All-trans-retinoic acid regulates aquaporin-3 expression and related cellular membrane permeability in the human amniotic environment.

INTRODUCTION: The aquaporins (AQP1, 3, 8, 9 and 11) are known to be expressed, and involved in the transport of water and small molecules through fetal membranes. To exert these crucial functions, these AQPs have to be finely regulated. All-trans-retinoic acid (atRA) was previously found to regulate some genes in this environment, raising the question of whether these AQPs were regulated by atRA. METHODS: Explants, and primary and established amniotic cells were cultured to determine which AQP were transcriptionally modified by atRA, using the qRT-PCR strategy. Immunohistochemistry and glycerol uptake tests were used to determine the impact of atRA on AQP protein expression and function. Specific agonists of retinoic acid receptors were used to identify the molecular mechanisms of AQP promoter activation. A classical gene AQP promoter study was also used to identify DR5 retinoic acid receptor elements (RAREs). RESULTS: Beyond these AQPs, only one specific atRA-dependent increase in AQP3 transcripts and proteins level was established in amnion (not in chorion) and in related primary and established cells. We found three DR5-RAREs essential for inducing this transcriptional AQP3 through RARα. This transactivation of the AQP3 coding gene was functionally related to an increase of AQP3 permeability tests by a glycerol uptake assay. DISCUSSION: Our data support an atRA regulatory model of AQP3 expression leading to an increased cellular permeability in the epithelial amniotic environment. We cast new light on AF regulation in healthy pregnancy, and advance new hypotheses for obstetrical complications linked to impairment of the retinoic signaling pathway.

[Rupture of membranes: pathophysiology, diagnosis, consequences and management]. / Rupture des membranes : physiopathologie, diagnostic, conséquences et prise en charge.

Rupture of membranes (ROM) depends on mechanical stretch, extracellular matrix components imbalance and increased apoptosis. It occurs in 2 to 3% of all pregnancies before 37 weeks' gestation (WG) and in up to 10% at term. Main consequences are labor induction and risk of maternal-fetal infection. ROM is associated with one third of preterm births and about 20% of perinatal mortality. This review deals with recent knowledge concerning ROM including diagnosis and management. In many cases, ROM is easily identified by clinical examination. In other cases, the use of vaginal pH appears to be less efficient than the use of immunochromatographic strips based on IGFBP-1 or PAMG-1 detection. Before 34WG, conservative management consists in in utero transfer, antibioprophylaxis and corticosteroids. After 37WG, delivery is the most appropriate option. Between 34 and 37WG, recent studies demonstrate that induction of labour does not improve pregnancy outcomes. Therefore, expectant management can be the first option between 34 and 37WG when no active infection is suspected especially in case of unfavourable cervix.

[Two immunoassays for antimullerian hormone measurement: analytical and clinical performances]. / Deux dosages de l'hormone antimüllérienne : performances analytiques et cliniques.

Today, serum antimullerian hormone (AMH) is considered as an interesting marker of fertility potential in women to determine follicular status and in men to evaluate testicular function. We study analytical and clinical characteristics of two AMH commercialized immunoassays: Immunotech and DSL methods. The detection limits (close to 0.13 ng/mL), functional sensitivities (close to 0.30 ng/mL) are equivalent, and imprecision results are acceptable for entirely manual assays. The Immunotech method is linear within the calibration range (up to 21 ng/mL) and the DSL method presents a lack of linearity making it accurate only up to 11 ng/mL (and not up to 14 ng/mL as it is indicated by the manufacturer). The two methods allow to measure human AMH, don't cross react with TGF-beta superfamily proteins and the DSL immunoassay recognize mouse (25%), rat (68%) and calf (100%) AMH. The comparison between the two methods (from 0.3 to 200 ng/mL) shows a good correlation (r = 0.979) with not statistically different results (p = 0.31). From a clinical point of view, the two methods allow the evaluation of follicular status in normo-ovulatory women and in women with polycystic ovary syndrome. Results are in agreement with studies showing that AMH serum concentration is strongly correlated with the number of antral follicles. In conclusion, the Immunotech method seems to be more efficient than the DSL method even if the two methods are suitable for clinical applications needing AMH measurements.

Components of the anti-Müllerian hormone signaling pathway in gonads.

Anti-Müllerian hormone (AMH) is a member of the Transforming Growth Factor-beta (TGF-beta) family implicated in the regression of Müllerian ducts in male fetuses and in the development and function of gonads of both sexes. Members of the TGF-beta family signal through two types of serine/threonine kinase receptors called type I and type II, and two types of Smad proteins, receptor-regulated Smad (R-Smad) and common Smad, Smad4. Components of the AMH signaling pathway have been identified in gonads and gonadal cell lines. The AMH type II receptor is highly specific. In contrast, the identity of the AMH type I receptor is not clear; three type I receptors of Bone Morphogenetic Proteins (BMPs), Alk2, Alk3 and Alk6 may transduce AMH signals, but none of them has all the characteristics of an AMH type I receptor. AMH activates BMP-specific R-Smads and reporter genes.

Persistence of Müllerian derivatives and intestinal lymphangiectasis in two newborn brothers: confirmation of the Urioste syndrome.

We describe two newborn brothers with a pattern of malformation characterized by the persistence of Müllerian duct derivatives, intestinal lymphangiectasia, hypertrophied alveolar ridges, and early death. Postmortem examination showed the presence of a rudimentary uterus, fallopian tubes, the upper third of a vagina, a prostate of normal shape, a dilated colon, and generalized intestinal and pulmonary lymphangiectasia. The syndrome was first delineated by Urioste and co-workers [1993: Am J Med Genet 47:494-503]. These cases confirm the existence of a definite and distinct entity.

Autosomal recessive segregation of a truncating mutation of anti-Müllerian type II receptor in a family affected by the persistent Müllerian duct syndrome contrasts with its dominant negative activity in vitro.

Anti-Müllerian hormone belongs to the TGFbeta family whose members exert their effects by signaling through two related serine/threonine kinase receptors. Mutations of the anti-Müllerian hormone type II receptor occur naturally, causing the persistent Müllerian duct syndrome. In a family with two members with persistent Müllerian duct syndrome and one normal sibling, we detected two novel mutations of the anti-Müllerian hormone type II receptor gene. One, transmitted by the mother to her three sons, is a deletion of a single base leading to a stop codon, causing receptor truncation after the transmembrane domain. The other, a missense mutation in the substrate-binding site of the kinase domain, is transmitted by the father to the two sons affected by persistent Müllerian duct syndrome, indicating a recessive autosomal transmission as in other cases of persistent Müllerian duct syndrome. Truncating mutations in receptors of the TGFbeta family exert dominant negative activity, which was seen only when each of the mutant anti-Müllerian hormone receptors was overexpressed in an anti-Müllerian hormone-responsive cell line. We conclude that assessment of dominant activity in vitro, which usually involves overexpression of mutant genes, does not necessarily produce information applicable to clinical conditions, in which mutant and endogenous genes are expressed on a one to one basis.

A novel mutation in the anti-müllerian hormone gene as cause of persistent müllerian duct syndrome.

UNLABELLED: Persistent müllerian duct syndrome is a relatively rare inherited defect of sexual differentiation characterised by failure of regression of the müllerian ducts in males. In affected individuals, uterus and tubes are present because of defects of synthesis or action of anti-müllerian hormone (AMH), normally produced by the Sertoli cells of the testis. Patients are normally virilised, although mono- or bilateral cryptorchidism may be present. We observed two brothers (chromosomes 46 XY), aged 11 years and 2 months and 8 years and 3 months respectively, with bilateral cryptorchidism. The diagnosis of persistent müllerian duct syndrome was made on the basis of laparoscopic evidence of uterus and tubes, undetectable plasma levels of AMH and a 23 base pair duplicative insertion in exon 5 of the AMH gene, causing the introduction of a premature stop codon, homozygous in the two brothers. The surgical correction of the genital abnormalities was successfully carried out by laparoscopic orchidopexy according to Fowler-Stephens. CONCLUSION: Persistent müllerian duct syndrome should be taken into consideration in all cases of bilateral cryptorchidism. Laparoscopy is the elective procedure for diagnosis of this disease and laparoscopic surgery for orchidopexy of intra-abdominal testes. Mutation analysis of the anti-müllerian hormone gene in these patients helps to understand the structure-function relationship of the anti-müllerian hormone protein, although it is not clear at present whether anti-müllerian hormone is necessary to maintain normal testicular function.

Detection of minimal levels of serum anti-Müllerian hormone during follow-up of patients with ovarian granulosa cell tumor by means of a highly sensitive enzyme-linked immunosorbent assay.

Granulosa cell tumors (GCT) are ovarian neoplasms that tend to recur and spread in the pelvis and the abdomen several years after the initial treatment. Anti-Mülerian hormone (AMH) is a reliable serum marker of these tumors. To enhance the availability and the sensitivity of serum AMH determination, we developed an ultrasensitive enzyme-linked immunosorbent assay. In this work we compare the results of serum AMH levels, obtained using the ultrasensitive and the traditional assays, in 31 patients with ovarian GCT followed up for up to 7 yr. The ultrasensitive enzyme-linked immunosorbent assay has a significantly higher sensitivity than the traditional one. This resulted in the detection of low serum AMH levels, which were undetectable with the traditional assay, in several cases including one patient in whom a recurrence of a GCT had developed and two patients in whom the treatment had not been completely successful. These cases highlight the importance of the availability of a highly sensitive assay allowing evaluation with high precision of the results of treatment and to detect the recurrences of GCT at an early, preclinical stage.

Evaluation of gonadal function in 107 intersex patients by means of serum antimüllerian hormone measurement.

Fetal male sexual differentiation is driven by two testicular hormones: testosterone (synthesized by interstitial Leydig cells) and antimüllerian hormone (AMH; produced by Sertoli cells present in the seminiferous tubules). Intersex states result either from gonadal dysgenesis, in which both Leydig and Sertoli cell populations are affected, or from impaired secretion or action of either testosterone or AMH. Until now, only Leydig cell function has been assessed in children with ambiguous genitalia, by means of testosterone assay. To determine whether serum AMH would help in the diagnosis of intersex conditions, we assayed serum AMH levels in 107 patients with ambiguous genitalia of various etiologies. In XY patients, AMH was low when the intersex condition was caused by abnormal testicular determination (including pure and partial gonadal dysgenesis) but was normal or elevated in patients with impaired testosterone secretion, whereas serum testosterone was low in both groups. AMH was also elevated during the first year of life and at puberty in intersex states caused by androgen insensitivity. In 46,XX patients with a normal male phenotype or ambiguous genitalia, in whom the diagnosis of female pseudohermaphroditism had been excluded, serum AMH levels higher than 75 pmol/L were indicative of the presence of testicular tissue and correlated with the mass of functional testicular parenchyma. In conclusion, serum AMH determination is a powerful tool to assess Sertoli cell function in children with intersex states, and it helps to distinguish between defects of male sexual differentiation caused by abnormal testicular determination and those resulting from isolated impairment of testosterone secretion or action.

Persistence of Müllerian derivatives in males.

The persistent müllerian duct syndrome is a rare, autosomal recessive disorder, characterized by the persistence of müllerian duct derivatives-uterus and fallopian tubes-in genetic males otherwise normally virilized. We have collected DNA from 69 families with this syndrome. In 45%, a mutation of the anti-müllerian hormone (AMH) gene was detected; 52% were homozygous. The level of circulating AMH was extremely low in the great majority of patients, even before puberty, when AMH levels are normally high. Single-strand conformation polymorphism (SSCP)-polymerase chain reaction (PCR) was a very effective screening method. In 39% of families, characterized by an AMH level normal for the age of the patient, a mutation of the type II receptor of AMH was detected by automatic sequencing, because SSCP-PCR was not very effective. Forty-eight percent of the mutations were homozygous. A 27-base-pair deletion in exon 10 was noted in 45% of the families. When this very common mutation is not taken into account, the proportion of recurrent mutations is 42% for the AMH gene and 33% for the AMH receptor type II gene. In 16% of families, no mutation of either the AMH or the AMH receptor gene was detectable; this group may correspond to mutations of unknown genes involved in AMH processing or in downstream AMH transduction.

Hormonal and cellular regulation of Sertoli cell anti-Müllerian hormone production in the postnatal mouse.

Anti-Müllerian hormone (AMH) is secreted by immature testicular Sertoli cells. Clinical studies have demonstrated a negative correlation between serum AMH and testosterone in puberty but not in the neonatal period. We investigated AMH regulation using mouse models mimicking physiopathological situations observed in humans. In normal mice, intratesticular, not serum, testosterone repressed AMH synthesis, explaining why AMH is downregulated in early puberty when serum testosterone is still low. In neonatal mice, AMH was not inhibited by intratesticular testosterone, due to the lack of expression of the androgen receptor in Sertoli cells. We had shown previously that androgen-insensitive patients exhibit elevated AMH in coincidence with gonadotropin activation. In immature normal and in androgen-insensitive Tfm mice, follicle stimulating hormone (FSH) administration resulted in elevation of AMH levels, indicating that AMH secretion is stimulated by FSH in the absence of the negative effect of androgens. The role of meiosis on AMH expression was investigated in Tfm and in pubertal XXSxrb mice, in which germ cells degenerate before meiosis. We show that meiotic entry acts in synergy with androgens to inhibit AMH. We conclude that AMH represents a useful marker of androgen and FSH action within the testis, as well as of the onset of meiosis.

A 27 base-pair deletion of the anti-müllerian type II receptor gene is the most common cause of the persistent müllerian duct syndrome.

The persistent müllerian duct syndrome, characterized by the lack of regression of müllerian derivatives, uterus and tubes in otherwise normally masculinized males, is a genetically transmitted disorder implicating either anti-müllerian hormone (AMH), a member of the transforming growth factor-beta superfamily, or its type II receptor, a serine/threonine kinase homologous to the receptors of other members of the transforming growth factor-beta superfamily. We have now performed molecular studies in a total of 38 families. The basis of the condition, namely 16 AMH and 16 AMH receptor mutations, was identified in 32 families. The type of genetic defect could be predicted from the level of serum AMH which is very low or undetectable in patients with AMH mutations and at the upper limit of normal in receptor mutations. Whereas AMH mutations are extremely diverse, patients from 10 out of 16 families with receptor mutations had a 27 bp deletion in exon 10 on at least one allele. This deletion is thus implicated in approximately 25% of patients with persistent müllerian duct syndrome. All AMH and AMH receptor mutations were consistent with an autosomal recessive mode of transmission.

[Molecular biology of the persistent Müllerian duct syndrome]. / Biologie moléculaire du syndrome de persistance des canaux de Müller.

The persistent Müllerian duct syndrome, characterized by the presence of uterus and tubes in otherwise normally masculinized 46,XY males, is a familial autosomal recessive disorder due to defects of synthesis or action of anti-Müllerian hormone. We have performed molecular studies in a total of 38 families and we have identified the basis of the condition, namely 16 anti-Müllerian hormone and 16 anti-Müllerian hormone receptor mutations, in 32 families.

Insensitivity to anti-müllerian hormone due to a mutation in the human anti-müllerian hormone receptor.

Anti-Müllerian hormone (AMH) and its receptor are involved in the regression of Müllerian ducts in male fetuses. We have now cloned and mapped the human AMH receptor gene and provide genetic proof that it is required for AMH signalling, by identifying a mutation in the AMH receptor in a patient with persistent Müllerian duct syndrome. The mutation destroys the invariant dinucleotide at the 5' end of the second intron, generating two abnormal mRNAs, one missing the second exon, required for ligand binding, and the other incorporating the first 12 bases of the second intron. The similar phenotypes observed in AMH-deficient and AMH receptor-deficient individuals indicate that the AMH signalling machinery is remarkably simple, consisting of one ligand and one type II receptor.

Molecular genetics of the persistent müllerian duct syndrome: a study of 19 families.

A rare form of familial male pseudohermaphroditism, the persistent Müllerian duct syndrome (PMDS) is characterized by persistence of uterus and Fallopian tubes in 46,XY phenotypic males and is ascribed to defects in the synthesis or action of anti-Müllerian hormone (AMH). Biologically, PMDS is heterogeneous: in some cases, bioactive AMH is normally expressed by testicular tissue while, in others, no AMH is produced, suggesting the possibility of an AMH gene mutation, several of which have already been described. Molecular analysis of the AMH gene has now been performed in 21 additional patients and their families. In 6 patients, with normal serum concentration of AMH, the AMH gene was normal or contained only polymorphisms and silent mutations, supporting the hypothesis that the condition is due to end-organ resistance. Nine novel mutations were discovered in the remaining subjects, with low or undetectable levels of serum AMH. These mutations, when present in homozygotes or compound heterozygotes, were associated with the PMDS phenotype, the same mutation never being observed in two different families. The three first exons of the AMH gene appear particularly mutation-prone, although they are less GC rich than the 2 last ones and code for the N-terminal part of the AMH protein, which is not in itself essential to bioactivity.