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Ovarian monitoring requires the determination of serum estradiol and progesterone levels. We investigated whole follicular steroidogenesis under rFSH in medically assisted procreation (MAP: 26 IVF, 24 ICSI) compared to 11 controls (IUI). Estrone, estradiol, Δ4-androstenedione, testosterone, progesterone and 17-hydroxyprogesterone were measured by immunoassay and mass spectrometry except for estrogens. At the start of a spontaneous or induced cycle, steroids levels fluctuated within normal ranges: estradiol (314–585 pmol/L), estrone (165–379 pmol/L) testosterone (1.3–1.6 nmol/L), Δ4-androstenedione (4.5–5.6 nmol/L), 17-hydroxyprogesterone (2.1–2.2 nmol/L) and progesterone (1.8–1.9 nmol/L). 17-hydroxyprogesterone, Δ 4-androstenedione and estradiol predominated. Then estradiol and oestrone levels rise, but less markedly for oestrone in IUI. In MAP, rFSH injections induce a sharp increase in estrogens associated with a rise in 17-hydroxyprogesterone and Δ4-androstenedione levels, disrupting oestrogen/androgen ratios. rFSH stimulation induces an ovarian hyperplasia and Δ4pathway which could become abnormal. Determining 17-hydroxyprogesterone and Δ4-androstenedione levels with LC-MS/MS may therefore be useful in managing recurrent MAP failures.

Summary: The effect of long term treatment with D-Trp6-LH-RH in microcapsules (GnRH-A) on pituitary gonadal axis was studied in a adolescent and adult Fox-terrier dogs. They received intramuscularly 50 μg/ GnRH-A/kg, on days 1 and 21 and every 4 weeks thereafter. Three adult dogs received 4 injections. cLH, cFSH and T levels were undetectable on day 7. Detectable then normal levels occurred 60 and 90 days respectively after the last injection on day 77. Testis thickness was respectively 22.1±0.8 mm and 16.3±0.8 mm on days 0 and 77; initial values were observed 90 days later. Spermatozoa disappeared from the ejaculate on day 21 in 2 dogs; reappearance and complete recovery were observed on days 161–175 and 252 respectively. Histological findings showed on day 91 atrophic lesions of testis and prostate and spermatogonia were present in all seminiferous tubules. After recovery a normal histological appearance was noticed. Three adolescent 29 weeks old dogs received 14 injections, the last one on day 357. cLH, cFSH and T levels were undetectable only from day 105. Testis thickness were respectively 15.8±0.7 mm, 18.1±0.7 mm, 12.5±0.3 mm and 21.4±0.7 mm on days 0, 21, 357 and 490. Initially, no spermatozoa were present in the ejaculates, they appeared in 2 dogs after 2 months for 20 to 40 days then disappeared until day 449. Normal semen caracteristics were observed in all three dogs on day 581. Histological findings on day 371 were comparable to those observed in the adult dogs. This study demonstrates that longterm treatment with D-Trp6-LH-RH in microcapsules leads to a reversible inhibition of spermatogenesis in dogs. The delayed response in adolescent dogs might be due to a transient resistance to therapy related to. Zusammenfassung: In einer experimentellen Studie wird an heranwachsenden und erwachsenen Fox-Terriern der Einflus einer Langzeit-Therapie mit D-Trp6-LH-RH in Mikrokapselform (GnRH-A) auf die Hypophysen-Gonaden-Achse untersucht. Die Tiere erhielten am 1. und 21. Tag, sowie alle 4 Wochen spater jeweils 50 μg/GnRH-A/kg i.m. Drei erwachsene Hunde erhielten 4 Injektionen. cLH, cFSH und Testosteron waren am 7. Tag nicht zu bestimmen. Die Normalwerte waren dagegen bestimmbar nach 60 bzw. 90 Tagen, wenn die letzte Inj. am 77. Tage gegeben war. Die Hodendicke betrug 22, 1±0,8 mm und 16.3± 0,8 mm an den Tagen 0 und 77; die Anfangswerte wurden 90 Tage spater bestimmt. Die Spermatozoen verschwanden am 21. Tag aus den Ejakulat bei 2 Hunden; sie kehrten zuruck an den Tagen 161–175 und 252. Die histologischen Befunde zeigten am 91. Tage eine Atrophie des Hodens und der Prostata, die Spermatogonien waren uberall nachweisbar. Nach einer Erholungsphase waren normale histologische Verhaltnisse gegeben. Drei heranwachsende Hunde im Alter von 29 Wochen erhielten 14 Injektionen, die letzte Inj. am 357. Tag. cLH, cFSH und Testosteron waren nicht zu bestimmen mit Ausnahme des 105. Tages. Die Hodendicke betrug 15,8±0,7 mm, 18.1±0,7 mm, 12,5±0,3 mm und 21,4±0,7 mm an dem Tage 0, 21, 357 und 490. Anfangs waren keine Spermatozoen im Ejakulat nachzuweisen; bei zwei Tieren erschienen sie nach 2 Monaten fur 20–40 Tage und verschwanden dann wieder bis zum 449. Tag. Die normalen Spermacharakteristika wurden bei allen drei Hunden am 581. Tage beobachtet. Die hostologischen Resultate waren am 371. Tage mit denen zu vergleichen, die bei erwachsenen Hunden erhoben waren. Diese Untersuchung belegt, das eine Langzeitbehandlung mit D-Trp6-LH-RH in Mikrokapselform zu einer reversiblen Hemmung der Spermatogenese bei Hunden fuhrt. Die verzogerte Reaktion bei heranwachsenden Hunden durfte auf eine vorubergehende Resistenz gegenuber der Therapie zuruckgehen.

Treatment with small doses of subcutaneous insulin is being investigated as a possible approach to prevent type 1 diabetes in humans. The mechanism of prophylactic insulin therapy could involve the inhibition of beta-cell secretory activity and/or the initiation of an active immunoregulatory process. To evaluate the pure metabolic effect of exogenous insulin, the present study assessed whether daily subcutaneous administration of ultralente insulin alters beta-cell function in normal adults. Fourteen healthy adults were randomized to receive 0.2 U/kg x d ultralente insulin (Ultratard; Novo Nordisk, Bagsvaerd, Denmark) or placebo subcutaneously once daily for 30 days. Plasma glucose, C-peptide, and insulin concentrations were measured in the fasting state and 1 hour after a standardized breakfast, during treatment and during a recovery period of 10 days. Insulin administration induced a 15% to 40% decrease of fasting plasma C-peptide. In contrast, postbreakfast plasma C-peptide increased by 40% to 90% in subjects receiving insulin. Fasting and postbreakfast C-peptide concentrations were significantly different between groups during the injection period after adjustment for baseline concentrations (P < .05, ANOVA with repeated measures). These alterations disappeared 3 days after cessation of insulin treatment. The present regimen of exogenous insulin alters endogenous insulin secretion in normal subjects. Instead of the expected beta-cell rest, the effect appeared to be dual, with insulin secretion decreasing in the basal state and increasing after meals.

The quinoline-3-carboxamide, linomide, protects non-obese diabetic mice from diabetes. The effects of linomide on insulin needs and beta cell function were studied in recent juvenile Type I diabetes in a double-blind trial. Patients with recent onset diabetes were randomly assigned to treatment with a fixed dose of 2.5 mg linomide (42 patients) or placebo (21 patients) for 1 year, in addition to insulin and diet. Glycated haemoglobin was 10–15 % lower at 9 months (p = 0.003) and 12 months (p < 0.05) in the linomide group. The insulin dose was 32–40 % smaller in the linomide group at 3 (p < 0.03), 6 (p < 0.02), 9 (p < 0.001) and 12 months (p = 0.01). Insulin doses correlated negatively with C peptide values (p = 0.001–0.002). The trend for higher C peptide values in the linomide group did not reach significance. In a post hoc subgroup analysis performed in 40 patients (25 from the linomide group and 15 from the placebo group) who still had detectable residual beta cell function at entry, linomide was associated with 45–59 % higher C peptide value at 6 months (p < 0.05), 9 months (p < 0.05) and 12 months (p < 0.05). The main adverse effects of linomide were mild transitory anaemia (45 vs 10 % in the linomide and placebo groups), thrombocytopenia (24 vs 10 %), and mild joint discomfort (45 vs 5 %) with no clinical signs. In conclusion, low-dose linomide reduced the insulin needs in patients with juvenile Type I diabetes of recent onset and improved beta cell function in patients who still had detectable beta cell function at entry. These results support further clinical and experimental studies to define the effects of linomide in Type I diabetes provided the safety of linomide is reliably established. [Diabetologia (1998) 41: 1040–1046]

Objectif Le syndrome de Turner (ST) est caracterise par l’absence de la totalite ou d’une partie d’un chromosome X. Son incidence est de 1/2500 naissances de sujets a phenotype feminin, 50 % sont des monosomies X, 25 % des mosaiques, les autres des duplications ou deletions partielles. La folliculogenese ovarienne est absente ou tres faible, mais une puberte spontanee est parfois possible. L’AMH serique est un marqueur fiable de la presence de follicules et notre but a ete de comparer l’efficacite de deux dosages, classique et ultrasensible (pico-AMH). Materiel et methodes Chez 39 patientes (11–25 ans), sous-ensemble d’une vaste etude sur la cryoconservation ovarienne, sept monosomies X0 (GI), 12 mosaiques X0/XX (GII), 20 autres mosaiques (GIII) ont ete dosees par les reactifs AnshLabs : inhibineB (limite de detection LD 10 ng/mL), AMH (LD 0,15 pmol/L) et pico-AMH (LD 0,028 pmol/L). Resultats Comme attendu, inhibineB et AMH sont indetectables en GI (sauf 1 cas) mais pico-AMH est detectable dans 4/7 cas. En GII, l’inhibibineB est indetectable dans 6/12 cas, AMH dans 5, et pico-AMH dans 3 seulement. En GIII, l’inhibineB est indetectable dans 13/20, AMH dans 11 cas et pico-AMH dans 9 cas seulement. Conclusion Chez les sujets turneriens âges de 11 ans ou plus, le nouveau dosage ultrasensible de l’AMH detecte une activite secretoire folliculaire dans un nombre de cas superieur au dosage classique : 24 vs 17. La confrontation a l’histologie demontre que les valeurs observees sont bien correlees, comme espere, au nombre de follicules etabli par biopsie.

The neonatal-midinfancy surge in pulsatile gonadotropin secretion is attributable to an increase in GnRH pulse amplitude and is associated with a rapid expansion of Leydig and Sertoli cell populations with concomitant surges in testosterone, inhibin, and anti-Mullerian hormone production as well as an increase in testicular volume. Boys with congenital hypogonadotropic hypogonadism (HH) do not activate these processes. A potential cause for azoospermia and infertility in adult life is deficient proliferation of immature Sertoli cells before and during puberty due to the absence of FSH.The objective of the study was to investigate whether early postnatal continuous sc infusion of gonadotropins could mimic the physiological growth of testes and to evaluate responses of the Leydig and Sertoli cells to early gonadotropin replacement.Two neonates (P1 with hypotuitarism and P2 with HH) with micropenis and microorchidism were treated for 6 months with high doses of recombinant LH and FSH (a gift of Luveris and Gonal-F from Serono, Lyon, France) delivered sc with an insulin pump.Gonadotropin continuous sc infusion increased mean serum LH and FSH to normal or supranormal levels. Mean testosterone increased from undetectable levels to 7.6 and 5.2 nmol/liter, respectively, in P1 and P2. Inhibin B and anti-Müllerian hormone increased to normal levels. Mean testicular volume increased from 0.45 to 0.57 ml at birth to 2.10 ml at 7 months. Stretched penile length increased from 8 to 30 mm (P1) and 12 to 48 mm (P2).The present regimen induced physiological postnatal testes growth and high-normal activation of Leydig and Sertoli cells.

BACKGROUND: In normo-ovulatory infertile women undergoing mild ovarian stimulation out of IVF, FSH stimulation regimen must be carefully adjusted to control the number of recruited follicles and to prevent multiple pregnancies. The aim of this prospective study was to assess the effect of the timing of FSH administration (fixed dose and duration) on the number of large follicles. METHODS: Women were prospectively randomized by means of sealed envelopes to receive daily 112.5 IU recombinant FSH (rFSH), either from cycle day (CD) 2-6 (Group A) or from CD 7-11 (Group B). Hormonal measurements and follicular ultrasound assessments were performed on CD 2, 7 and 12. RESULTS: On CD 12, the development rate of exactly two follicles >or=14 mm in diameter was significantly lower in Group A than in Group B (4% of women versus 42%, P = 0.002). Although the pattern of serum estradiol (E(2)) concentrations in Group A displayed a plateau from CD 7, the cancellation rate for overstimulation (more than three follicles >or=14 mm in diameter) was significantly increased (P = 0.009). CONCLUSIONS: Preventing the closure of the FSH window by mid to late follicular phase FSH administration better fulfils the objective of obtaining a limited number of large follicles than surpassing the FSH threshold by an early administration.

Since 1981, GnRH agonist administration has been the treatment of choice for central precocious puberty. Continuous administration of the agonist, instead of permanently stimulating gonadotropin secretion, deeply suppresses LH and FSH levels and induces a marked inhibition of gonadal activity and regression of clinical symptoms. This inhibitory effect is due both to specific kinetic parameters relative to natural GnRH, and to marked alterations of the biosynthetic pathways of gonadotropin subunits. The half disappearance time of infused agonists is 3-10 fold that of natural GnRH. This means that the residence time of GnRH agonists is significantly longer than that of GnRH. The resistance of agonist to enzymatic degradation, mainly due to the substitution of a hydrophobic D-amino acid for glycine 6, is one of the factors involved in the increased availability of GnRH superagonists. The paradoxical effects of GnRH superagonists are still incompletely understood. In children long-term treated with depot formulations of triptorelin or leuprorelin, alpha-subunit secretion is markedly increased, and remains sensitive to exogenous GnRH, which demonstrates that the gonadotrophs are not totally desensitized. Despite the sustained stimulation of a-subunit secretion, no deleterious side effects, either during therapy or during post-therapy follow-up, have been reported in children treated with GnRH agonists. It should be noted that alpha-subunit responsiveness to exogenous GnRH decreases progressively after several years of treatment, although it is never completely abolished. On the other hand, LH beta-subunit secretion is suppressed as evidenced by radioimmunoassay of LH beta-subunit in serum chromatographic fractions from children treated with triptorelin. This differential pattern of secretion parallels that of mRNA levels in rat pituitary after in vivo exposure to triptorelin. Both pharmacodynamic and pharmacokinetic data can help diagnose the situations of resistance or escape. The lack of clinical effect of GnRH in the treatment of precocious puberty can be due to true resistance, or to an inappropriate injection schedule, or to abnormal metabolism. Measurement of serum alpha-subunit level, and, if needed, of serum agonist level, generally provides the answer.

Treatment of prostatic cancer with GnRH agonist is a medical alternative to surgical castration, although hyperstimulation of the tumor can occur. We describe an unusual unwanted effect of such a treatment which unmasked a clinically silent gonadotroph adenoma. A 62-year-old man developed after the first injection of leuprorelin-depot a sudden intracranial hypertension, which was related to apoplexy of an unknown pituitary adenoma. Its gonadotroph origin was recognized after surgery by immunocytochemistry. Retrospectively, the tumor was shown to secrete in vivo both FSH and LH when on therapy with the agonist, demonstrating the lack of desensitization. Testosterone levels were also markedly and sustainly high when on therapy, a particularly unwanted effect in prostatic cancer. As gonadotroph adenomas occur in men in the same age group as prostatic cancer, the question is raised whether hormonal testing and pituitary imaging should be performed before starting a therapy with GnRH agonist in men.

Long term administration of GnRH agonists is currently considered as the treatment of choice for precocious puberty. For the first time, we have at our disposal an efficacious and specific treatment of the disorder responsible for the syndrome called precocious puberty. Whether of idiopathic or neurogenic origin, true precocious puberty is primarily a hypothalamo-pituitary disorder. It is at this location that the GnRH agonists exert their effect. Knobil’s experiments in monkeys [1] provided a rationale for agonist treatment by showing that continuous infusion of GnRH caused suppression of gonadotropin release. Our understanding of the mechanism of action of GnRH agonists is however incomplete and many questions arise when the gonadotropin secretory patterns during agonist treatment are considered.

Thirty cryptorchid boys with pre-scrotal testes were divided in two groups: Group 1 (3 bil/12 unil)received hCG (750U/m2/dose)+hMG (100U/m2/dose) 2 times weekly, during 6 weeks; Group II (5 bil/10 unil) received only hCG. Surgical correction was necessary in 28 cases, 22 of wich had a testicular biopsy and subsequent hystomorphological analysis. Results were obtained in basal condition and at 3rd. and 6th. weeks of treatment. Peak values are showed: T=topic C=cryptorch. Sperm=spermatogonia/50 tubes At=tubular area. LH(mUI/ml) FSH(mUI/ml) Testosterone (ng/dl) inhibin (UI/L). There was no advantage in hMG association. The hormonal level fell between 3rd 6th weeks (down-regulation). There was no correlation between inhibin levels and testosterone, spermatogonia or tubular area. Tubular area and number of spermatogonias were decreased on the affected side in relation to the topic testes and in bilateral cases in relation to the normal patterns.

To investigate the efficacy of the different routes of luteinizing hormone releasing hormone (LHRH) administration upon pituitary responsiveness, we compared plasma LHRH concentrations and pituitary LH responses in four patients with hypothalamic amenorrhea treated with pulsatile LHRH. A portable computerized infusion pump delivered sc or iv LHRH pulses of 5, 10 or 20 micrograms every 90 min. Comparison of the two modes of LHRH delivery was performed using radioimmunoassay of exogenous LHRH and studying its pharmacokinetics for a 3 pulses period. With 10 micrograms of LHRH given iv, plasma LHRH levels increased between 700 and 1000 pg/ml within 3 min and returned to basal levels in 30 min. When given sc (10 micrograms), plasma LHRH levels peaked between 80 and 100 pg/ml in 15 min and returned to basal levels 60 min later. In one patient treated with 5 micrograms per pulse iv or sc, plasma LHRH increased to 380 and 60 pg/ml respectively. In all patients, computerized analysis of LH pulses was performed during sc and iv LHRH administration. LH pulsatile release displayed a similar rhythm period with both routes. However, for the same dose of LHRH (10 micrograms), the adjusted mean of LH plasma levels was lower with the sc route. In conclusions, the pharmacokinetics of LHRH administered sc or iv displayed a similar pattern but, with equivalent doses, higher plasma LHRH levels are attained with the iv route. Concomitantly, the mean LH levels were also greater after iv administration. Ovulation can be successfully induced by both pulsatile iv and sc LHRH therapy. However, with the sc route, a higher dose of LHRH should be used to prevent a delay of ovulation or a luteal deficiency.

hCG is known to be produced by trophoblastic cells as well as by many normal tissues and malignancies. hCG is considered as a reliable marker for gonadal and extra gonadal choriocarcinomas; moreover it has been evidenced by immunohistochemical techniques in other malignancies. In this study, hCG and/or its alpha and beta subunits were detected in 73 males. Intact hCG was detected in 48 patients with gonadal tumors (n = 37) or extragonadal malignancies (n = 11), with or without free alpha or beta subunits. In the other 25 patients, native hCG was not detected but there were significant amounts of free beta subunit in 8 germ cells tumors and 17 extragonadal tumors. The production of free beta chains is correlated with a severe evolution in extragonadal tumors. hCG should be considered as a good marker for many tumors, but it is absolutely necessary to evaluate intact hCG as well as free beta chains.

DIFFERENTIAL EFFECT OF D-TRP-6-LH-RH IN MICROCAPSULES (LRH-M) UPON LH AND LHα RELEASE DURING LONG TERM TREATMENT OF PRECOCIOUS PUBERTY (PP)

Gnα is secreted in synchronism with LH pulses and is responsive to LH-RH administration. The changes of the Gnα response to LH-RH test were investigated throughout puberty in boys and girls. Gnα was measured by RIA, the assay specificity being assessed by chromatography on Sephadex G100. Basal and peak responses to LH-RH (±sem, ng/ml) were respectively, in prepubertal boys (n=4) = 0.54 ± 0.22 and 3.4± 0.19, in pubertal boys (stage 2-3, n=4) : 0.88 ± 0.21 and 5.68 ±0.71, in prepubertal girls (n=4) : 0.30±0.15 and 4.30 ± 1.67, in pubertal girls (stage 2-3, n-4) = 0.44 ± 0.29 and 6.50 ± 1.96, in follicular phase (n=4) 1.40 ±0.39 and 7.40 ± 1.18. Peak values were significantly higher in pubertal than in prepubertal subjects. In 13 girls with precocious puberty basal and peak values were respectively 0.77 ± 0.21 and 12.1 ± 1.5 before treatment, 2.6 ± 0.85 and 7.86 ± 1.19 after 6 months of therapy with D-TRP-6-LH-RH (Deca-peptyl microcapsules). In adolescents with hypopituitarism (n=4), Gnα peak after LH-RH was only 0.28 + 0.18, in contrast with the high response in Klinefelter syndrome (n=4, 10,18 ± 2,71) and in Turner syndrome (n=4, 32.7 ± 6.33). This study confirm the parallelism of Gnα and LH secretions in various situations except in subject treated with a LH-RH agonist.

The release of the free TSH α-subunit (F-TSH-A) during TRH stimulation (200 μg IV/m2) was investigated in 8 prepubertal girls (4-9 years of age, group I), 8 girls with precocious puberty (4-8 years of age, pubertal stages 2-3, group II) and 12 menstruating women (group III). F-TSH-A was measured by a polyclonal RIA. Results were expressed as mean ± SEM (ng 1°-IRP-hCGα/ml). Basal values in group I, II, III were respectively 0.14 ± 0.02, 0.33 ± 0.08 (p = 0.02) and 0.47 ± 0.05 (not different from II). TRH peak values were respectively 0.59 ± 0.12, 0.75 ± 0.08, and 0.71 ± 0.06 (differences not significant). The increment between basal and peak values was significantly higher (p < 0.02) in group I (0.45 ± 0.07) than in group III (0.24 ± 0.03). Girls from group II were treated by IM injections every 4 weeks of the LH-RH agonist triptorelin (Decapeptyl microcapsules, 60 μg/kg body weight). After 6 months basal and peak levels increased respectively to 1.1 ± 0.12 and 2.0 ± 0.20 (p < 0.001 relative to pretreatment values). The increment (0.84 ± 0.11) was significantly higher than in groups I, untreated II and III. These data suggest that : 1-ovarian secretion might exert a suppressive effect on the release of F-TSH-A; 2-long term LH-RH agonist administration might increase α-subunit secretion not only by the gonadotroph cells, but also by the thyreotroph cells, provided a direct effect of TRH on the gonadotrophs can be precluded.

Periodic intramuscular injection of LRH-M has been shown to suppress pituitary-gonadal axis in children with precocious puberty (Roger et al. ESPE 1984). In order to assess the effects of LRH-M on spermatogenesis, 50 μg/kg were given to Fox dogs on days 1 and 21, then every 28 days. In the adult dogs treated for 15 weeks spermatogenesis was inhibited during therapy and recovered 90 days after the last injection. A one-year treatment was started in 3 adolescent dogs just before completion of puberty. LH level (ng/ml, ±sem) decreased from 4.6 ± 0.6 to 1.2 ± 0.1 on day 21 (p