the prostate also secretes ILM and further suggest that the prostate along with testis, may play an important role in the regulation of pituitary secretion of FSH.     

The value of basal serum follicle stimulating hormone, luteinizing hormone and oestradiol concentrations following pituitary down-regulation in predicting ovarian response to stimulation with highly purified follicle stimulating hormone.

The value of gonadotrophin and oestradiol concentrations following pituitary down-regulation with leuprolide acetate in predicting ovarian response to stimulation was evaluated in three groups of women undergoing ovarian stimulation for in-vitro fertilization with highly purified follicle stimulating hormone (FSH). Leuprolide acetate was started in the midluteal phase, and either stopped at menses (IVF-SL group, n = 3), or continued throughout stimulation (IVF-LL group, n = 38; oocyte donors, n = 58). Ovarian stimulation was started on cycle day 3, after blood was drawn for down-regulated FSH, luteinizing hormone (LH) and oestradiol. Higher down-regulated LH was predictive of higher oestradiol on day 5 of stimulation in both IVF groups, and of need for fewer ampoules in the IVF-LL group, but not of oestradiol on day of human chorionic gonadotrophin (HCG) administration or number of oocytes retrieved. Higher FSH after down-regulation predicted yield of fewer oocytes in the donor and IVF-LL groups, and higher oestradiol on day 5 of stimulation, need for fewer ampoules and a shorter duration of therapy in both IVF groups. Higher oestradiol after down-regulation was associated with higher oestradiol on day 5 of stimulation and on day of HCG administration, a shorter duration of therapy and need for fewer ampoules in all groups. Whereas these results do not ascribe any predictive significance to LH, they suggest that oestradiol and FSH concentrations after down-regulation are predictive of the pattern of ovarian response to stimulation and of oocyte yield.     

Serum concentrations of follicle stimulating hormone may predict premature ovarian failure in FRAXA premutation women.

It is now recognized that female carriers of fragile X premutations are at increased risk of premature ovarian failure. We have studied 51 premenopausal women from fragile X families, to determine whether premutation carriers have variations in the hormonal markers of menopause, compared to full mutations and controls. We found a significant increase in serum follicle stimulating hormone in premutation carriers, suggesting that as a group they will enter menopause before full mutation carriers and unaffected controls. These results have important implications for fertility in these women.     

The effect of recombinant follicle stimulating hormone (Gonal-F) on endogenous luteinizing hormone secretion in women

Parenteral administration of follicle stimulating hormone (FSH) has been shown to lower luteinizing hormone (LH) concentrations in women undergoing ovulation induction. This study was designed to explore the physiological mechanism of this effect. Seven healthy women were recruited into a double-blind placebo-controlled study. LH secretion, after the administration of variable i.v. boluses (37.5, 75 and 150 IU) of recombinant FSH (Gonal-F), was evaluated. LH was measured at 10 min intervals for 2 h before and 4 h after the FSH/placebo infusion. LH pulse frequency and amplitude were evaluated and there was no significant difference between control and trial cycles for each subject. A linear regression analysis revealed that in the group receiving 150 IU FSH, the mean plasma LH concentration decreased significantly due to a reduction tonic LH secretion. This could be a result of the suppression of secretion or an alteration of clearance. This decrease was not seen in the other dosage groups, revealing that above a dosage threshold, FSH reduced non-pulsatile LH secretion. Therefore the effect of FSH in this study exposed the likely presence of two components of LH concentration: an FSH-sensitive, non-pulsatile tonic secretion and a gonadotrophin-releasing hormone-stimulated, pulsatile release that is unaffected by FSH. Although an indirect effect involving ovarian regulation is not excluded, the rapidity of the effect suggests that FSH acts directly on the pituitary gland.      

Immunocytochemical localization of follicle stimulating hormone in normal human stomach.

Immunoreactive follicle-stimulating hormone (IR-FSH) is detected in sections of formalin-fixed and paraffin-embedded gastric mucosal tissue of normal men, using the immunoperoxidase staining technique and specific antisera to hFSH (NIDDK, NIH). Positive staining for IR-FSH was detected in the parietal cells lining the gastric glands of the intermediate zone. The staining was intracytoplasmic and distributed throughout the cytoplasm. IR-FSH was also found to be present in the basal part of the foveolar epithelium. Stromal tissue and nuclei were devoid of the stain. The zymogen cells in the deeper region of the mucosa did not show any detectable staining for IR-FSH. The presence of IR-FSH in gastric mucosa was also detected by radioimmunoassay. Gel chromatography of the gastric tissue extract showed a single peak of FSH immunoreactivity that coeluted with the 125I-labeled highly purified FSH preparation (NIDDK, NIH). Furthermore, the FSH in the pituitary tissue extract had a chromatographic profile similar to that of IR-FSH from gastric tissue, and 125I-FSH labeled highly purified FSH, indicating a close resemblance in their molecular sizes. These results demonstrate that IR-FSH is present in the normal human gastric mucosa. The role of this regulatory petpide in gastric tissue, if any, needs to be investigated.     

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.     

Endocrinology: Effects of recombinant human follicle stimulating hormone on cultured human granulosa cells: comparison with urinary gonadotrophins and actions in preovulatory follicles

The effects of recombinant human follicle stimulating hormone(rFSH; Org 32489) have been examined in human granulosa cellsfrom ovaries obtained from women with spontaneous menses. Inthe first series of experiments the actions of rFSH on productionof oestradiol and progesterone were compared with those of urinary-derivedgonadotrophins. Recombinant FSH induced dose-dependent increasesin production of both oestradiol and progesterone which weresimilar to the effects of ’pure‘ FSH (Metrodin®)and the International Standard IS 71/223. In further studies,the actions of rFSH on oestradiol production by individual preovulatoryfollicles were investigated; rFSH increased oestradiol accumulationfrom cells obtained from follicles before the luteinizing hormone(LH) surge. In contrast, rFSH inhibited oestradiol productionby granulosa cells derived from a follicle after the onset ofthe LH surge, whereas the gonadotrophic action of growth hormonewas maintained. Following preliminary reports of the in-vivoeffects of rFSH in women, these findings provide further validationof the efficacy of rFSH in the human ovary. The results of studiesof the preovulatory follicle illustrate the experimental importanceof the availability of recombinant preparations of pure gonadotrophins,produced by recombinant technology, in the understanding ofhuman ovarian function.     

In vitro pituitary prolactin, growth hormone and follicle stimulating hormone secretion during sexual maturation of female rats primed with melatonin

The influence of in vivo melatonin administration on in vitro pituitary follicle stimulating hormone (FSH), growth hormone (GH) and prolactin secretion, as well as the possible influence of dopamine (DA) were evaluated in prepubertal (31-day-old), pubertal (33-day-old) and adult female rats at diestrus phase of the sexual cycle. The in vitro pituitary hormone secretions were evaluated at basal rate for the first hour of incubation only, in Krebs Ringer phosphate (KRP) (I1) and after a second hour of incubation with KRP (I2) or with KRP+DA (I2 plus DA). I1PRL secretion was significantly higher in 33-day-old control and melatonin treated (MEL) rats as compared to I2 periods. However, in 31-day-old rats I1 secretion was higher than in the I2 or I2+DA periods, in MEL rats. In vitro GH secretion was significantly higher at I1 than during I2 periods in the control 31- and 33-day-old groups, but not in MEL rats. The only significant effect of DA was the elevation of GH in prepubertal MEL rats. In vitro FSH release was increased by melatonin in 31- and 33-day-old female rats. No differences in PRL, GH and FSH secretion were found in adult rats. In conclusion, the results show that melatonin effects upon in vitro pituitary gland activity are reproductive-stage-dependent modifying the secretory capacity of the lactotrop, gonadotrop and somatotrop during prepubertal and pubertal ages but not in adult rats studied at a quiescent phase of the sexual cycle.     

Pregnancies after oocyte donation in women with ovarian failure caused by an inactivating mutation in the follicle stimulating hormone receptor.

BACKGROUND: An inactivating point mutation (Ala189Val) in the FSH receptor (FSHR) causes primary ovarian failure. It has not been known if FSH action is necessary during pregnancy and childbirth. METHODS: In 1991-2001, donated oocytes were used to treat the infertility of 12 women with ovarian failure due to this mutation. RESULTS: When 30 fresh and 15 frozen-thawed embryo transfers were performed, 14 clinical and two biochemical pregnancies resulted. To date, 12 children have been born to eight women, while one pregnancy ended in miscarriage. Three women had twin pregnancies, and one woman has delivered twice. Additionally, there are three ongoing pregnancies, of which two are second pregnancies of women who previously had a normal delivery after similar treatment. In all, 10 out of the 12 women became pregnant. Two deliveries were by Caesarean section. The rate of complications was comparable with that in pregnancies resulting from oocyte donation in general. CONCLUSIONS: Achieving and undergoing a successful pregnancy is possible when FSH action is severely decreased. Oocyte donation is an effective infertility treatment for women with FSHR mutations.     

Luteinizing hormone response to gonadotrophin-releasing hormone in normal women undergoing ovulation induction with urinary or recombinant follicle stimulating hormone

Oestradiol enhances pituitary sensitivity to gonadotrophin-releasing hormone (GnRH) in normal women, while in women undergoing ovulation induction the putative factor gonadotrophin surge attenuating factor (GnSAF) attenuates the response of luteinizing hormone (LH) to GnRH. To study the relationships between oestradiol and GnSAF during ovulation induction, 15 normally ovulating women were investigated in an untreated spontaneous cycle (control, first cycle), in a cycle treated with daily i.m. injections of 225 IU urinary follicle-stimulating hormone (FSH) (Metrodin HP, uFSH cycle) and in a cycle treated with daily s.c. injections of 225 IU recombinant FSH (Gonal-F, rFSH cycle). Treatment with FSH started on cycle day 2. The women during the second and third cycle were allocated to the two treatments in an alternate way. One woman who became pregnant during the first treatment cycle (rFSH) was excluded from the study. In all cycles, an i.v. injection of 10 microg GnRH was given to the women (n = 14) daily from days 2-7 as well as from the day on which the leading follicle was 14 mm in diameter (day V) until mid-cycle (n = 7). The response of LH to GnRH at 30 min (deltaLH), representing pituitary sensitivity, was calculated. In the spontaneous (control) cycles, deltaLH values increased significantly only during the late follicular phase, i.e. from day V to mid-cycle, at which time they were correlated significantly with serum oestradiol values (r = 0.554, P < 0.01). Initially during the early follicular phase in the uFSH and the rFSH cycles, deltaLH values showed a significant decline which was not related to oestradiol (increased GnSAF bioactivity). Then, deltaLH values increased significantly on cycle day 7 and further on day v with no change thereafter up to mid- cycle. On these two days, deltaLH values were correlated significantly with serum oestradiol values (r = 0.587 and r = 0.652 respectively, P < 0.05). During the pre-ovulatory period, deltaLH values in the FSH cycles were significantly lower than in the spontaneous cycles. Significantly higher serum FSH values were achieved during treatment with uFSH than rFSH. However, serum values of oestradiol, immunoreactive inhibin, and deltaLH as well as the number of follicles > or = 12 mm in diameter did not differ significantly between the two FSH preparations. These results suggest that in women undergoing ovulation induction with FSH, oestradiol enhances pituitary sensitivity to GnRH, while GnSAF exerts antagonistic effects. The rFSH used in this study (Gonal-F) was at least as effective as the uFSH preparation (Metrodin-HP) in inducing multiple follicular maturation in normally cycling women.      

Ovarian sensitivity to follicle stimulating hormone is blunted in normo- ovulatory women with Down's syndrome

Ovarian sensitivity to follicle stimulating hormone (FSH) during the early follicular phase of the human menstrual cycle was studied in six post-menarchal patients with Down's syndrome and 12 normo-ovulatory women. Pure FSH (75 IU) was given i.v. to six controls and six Down's syndrome patients, while saline was administered to the remaining six controls. Plasma concentrations of luteinizing hormone (LH), FSH, oestradiol, testosterone and growth hormone (GH) in samples collected for a period of 26 h after the injection were assayed. In control patients FSH injection increased oestradiol stimulated area under the curve (AUC). This value was significantly higher than that found in Down's syndrome patients (P < 0.02), who exhibited an oestradiol- stimulated AUC equivalent to saline-treated controls. In Down's syndrome, GH plasma concentrations were significantly lower than in the control group (P < 0.05). These results indicate that the ovarian sensitivity to FSH in patients with Down's syndrome is blunted. Lower GH plasma concentrations found in this group may in part account for this biological feature.      

Endocrinology and Paracrinology: Interference with follicle stimulating hormone regulation of human ovarian function

This review summarizes observations on the background and potentialclinical significance of interference with follicle stimulatinghormone (FSH) regulation of human ovarian function. This interferencemay occur at the level of the pituitary by the secretion ofFSH isoforms with reduced or absent bioactivity. In addition,interference with FSH may occur in the circulation, or withinthe ovarian follicular compartment. Although the full rangeof its significance remains to be elucidated, there are distinctindications that these mechanisms may be involved in normalovarian physiology, as well as in abnormal response of the ovaryto stimulation by endogenous FSH or by exogenously-administeredgonadotrophin preparations. Moreover, recent advances in thedetermination of the structure-function relationship of FSHand FSH-receptor interaction, in combination with new developmentsin recombinant DNA technology, will allow the production ofmodified FSH-or FSH receptor-like molecules with altered bioactivity.The availability of FSH agonists and antagonists in the nearfuture should provide a challenge for clinicians to improvetreatment outcome and to find new indications for the use ofthese compounds. anti-FSH/bioactivity/FSH/follicle development/gonadotrophins ^*Presented as an invited plenary lecture during the 11th AnnualMeeting of the European Society for Human Reproduction and Embryology,Hamburg, 1995.     

Endocrinology: Evidence suggesting an additional control mechanism regulating episodic secretion of luteinzing hormone and follicle stimulating hormone in pre-pubertal children and post-menopausal women

The possible differential regulation of pulsatile follicle stimulatinghormone (FSH) and luteinizing hormone (LH) secretion in pre-pubertalchildren and in post-menopausal women was investigated. Childrenwere studied for 4 h and post-menopausal women for 6 h; bloodsamples were taken every 10 min. Post-menopausal women werestudied before and 21 days after administration of a singlei.m. dose of gonadotrophin-releasing hormone(GnRH) analogue.Eight post-menopausal women and 18 children (nine boys and ninegirls) were enrolled. The children were divided into two groups:A, at Tanner stages 0–1 (four boys and three girls); B,at Tanner stage 2–3 (five boys and six girls). PlasmaLH and FSH concentrations were determined using an immunofluorimetricassay. Time series were analysed and the specific concordance(SC) index was computed to determine the degree of concordancebetween episodes of LH and FSH secretion. While children ofgroup A had LH concentrations below the minimal detectable doseof 0.1 IU/I, group B showed measurable LH plasma concentrations(1.4 ± 0.3 IU/1, mean ± SEM). Plasma FSH concentrationswere detectable in both groups. Group A showed FSH plasma concentrationssignificantly lower than those of group B (0.75 ± 0.2and 1.95 ± 0.4 IU/1 respectively; P < 0.05), but FSHpulse frequency was higher in group A (P < 0.05). Childrenof group B showed significant concomitance of LH and FSH secretoryevents at time 0 (P < 0.05). Post-menopausal women showedboth LH and FSH plasma concentrations to be reduced after GnRHanalogue administration (P < 0.01), but only LH pulse frequency,not FSH pulse frequency, was significantly decreased (P <0.01). In these patients LH and FSH were co-secreted before(P < 0.05) but not after GnRH analogue administration. Inconclusion, this study confirms that during pre-puberty FSHis episodically secreted while LH is not. Following pubertaldevelopment, plasma LH concentrations increase and pulses ofthe two gonadotrophins are synchronized. The co-secretion ofLH and FSH is also observed in post-menopausal women, but notafter the single administration of a GnRH analogue. These datasuggest that FSH might have an additional stimulatory pathwaythat is independent of GnRH.     

Incipient ovarian failure associated with raised levels of follicle stimulating hormone and reduced levels of inhibin A in older sheep

In women there is a gradual rise in the basal level of follicle stimulating hormone (FSH) in the years prior to the menopause (pre- menopause) which is thought to be due to a relative lack of ovarian factors reflecting the number of antral follicles present in the ovaries. Experimental animal models for this phenomenon, particularly in mono-ovulatory species, have been lacking due to most animals' relatively short life span. We have available a group of experimental ewes in which the right ovary was removed and the left ovary was autotransplanted to the neck at least 10 years previously, which have been maintained in good health until an age of 12-13 years. Two experiments were conducted with these animals to determine the endocrine and follicular effects of age: a retrospective experiment in the same Finn-Merino ewes (n = 5) when the animals were 6-7 or 12-13 years of age; and a cohort experiment in old (12-13 years, n = 6) and young (2 years, n = 5) ewes of the same breed. In both retrospective and cohort experiments, the concentrations of FSH were significantly higher (P < 0.05) in older animals during the luteal phase when oestradiol secretion was low. This increase in FSH was associated with a decrease in the concentration of inhibin A (P < 0.05) in older animals in both the follicular and luteal stages of the cycle but the concentrations of oestradiol were similar between ages. Although there were significantly fewer antral follicles (P < 0.05) available for development in older ewes during the early luteal phase of the cycle, the ovulation rate was similar to that observed in younger animals (2.0+/-0 vs 2.0+/-4; P > 0.05) but the interval from luteal regression to the onset of the LH surge was longer (P < 0.05) in older animals. In conclusion, the endocrine changes associated with increasing reproductive age in sheep are therefore similar to those observed in women, suggesting that the sheep could be a useful animal model to study the effect of age on human fertility.      

Effect of follicle stimulating hormone treatment on the pituitary response to luteinizing hormone-releasing hormone in post-menopausal women

To study the role of exogenous follicle stimulating hormone(FSH) in the attenuation of luteinizing hormone (LH) responseto luteinizing hormone-releasing hormone (LHRH) during ovulationinduction in women, 10 healthy post-menopausal women were treatedwith FSH (225 IU/day) for 5 days and normal saline (2 ml/day)for another 5 days. The two regimens were given consecutivelyin a 10 day experiment. The regimen for the first 5 days wasrandomly chosen and was given to the women in an alternate way.The response of LH to an i.v. injection of 10 µg LHRHwas investigated twice on day 1 (i.e. before the onset of treatmentand 12 h later) and once on days 2, 5 and 10 of the experiment(0900 h). Basal FSH and LH values before the onset of treatmenton day 1 were similar in the five women who started with thesaline and the five who started with the FSH regimen. BasalFSH values increased significantly during treatment with FSH,while LH and oestradiol values remained unchanged throught thewhole experiment. LH increment 30 min post –LHRH did notchange significantly either during the first 24 h or duringthe whole experiment regardless of the starting regimen. Theseresults demonstrate that in post-menopausal women the responseof LH to LHRH is not affected by exogenous administration ofFSH. It is suggested that exogenous FSH does not show activitieson gonadotrophin secretion similar to those ascribed to a gonadotrophinsecretion similar to those ascribed to a gonadotrophin surgeattenuating factor.     

Endocrinology: Development of a human granulosa cell culture model with follicle stimulating hormone responsiveness

In order to study the effects of follicle-stimulating hormone(FSH) on differentiation of granulosa cells, a well-definedand validated in-vitro culture system is indispensable. In thisstudy, pooled follicular aspirates were stimulated in vitrowith FSH and luteinizing hormone (LH) for 2, 4 and 6 days, eitherimmediately after plating or after 7 days of preincubation.Cultures were assayed for progesterone and oestradiol production.Fresh cells displayed very high basal progesterone productionwhich could be stimulated with LH but not FSH. After preincubation,addition of LH and FSH resulted in dose-dependent increasesof progesterone and oestradiol. When cultured on human fibronectin-coatedwells, similar basal but higher progesterone concentrationsafter stimulation were observed. In comparison with serum-freemedia, addition of Serum-Plus^TM resulted in higher basal andstimulated progesterone concentration, possibly due to the presenceof serum factors. This study demonstrates firstly that after7 days preincubation, cultures gained responsiveness to FSHbut remained responsive to LH during 4 days of stimulation.This suggests a persisting differentiated cell population invitro. Secondly, the use of human fibronectin extracellularmatrix and serum promotes steroid production, either due tofactors promoting cell growth and function or to availabilityof steroid precursors. Therefore one has to be cautious withinterpretation of data obtained from this widely used culturesystem, employing highly differentiated cells obtained afterovarian stimulation for in-vitro fertilization for study oflocal regulation of granulosa cell function.     

Responsiveness of human male volunteers to immunization with ovine follicle stimulating hormone vaccine: results of a pilot study

A study of 140 days duration was performed to examine if human male volunteers (n = 5) respond to ovine follicle stimulating hormone (oFSH) immunization (administered adsorbed on Alugel on days 1, 20, 40 and 70) by producing antibodies capable of both binding and neutralizing bioactivity of human FSH. The kinetics of antibody production for both the immunogen (oFSH) and the cross-reactive antigen (hFSH) were essentially similar. The volunteers responded only to the first two immunizations. The boosters given on days 40 and 70 were ineffective, probably because of the presence of substantial amounts of circulating antibody to oFSH. Of the antibodies generated to oFSH, 25-45% bound hFSH with a mean binding affinity of 0.65 x 10(9) +/- 0.53 M(-1). The binding capacities at the time of high (30-80 days of immunization) and low (>110 days) titres were 346 +/- 185 and 10.5 +/- 5.8 ng hFSH/ml respectively. During the period of high titre, free serum FSH (value in normal males 1-5 ng/ml) was not monitorable. A 50 microl aliquot of the antiserum obtained from different volunteers between days 30 and 80 and on day 140 blocked binding of (125)I-labelled hFSH to its receptor by 82 +/- 9.7 and 53 +/- 12.2% respectively. The antibody produced was specific for FSH, and no significant change in the values of related glycoprotein hormones (luteinizing hormone/testosterone and thyroid stimulating hormone/thyroxine) were recorded. Seminal plasma transferrin, a marker of Sertoli cell as well as of seminiferous tubular function, showed marked reduction (30-90%) following immunization with oFSH. Considering that endogenous FSH remained neutralized for approximately one sperm cycle only (65 days), the reduction in sperm counts (30-74%) exhibited by some volunteers is encouraging. Immunization with oFSH did not result in any significant changes in haematology, serum biochemistry or hormonal profiles. There was no production of antibodies capable of interacting with non- specific tissues. It is concluded that it should be possible to obtain a sustained long-term blockade of endogenous FSH action in men by using oFSH as an immunogen. This is a prerequisite for obtaining significant reduction in the quality and quantity of spermatozoa produced, thus leading to infertility.