Jérôme Dulon

Phenotyping and genetic studies of 357 consecutive patients presenting with premature ovarian failure

OBJECTIVE Premature ovarian failure (POF) encompasses a heterogeneous spectrum of conditions, with phenotypic variability among patients. The etiology of POF remains unknown in most cases. We performed a global phenotyping of POF women with the aim of better orienting attempts at an etiological diagnosis. DESIGN AND METHODS We performed a mixed retrospective and prospective study of clinical, biological, histological, morphological, and genetic data relating to 357 consecutive POF patients between 1997 and 2008. The study was conducted at a reproductive endocrinology referral center. RESULTS Seventy-six percent of the patients presented with normal puberty and secondary amenorrhea. Family history was present in 14% of the patients, clinical and/or biological autoimmunity in 14.3%. Fifty-six women had a fluctuating form of POF. The presence of follicles was suggested at ultrasonography in 50% of the patients, and observed in 29% at histology; the negative predictive value of the presence of follicles at ultrasonography was 77%. Bone mineral density alterations were found in 58% of the women. Eight patients had X chromosomal abnormalities other than Turners syndrome, eight other patients evidenced FMR1 pre-mutation. Two other patients had autoimmune polyendocrine syndrome type 2 and 1. CONCLUSION A genetic cause of POF was identified in 25 patients, i.e. 7% of the whole cohort. POF etiology remains most often undiscovered. Novel strategies of POF phenotyping are in such content mandatory to improve the rate of POF patients for whom etiology is identified.

Classical Forms of Congenital Adrenal Hyperplasia due to 21-Hydroxylase Deficiency in Adults

During childhood, the main aims of the medical treatment of congenital adrenal hyperplasia (CAH) secondary to 21-hydroxylase are to prevent salt loss and virilization and to attain normal stature and normal puberty. As such, there is a narrow therapeutic window through which the intended results can be achieved. In adulthood, the clinical management has received little attention, but recent studies have shown the relevance of long-term follow-up of these patients. The aims here are to review the multiple clinical, hormonal and metabolic abnormalities that could be found in adult CAH patients as such a decrease in bone mineral density, overweight and disturbed reproductive functions. In women with classic CAH, a low fertility rate is reported, and is probably the consequence of multiple factors including neuroendocrine and hormonal factors, feminizing surgery, and psychological factors. Men with CAH may present hypogonadism either through the effect of adrenal rests or from suppression of gonadotropins resulting in infertility. Therefore a multidisciplinary team with knowledge of CAH should carefully follow up these patients, from childhood through to adulthood, to avoid these complications and to ensure treatment compliance and tight control of the adrenal androgens.

Bone health should be an important concern in the care of patients affected by 21 hydroxylase deficiency.

Osteoporosis has been an understandable concern for children and adult patients with congenital adrenal hyperplasia (CAH) who may receive or have received supraphysiological doses of glucocorticoids. Some previous reports on bone mineral density (BMD) in adult CAH patients showed no significant differences in BMD between patients with CAH and controls, but others have found lower BMD in CAH patients. In reports documenting the BMD reduction, this outcome has been attributed to an accumulated effect of prolonged exposure to excess glucocorticoids during infancy and childhood. We recently conducted a trial to establish the role of the total cumulative glucocorticoid dose on BMD. We established for the first time that there was a negative relationship between total cumulative glucocorticoid dose and lumbar and femoral BMD. Women might benefit from the preserving effect of estrogens compared to men. BMI (Body Mass Index) also appeared to protect patients from bone loss. In light of this, physicians should bear in mind the potential consequences of glucocorticoids on bone and therefore adjust the treatment and improve clinical and biological surveillance from infancy. Furthermore, preventive measures against corticosteroid-induced osteoporosis should be discussed right from the beginning of glucocorticoid therapy.

NR5A1 (SF-1) Mutations Are Not a Major Cause of Primary Ovarian Insufficiency

CONTEXT Primary ovarian insufficiency (POI) is a disorder affecting approximately 1% of women under the age of 40 years. NR5A1 (SF-1) mutations have been recently reported in association with POI. OBJECTIVE Our objective was to evaluate the frequency and functional impact of NR5A1 variants in POI. PATIENTS AND METHODS One hundred eighty patients diagnosed with idiopathic POI were screened for NR5A1 mutations and functional analysis was performed for the identified variants. The DNA-binding capacity of the variants was evaluated by means of EMSA, while their transcriptional activity was assessed using luciferase reporter assays. RESULTS Sequencing the NR5A1 gene revealed 4 missense variants in 3 patients. These patients were aged 20, 25, and 33 years at diagnosis and presented with secondary amenorrhea. None of them presented a syndromic form, although 2 had a familial history of POI. The functional analysis carried out for these missense variants showed no significant difference in DNA binding capacity or in transcriptional activity compared to wild-type NR5A1. CONCLUSIONS Our study in a large cohort of patients with POI showed the prevalence of NR5A1 mutations to be low (1.6%, upper 95% confidence interval 3.5%). Moreover, no functional impact was observed. Overall, in contrast with the initial report, our results exclude NR5A1 mutations as a major genetic cause of POI.

Determining clinical and biological indicators for health outcomes in adult patients with childhood onset of congenital adrenal hyperplasia.

AIM Adverse outcomes in adult congenital adrenal hyperplasia (CAH) patients are frequent. The determinants of them have not yet been established. OBJECTIVE To establish the prevalence of adverse outcomes and to find determining factors for each of them. DESIGN, PATIENTS, AND METHODS Cross-sectional monocentric study of 104 patients with childhood onset of CAH (71 women, 33 men). Analysis established first the determinants of clinical, hormonal, genetic variables and second a composite criterion for some of the outcomes and determinants. RESULTS BMI was above 25 kg/m(2) in 44% of the cohort, adrenal hyperplasia and/or nodules were present in 45% of the patients, and irregular menstrual cycles and hyperandrogenism were found in 50 and 35% of the women respectively. In univariate analysis, the determinants of these outcomes were all linked to disease control, especially 17-hydroxyprogesterone (17OHP) and androstenedione concentrations. Low weight was a determinant of abnormal bone mineral density (BMD) (60% of the cohort). Multivariate analysis confirmed these data. A classic form (CF) of CAH was a determinant of testicular adrenal rest tumors (TARTs) (36% of the men). Total cumulative glucocorticoid dose was a determinant of BMI and TART, whereas fludrocortisone dose was a determinant of TART (P=0.03). In men, the composite criterion was associated with androstenedione concentration and CF. In women, the composite criterion was associated with total testosterone concentration. CONCLUSION The present study confirms the high prevalence of adverse outcomes in CAH patients. These are, most often, related to disease control. The impaired health status of adults with CAH could therefore be improved through the modification of treatment.

A common African variant of human connexin 37 is associated with Caucasian primary ovarian insufficiency and has a deleterious effect in vitro

Folliculogenesis requires communication between granulosa cells and oocytes, mediated by connexin-based gap junctions. Connexin 37 (Cx37)-deficient female mice are infertile. The present study assessed Cx37 deficiency in patients with primary ovarian insufficiency (POI). A candidate gene study was performed in patients and controls from the National Genotyping Center (Evry, France) including 58 Caucasian patients with idiopathic isolated POI and 142 Caucasian controls. Direct genomic sequencing of the coding regions of the GJA4 gene (encoding Cx37) was performed with the aim to identify a deleterious variant associated with POI and absent in ethnically matched controls. A single Cx37 variant absent in the control population was identified, namely a c.946G>A heterozygous substitution leading to a p.Gly316Ser variant that was present in two POI patients. This variant was absent in all Caucasian controls from various databases, and has been observed exclusively in African populations. This variant was identified to have a dominant negative effect in HeLa cells in vitro to alter connexon function (by 67.2±7.17%), as determined by Gap-fluorescence recovery after photobleaching. The alteration principally resulted from a decrease of cell surface connexons due to altered trafficking (by 47.73±8.59%). In marked contrast to this observation, a p.Pro258Ser variant frequent in all ethnic populations in databases had no functional effect in vitro. In conclusion, the present study reported on a Cx37 variant in two Caucasian POI patients, which was absent in control Caucasian populations, and which had a deleterious effect in vitro. It is therefore suggested that in the genetic context of the Caucasian population, this variant may contribute to POI.

Poor Compliance to Hormone Therapy and Decreased Bone Mineral Density in Women with Premature Ovarian Insufficiency

Premature ovarian insufficiency leads to through infertility and estrogen deficiency. Optimal management encompasses estrogen replacement therapy. Long-term outcome of women with POI is not known. We design a study to evaluate the medical care, hormone replacement therapy compliance and bone mineral density (BMD) in POI women with at least a five-year follow-up after the first evaluation. One hundred and sixty-two patients (37.3±8.0 years) were evaluated (follow-up 7.9±2.8 years). Sixty-nine patients (42.6%) had stopped their hormone replacement therapy (HRT) for at least one year during the follow up period. BMD determination at initial evaluation and at follow-up visit was completed in 92 patients. At first evaluation, 28 patients (30%) had osteopenia and 7 (8%) had osteoporosis. At follow up, 31 women (34%) had BMD impairment with osteopenia in 61% and osteoporosis in 5%. In univariate analysis and multivariate analysis, there was a significant loss of femoral BMD in women who had stopped their HRT for over a year. In conclusion, this first study concerning long-term follow-up of POI patients shows the poor compliance to their HRT, despite its importance in the prevention of bone demineralization. This study reinforces the need for follow up and specific care for POI women.

Fertility and Pregnancy in Patients With 21-Hydroxylase Deficiency

21-Hydroxylase deficiency (21OHD) is the most common cause of congenital adrenal hyperplasia. Sexuality and fertility in women with classic 21OHD is impaired, due to several issues such as disrupted gonadotropic axis due to androgen and progesterone overproduction, and mechanical, psychological factors related to genital surgery. Fertility and fecundity in these women gets better over the years. Subfertility seems contrariwise to be relative in nonclassic CAH women. Before pregnancy, genotyping the partner and genetic counseling is mandatory. The use of dexamethasone during pregnancy in order to prevent the virilization of the young affected CAH girl remains a matter of debate.