Frédérique Kuttenn

Molecular Pathology of the FSH receptor: new insights into FSH physiology

Manipulations of mouse genome have helped to elucidate gonadotrophin function but important differences subsist between rodent and human reproduction. Studies of patients with mutations of gonadotrophins or gonadotrophin receptors genes allow understanding their physiological effects in humans. The correlation of the clinical phenotypes of patients with in vitro studies of the mutated receptor residual function and histological and immunohistological studies of the ovarian biopsies permits to understand which stages of follicular development are under FSH control. Total FSH receptor (FSHR) inactivation causes infertility with an early block of follicular maturation remarkably associated with abundant small follicles as in prepubertal ovaries and demonstrates the absolute requirement of FSH for follicular development starting from the primary stage. Partial FSHR inactivation, characterized by normal-sized ovaries, can sustain follicular development up to the early antral stages but incremental levels of FSH stimulation seem to be required for antral follicular growth before selection. These findings contrast with the traditional view of an initial gonadotrophin-independent follicular growth prior to the preantral-early antral stages. The presence of numerous reserve follicles in the ovaries of these patients may permit a future treatment of their infertility. The study of reduced FSHbeta or FSHR activity in genetically modified male mice models and in men suggests a minor impact of the FSHR on masculine fertility. Further studies on patients with a demonstrated total FSHbeta or FSHR inactivation are required to elucidate reported differences in spermatogenesis impairment. Finally, the studies of mutations of gonadotrophins and their receptors demonstrate differences in gonadotrophin function between genetically modified rodents and humans which suggest prudence in extrapolating observations in rodents to human reproduction. Ovarian hyperstimulation syndrome (OHSS) can infrequently arise spontaneously during pregnancy, but most often it is an iatrogenic complication of ovarian stimulation treatments with ovulation drugs for in vitro fertilization. The first genetic cause of familial recurrent spontaneous OHSS was identified as a broadening specificity of the FSHR for hCG due to naturally occurring heterozygous mutations located unexpectedly in the transmembrane domain of the FSHR. Broadening specificity of a G protein-coupled receptor is extremely rare. These observations led to the identification of the etiology of this previously unexplained syndrome and permitted to conceive novel models of FSHR activation. Susceptibility to iatrogenic OHSS or its clinical severity may be associated with FSHR polymorphisms with slightly different activities in vivo as suggested by several studies. The study of larger cohorts is needed to evaluate the clinical impact of these observations in the management of patients undergoing IVF protocols.

Studies on clinical, hormonal and pathological correlations in breast fibroadenomas

This study was undertaken to determine if the observation made in vito in women with benign breast diseases of an abnormal hormonal status characterized by an unopposed ovarian estrogenic secretion was correlated in vitro with an abnormal concentration of cytosolic estradiol receptors (ER) in breast tissue. Plasma progesterone (P) measured throughout the menstrual cycle in 84 women for all the patients (11.7+ or -2.8 ng/ml) were significantly lower (P 20 fmol/mg of protein; ll cases) was also found to have the highest cell proliferation; whereas the 49 patients with the lowest ER concentration (< 7 fmol/mg of protein) showed an important stromal reaction. In 28 cases where ER binding site levels varied from 7-20 fmol the cellular density was intermediate between both groups. The presence of positive ER protein in fibroadenomas where the epithelial cellularity was most important emphasized the role of E2 in cellular multiplication and hyperplasia. The in vivo E2-P imbalance in all patients studied indicated that this was the primary cause favoring development of benign dystrophy in mammary glands.

The Application of a New Highly-Sensitive Radioimmunoassay for Plasma 21-Deoxycortisol to the Detection of Steroid-21-Hydroxylase Deficiency

21-deoxycortisol (21-DF) is a steroid of strictly adrenal origin formed by the 11-hydroxylation of 17-hydroxyprogesterone. This metabolic pathway is minor in normal subjects, in whom basal plasma concentrations range from 0·03 to 0·63 nmol/L and from 0·865 to 1·50 nmol/L after adrenocorticotropic hormone (ACTH; Synacthène Immédiat, Ciba/Geigy, France). However, this metabolic pathway becomes major in 21-hydroxylase-deficient patients: in those who have the classical form of congenital adrenal hyperplasia (CAH) basal plasma 21-DF levels can attain more than 144 nmol/L. The synthesis of two isomers, E and Z, of the 21-deoxycortisol-3-carboxymethyloxime (CMO) hapten enabled us to prepare the corresponding E and Z immunogens by coupling them to bovine serum albumin (BSA), as well as the corresponding iodinated E and Z 21-DF-3-CMO-histamine tracers. We developed a very sensitive radioimmunoassay for 21-DF in plasma by associating an anti-21-DF-3-CMO-BSA-E isomer antibody to an iodinated 21-DF histamine-Z isomer (standard curve IC 50 = 8 pg/tube). This plasma 21-DF radioimmunoassay allowed diagnosis of the classical form of CAH in untreated newborn (basal 21-DF levels greater than 144 nmol/L), as well as the late-onset form (post-ACTH 21-DF levels greater than 11 · 54 nmol/L), and also permitted detection of 21-hydroxylase-deficient heterozygotes of both forms of CAH among the general population (post-ACTH 21-DF levels between 2·02 and 9·52 nmol/L).

Evaluation of different markers of the ovarian reserve in patients presenting with premature ovarian failure

Premature ovarian failure (POF) is a heterogeneous syndrome, possibly due to mutations of genes involved in the normal development of the ovary and/or the follicles. Based essentially on animal models, these mutations are associated with various ovarian histological phenotypes, from a complete absence of to a partial follicular maturation. The aims of our work were in one hand to determine if ovarian histology, compared to pelvic ultrasonography, would be helpful either in identifying which patients display an impaired follicular growth or in the orientation of the POF etiology; on the other hand, since developing follicles up to the antral stage are reported in POF and that Anti-Müllerian hormone (AMH) might be a good indicator of follicular presence, we decided to determine whether AMH should be a better marker to determine the presence of an ovarian reserve in POF patients. To try to answer to the first question, we studied first 166 patients suffering from POF with a normal karyotype. Vaginal ultrasonography (US) was performed in 134 patients and an ovarian biopsy was obtained in 67 women. The presence of follicles suggested at US was confirmed at histology in only 56% of the patients. Ovarian histology led to the distinction of two phenotypes (a) small-sized ovaries, deprived of follicles, and (b) normal-sized ovaries with partial follicular maturation. To confirm the value of ovarian biopsies, samples from 20 normal women have been studied, confirming that ovarian biopsy at random allow reliable assessment of follicular activity. To try to answer to the second question of our work, a cross sectional study analyzing serum AMH, ovarian histology and AMH immunoexpression in 48 POF patients, was performed. Serum AMH was significantly higher in women with more than 5 follicles at ovarian histology. Ovarian AMH immunostaining revealed a normal AMH expression in POF preantral follicles but a decrease expression at the early antral stages. In conclusion, ovarian histology appears to be a reliable tool to appreciate the follicular reserve, and helpful and complementary to clinical and hormonal phenotyping in order to orient the search for various genetic causes of POF syndrome. Finally, AMH levels in POF patients could identify women with persistent follicles.

Progesterone effect on cell growth, ultrastructural aspect and estradiol receptors of normal human breast epithelial (HBE) cells in culture.

The stimulating effect of estradiol (E2) on breast cell growth is well documented. However, the actions of progesterone (P) and its derivatives remain controversial. Additional information is therefore necessary. On a culture system of normal human breast epithelial (HBE) cells, we observed an inhibitory effect on cell growth of a long-term P treatment (7 days) in the presence or absence of E2, using two methods: a daily cell count providing a histometric growth index, and [3H]-thymidine incorporation during the exponential phase of cell growth. A scanning electron microscopy study confirmed these results. Cells exhibited a proliferative appearance after E2 treatment, and returned to a quiescent appearance when P was added to E2. In both studies, P proved to be as efficient as the synthetic progestin R5020. Moreover, the immunocytochemical study of E2 receptors indicated that E2 increases its own receptor level whereas P and R5020 have the opposite effect, thus limiting the stimulatory effect of E2 on cell growth. In the HBE cell culture system and in long-term treatment, P and R5020 appear predominantly to inhibit cell growth, both in the presence and absence of E2.

17β-estradiol dehydrogenase (E2DH) activity in T47D cells

Abstract Activity of NAD-dependent 17β-hydroxysteroid dehydrogenase (E2DH), the enzyme which converts estradiol (E2) into its less active metabolite estrone (E1), has been previously characterized in normal human breast cells in culture and in benign and malignant breast tumors. E2DH activity is far greater in epithelial cells than in fibroblasts. Moreover, it is progesterone dependent in epithelial cells. It was therefore interesting to explore E2DH in the progesterone receptor (PR)-rich T47D cell line as a possible marker of hormone dependence in breast cancer cells. In T47D cells, transformation of [ 3 H]E2 to E1 is limited. The metabolism seems to be preferentially oriented in the way E1 → E2 in these cells. However, in the presence of the cofactor NAD the conversion of E2 into E1 increases. Moreover, treatment of T47D cells in culture by the progestin R5020 stimulates E2 to E1 conversion 2- to 3-fold. Stimulation of E2DH (E2 → E1) activity reflects both the presence and the operability of PR. This observation underlines the possible interest of E2DH assay in parallel to estradiol receptor and PR to evaluate hormone-dependence of breast cancer.

Selective venous catheterization in the evaluation of hyperandrogenism

Retrograde bilateral ovarian-adrenal vein catheterization was carried out in 16 patients with plasma testosterone levels exceeding 1.4 ng/ml (4.85 nmol/l). While pelvic ultrasonography and computerized axial tomographic scan failed to locate the androgen-producing ovarian tumors, catheterization led to a diagnosis of occult ovarian tumor in 5 patients, based on the observation of an abnormally-high and unilateral ovarian-peripheral vein testosterone gradient, which was subsequently confirmed histopathologically. In one case, unilateral elevation of the adrenal-peripheral vein testosterone gradient was found, complementing the ultrasonographic finding of an adrenal mass and confirming the diagnosis of a virilizing adrenal tumor. In the other 10 patients, gradient analysis ruled out an androgen-producing tumor, leading to the identification of nontumoral hyperandrogeny, such as a severe form of the polycystic ovary syndrome in the 6 premenopausal patients and of ovarian stromal and hilus cell hyperplasia in the 4 menopausal patients. In conclusion, appropriate indication of selective catheterization may considerably reduce the need for exploratory surgery and may help in selecting the adequate surgical approach.

Long‐term follow‐up of 246 hyperprolactinemic patients

Background. We wanted to evaluate the very long‐term effects of bromocriptine on prolactin (PRL) levels and pituitary tumor size in a large cohort of hyperprolactinemic patients.

Estradiol stimulates c-myc proto-oncogene expression in normal human breast epithelial cells in culture

The proto-oncogene c-myc is involved in the stimulation of cell proliferation, and its expression is known to be stimulated by estradiol (E2) in human breast cancer cell lines and various non-cancerous E2-dependent tissues. However, little information is currently available concerning its expression and regulation in normal human breast tissue. We therefore studied c-myc expression and hormone modulation in normal human breast epithelial (HBE) cells in culture, routinely obtained in our laboratory and which remain hormone-dependent. On these normal HBE cells, E2 induced a biphasic increase in c-myc mRNA level, with a first peak as early as 30 min, and a secondary increase after 2 h of treatment; this stimulation was dose-dependent, with an optimal concentration of 10 nM E2. Its primary action is probably at the transcriptional level since the half-life of c-myc mRNA measured in the presence of actinomycin D (12 +/- 3 min) was not modified by E2 treatment. In addition, E2 stimulation of c-myc mRNA does not require protein synthesis since it was not suppressed by cycloheximide treatment. Western blot studies of c-myc protein in HBE cells revealed the same biphasic pattern of stimulation, with a first peak after 60 min and a second one after 2 h of E2 treatment. In conclusion, the c-myc proto-oncogene is expressed in normal HBE cells, as in breast cancer cells. Moreover, E2 stimulates c-myc expression which, therefore, may partly mediate the growth-promoting effect of E2.

Progesterone receptors in breast fibroadenomas

Abstract Cytosol progesterone receptors (P-R) were measured in breast fibroadenomas from 88 women, and their levels were compared to the tumor epithelial cell density and estradiol receptor levels (E-R). Three groups of fibroadenoma were distinguished: type I with a high epithelial cell density (n= 18), type III (n = 46) with low epithelial cell density and extensive fibrosis, and type II with cell density intermediate between the two other groups (n = 24). Whereas E-R levels correlated well with cellular density, P-R levels were elevated in group I and absent in group III, but in contrast to E-R, the low P-R levels observed in group II could not be only explained by cellular density. Since P-R is an estrogen-dependent protein and an hormonal marker, its decrease in type II fibroadenoma might be interpreted as reflecting a rapid decrease in hormone dependence.