The effect of CRH and its inhibitor, antalarmin, on in vitro growth of preantral mouse follicles, early embryo development, and steroidogenesis.

In vitro growth systems of preantral follicles allow studying the effect of various endocrine, paracrine, and autocrine factors on follicular growth and oocyte maturation. CRH is a 41-amino-acid neuropeptide responsible for endocrine, autonomic, immunological, and behavioral responses of mammals to stress and has two receptors, CRH receptor type 1 (CRH-R1) and CRH-R2. Antalarmin, a CRH-R1 antagonist, has been used to elucidate the role of CRH in stress, inflammation, and reproduction. The present study describes in vitro growth of mouse preantral follicles, early embryo development, and steroidogenesis in the presence of CRH and its antagonist antalarmin. We cultured 732 follicles in control media, 1306 in CRH 10(-7) mol/liter, and 1202 in CRH 10(-7) plus antalarmin 10(-6) mol/liter. The culture medium was assayed on alternate days for 17ß-estradiol, progesterone, and ß-human chorionic gonadotropin. Total RNA was extracted from preantral follicles as well as early preimplantation embryos and was assessed by real-time RT-PCR for the expression of CRH-R1 and CRH-R2 mRNAs. Hormone analysis showed that the CRH group had lower levels of 17ß-estradiol, progesterone, and ß-human chorionic gonadotropin as the culture progressed, in comparison with the other two groups. RT-PCR demonstrated the presence of CRH-R1 and CRH-R2 in all stages of preantral follicle culture. Morula/blastocyst-stage embryos expressed only CRH-R1. In conclusion, CRH has an inhibitory effect on in vitro fertilized oocytes, resulting from cultured preantral follicles at all stages of preimplantation embryo development. Furthermore, the presence of CRH in the culture medium inhibits steroidogenesis by preantral mouse follicles cultured in vitro.

Genetic profile of SNP(s) and ovulation induction.

Obtaining an adequate number of good quality oocytes while minimizing adverse drug reactions (ADRs) and cycle cancellation rates is considered the gold standard in controlled ovarian hyperstimulation (COH) for fertility treatment. Patients who undergo IVF/ICSI cycles tend to present with different responses to exogenous gonadotrophin administration. Research has shown that the secret probably lies in the various single nucleotide polymorhisms (SNPs) in their receptor genes. The decryption of human genome provided specialists with additional information in assessing and even predicting ovarian response to COH. In this context, the study of Pharmacogenomics, Pharmacogenetics and SNPs unravels as a promising field in optimizing fertility treatment. Several SNPs in FSH and estrogen receptor genes have been detected so far, but only three of them, one in FSH receptor and two in estrogen receptor genes have been associated with ovarian response to COH. It seems that the Asn/Ser variant of the FSH receptor functions more efficiently, while the Ser/Ser and Asn/Asn variants have a tendency to resist to FSH stimulation. With regards to estrogen receptor 1 (ESR1), the Pvull and the Xbal polymorphisms seem to be associated with differences in the response to ovarian stimulation, while the Rsal polymorphism in estrogen receptor 2 (ESR2) is currently under investigation. There exists evidence supporting the hypothesis that a set of genes, all related to the FSH hormone mechanism of action, may participate along with other factors to the control of ovarian response to FSH, thus a cautious interpretation of polymorphism detection results is considered mandatory. However, identifying potential genetic markers that could predict ovarian response and implementing them in routine screening tests for every woman entering an IVF/ICSI cycle, would be able to tailor fertility treatment to each patients needs thus maximizing the success rate and eliminating potential side-effects of fertility drugs.

ESR1, ESR2 and FSH receptor gene polymorphisms in combination: a useful genetic tool for the prediction of poor responders.

PURPOSE: Previous studies in humans concluded that a multigenic model including specific FSHR, ESR1 and ESR2 genotype patterns may partially explain the poor response to FSH. The aim of our study is to analyse three different loci -polymorphisms in ESR1 Pvu II, ESR2 Rsa I and Ser680Asn FSH receptor gene- in a Greek population and their involvement in stimulation outcome and pregnancy rates. METHODS: Each locus was studied alone, and in combination with the others. We performed both restriction fragment length polymorphism analysis and real-time polymerase chain reaction. A total of 109 normally ovulating female patients underwent IVF or ICSI. RESULTS: Studying each locus alone, no significant results were drawn for ESR1 and ESR2 genes. Concerning the FSHR polymorphism, the women carrying the AA variant presented higher total amount of gonadotrophins used (P=0,048) and tended to have higher number of stimulation days (P=0,057). Considering the ESR1 and FSHR gene polymorphisms in combination, the TC/SA combination presents the highest number of pregnancies in poor responders group (3/4 pregnancies carried this genotype), in good responders group (4/12 pregnancies carried this genotype) and in the total population (10/26 pregnancies carried this genotype). Except the CC/AA combination, all other genotype combinations presented incidence of pregnancy, with TC/SA having the highest incidence. The CC/AA genotype presents the worst profile of ovulation induction, confirming a poor responder profile: the total amount of gonadotrophins used was highest in CC/AA group (P < 0,05). The peak E2, the number of follicles and of retrieved oocytes and the pregnancy rate were significantly lower (P < 0,05). This genotype combination seems to be over-presented in the poor responders group in a statistically significant way (P=0,038). Women with CC/AA combination have 1,5-2,4 times more risk to be poor responders in comparison with women that do not carry that combination. CONCLUSION: This study supports the hypothesis that a multigenic model, including the well studied ESR1 and FSHR genes is involved in the controlled ovarian stimulation outcome indicating that the CC/AA genotype presents the worst ovulation induction profile, while the TC/SA genotype presents the higher number of pregnancies in our population.

FLT3 overexpression in acute promyelocytic leukemia patients without detectable FLT3-ITD or codon 835-836 mutations: a pilot study.

BACKGROUND: Activating mutations of the FLT3 receptor tyrosine kinase are common in acute promyelocytic leukemia (APL) but have uncertain prognostic significance. Information regarding FLT3 expression levels in APL without FLT3 mutations is lacking. MATERIALS AND METHODS: Using RT-PCR, mutation analysis of the FLT3 gene, regarding internal tandem duplications (ITDs) and codon 835-836 point mutations, was performed and real-time PCR was carried out to determine the level of FLT3 expression in 11 APL patients at diagnosis and 5 in haematological remission with molecularly detectable disease. RESULTS: High levels of FLT3 transcript, at least a 10-fold increase compared to the normal controls, were found at diagnosis in all 3 mutated cases and in 2 patients without detectable FLT3 mutations. CONCLUSION: FLT3 overexpression can be documented in patients without FLT3 mutations. These patients might benefit from treatment using specific FLT3 tyrosine kinase inhibitors. Larger studies are needed to evaluate the clinical and biological significance of FLT3 overexpression in the absence of FLT3 mutations.

Familial Mediterranean fever associated pyrin mutations in Greece.

OBJECTIVE: To search for pyrin mutations associated with familial Mediterranean fever (FMF) in Greece. PATIENTS AND METHODS: 62 patients fulfilling the Tel Hashomer diagnostic criteria for definite (33) or probable (29) FMF diagnosis were studied. Eight point mutations of pyrin gene were tested by standard methods. Of the 62 patients tested, 48 were Greek, four were Jewish, seven were Armenian, and three were Arab. RESULTS: 42 patients were found to be homozygotes for pyrin mutations; 11 patients were found to carry only one of the tested mutations; in nine patients no mutations were detected. CONCLUSION: Molecular detection of pyrin gene mutations seems useful in confirming suspected cases, and in detecting asymptomatic cases, of Mediterranean fever in Greece. It may also be used as a screening tool within affected families.

Detection of CD55 and/or CD59 deficient red cell populations in patients with aplastic anaemia, myelodysplastic syndromes and myeloproliferative disorders.

Paroxysmal nocturnal haemoglobinuria (PNH) is an acquired clonal stem cell disorder characterized by intravascular haemolysis, venous thrombosis, marrow hypoplasia, frequent episodes of infection, and rarely leukaemic conversion. At the cellular level, PNH is characterized by the decrease or absence of glycosylphosphatidylinositol (GPI)-anchored molecules, such as CD55 and CD59, from the cell surface. PNH-like clones have been described in various haematological disorders. The link between PNH and aplastic anaemia (AA) has been established but the relationship of PNH with myelodysplastic syndromes (MDS) or myeloproliferative disorders (MPD) remains unclear. In this study, the presence of CD55 and/or CD59 defective (PNH-like) red cell populations was evaluated in 21 patients with AA, 133 with MDS, 197 with MPD, 7 with PNH and in 121 healthy blood donors using the Sephacryl Gel Test microtyping system. Red cell populations deficient in both molecules CD55 and CD59 were detected in 33.3% of AA patients, in 16.5% of MDS patients (50% with hypoplastic bone marrow), in 14.2% of MPD patients (more often in essential thrombocythemia, 21.2%) and in all PNH patients. CD55 deficient red cell populations were found in 14.2% of patients with AA, 12.7% of patients with MDS and 21.3% of patients with MPD. CD59 deficient populations were found in 9.5% of AA patients, 2.2% of MDS patients and 2% of MPD patients. These results indicate an association between PNH, AA and MDS or even between PNH and MPD. Further investigation is necessary to work out the mechanisms of this association, and to define classification criteria for borderline cases, where diagnosis is difficult.

Detection of CD55- and/or CD59-deficient red cell populations in patients with lymphoproliferative syndromes.

INTRODUCTION: Paroxysmal nocturnal hemoglobinuria is an acquired clonal stem cell disorder characterized by the decrease or absence of glycosylphosphatidylinositol-anchored molecules from the surface of the affected cells, such as CD55 and CD59, resulting in chronic intravascular hemolysis, cytopenia and increased tendency to thrombosis. PNH-phenotype has been described in various hematological disorders, mainly in aplastic anemia and myelodysplastic syndromes, while it has been reported that complete deficiency of CD55 and CD59 has also been found in patients with lymphoproliferative syndromes, like non-Hodgkin's lymphomas. MATERIALS AND METHODS: The presence of CD55- and/or CD59-defective red cell populations was evaluated in 217 patients with lymphoproliferative syndromes. The study population included 87 patients with NHL, 55 with HD, 49 with CLL, 22 with ALL and four with hairy cell leukemia. One hundred and twenty-one healthy blood donors and seven patients with PNH were also studied as control groups. The sephacryl gel microtyping system was performed for the detection of CD55- and CD59-deficient red cell populations. Ham and sucrose lysis tests were also performed in all samples with CD55 or CD59 negative populations. RESULTS: Red cell populations deficient in both CD55 and CD59 molecules were detected in 9.2% of patients with lymphoproliferative syndromes (more often in ALL and nodular sclerosis type of HD) and in all PNH patients. CD55-deficient red cell populations were found in 8.7% of LPS patients (especially in low grade B-cell NHL), while CD59-deficient populations were found in only two patients with low grade B-cell NHL. CONCLUSION: These data indicate a possible association between paroxysmal nocturnal hemoglobinuria phenotype and lymphoproliferative syndromes, while further investigation is necessary to work out the mechanisms and the significance of the existence of this phenotype in these patients.