Expression of Stemness Markers in the Cervical Smear of Patients with Cervical Insufficiency.

The presence of stem cells has been previously described in human precancerous and malignant cervical cultures. Previous studies have shown a direct interplay of the stem cell niche, which is present in practically every tissue with the extracellular matrix. In the present study, we sought to determine the expression of stemness markers in cytological specimens collected from the ectocervix among women with cervical insufficiency during the second trimester of pregnancy and women with normal cervical length. A prospective cohort of 59 women was enrolled of whom 41 were diagnosed with cervical insufficiency. The expression of OCT-4 and NANOG was higher in the cervical insufficiency group compared to the control group (-5.03 (-6.27, -3.72) vs. -5.81 (-7.67, -5.02) p = 0.040 for OCT4) and (-7.47 (-8.78, -6.27) vs. -8.5 (-10.75, -7.14), p = 0.035 for NANOG. Differences in the DAZL gene were not significantly different (5.94 (4.82, 7.14) vs. 6.98 (5.87, 7.43) p = 0.097). Pearson correlation analysis indicated the existence of a moderate correlation of OCT-4 and Nanog with cervical length. Considering this information, the enhanced activity of stemness biomarkers among pregnant women diagnosed with cervical insufficiency may be predisposed to cervical insufficiency, and its predictive accuracy remains to be noted in larger population sizes.

Individual and Combined Assessment of Ser680Asn FSH Receptor and FSHß-211 G>T Gene Polymorphisms in Ovarian Response in IVF/ICSI Program.

BACKGROUND: Molecular biology tools, such as the detection of Single Nucleotide Polymorphisms (SNPs), have been considered to assist in the management of ovarian stimulation protocols. PURPOSE: The aim of this study was to evaluate the impact of two polymorphisms, the Asn680Ser polymorphism of the FSHR gene, and the FSH ß subunit (FSHß) gene polymorphism -211 G>T, in a Greek population of women undergoing IVF/ICSI program in our center. In addition, a control group of fertile women was studied to verify whether there are differences in the genotype distribution between fertile and infertile population for both polymorphisms, as the FSHß gene polymorphism -211 G>T is studied for the first time in the Greek population. RESULTS: The FSH ß-211 G>T polymorphism, studied for the first time in the infertile Greek population, appears to be quite rare. When studying the two polymorphisms separately, statistically significant differences were obtained that concerned the LH levels. DISCUSSION: According to the combination analysis of the two polymorphisms by the number of alleles, women with 2-3 polymorphic alleles needed more days of stimulation, but there were no differences in pregnancy rates. CONCLUSION: This molecular genetic study helps to elucidate whether the polygenic combination of the Asn680Ser and FSH ß subunit -211 G>T gene polymorphisms is of additive value in the prediction of ovarian response to exogenous gonadotropins.

The impact of genetics profile (gene polymorphisms) in obese non-PCOS women entering an IVF/ICSI program.

Data concerning the effects of increased body mass index (BMI) on ovarian and pregnancy outcome are rich, but the results are rather controversial. Regarding pharmacogenetics, gene polymorphisms of hormonal receptor genes, such as Estrogen Receptor alpha (ESR1), Estrogen Receptor beta (ESR2) and FSH receptor (FSHR) genes, are associated with ovarian stimulation and pregnancy outcome and may constitute a useful tool for ART experts for the prediction of this outcome. The aim of this study is to track differences in the distribution of gene polymorphisms among obese non-PCOS and non-obese patients concerning three distinct genes which are involved in the ovarian stimulation mechanism: PvuII polymorphism of ESR1 gene, RsaI polymorphism of ESR2 gene and Ser680Asn variation of FSHR gene, using restriction fragment length polymorphism analysis and real-time polymerase chain reaction. A total of 151 normally ovulating female patients underwent IVF or ICSI. Interestingly, the pregnancy rate in the BMI≥30 kg/m² group was higher in a statistically significant way (40.9% versus 17.8%, p=0.023). The obese patients of this study were in need of increased total FSH dose in order to achieve a satisfactory oocyte number (p<0.001) and needed more days of stimulation (p=0.002), but also presented lower basal FSH levels (p=0.032), which may explain, to an extend, the better pregnancy outcome. Concerning the polymorphisms of ESR1, ESR2 and FSHR genes, we did not observe differences in the genotype distribution when we compared the obese non-PCOS population with the non-obese population. Thus, obesity does not constitute an additional indication to perform a genetic analysis before entering an IVF/ICSI program.

Do estrogen receptor alpha polymorphisms have any impact on the outcome in an ART program?

PURPOSE: To investigate two of the most studied estrogen receptor alpha polymorphisms (PvuII and XbaI) in combination, in order to evaluate their impact on an ART program outcome. METHODS: 203 normally ovulating women who underwent IVF or ICSI treatment were genotyped for PvuII and XbaI polymorphisms in ESR1 intron 1 using Real-Time PCR. The relationship between the presence of polymorphic alleles and the ovulation induction parameters and outcome was examined. RESULTS: Women were grouped according to the number of polymorphic alleles they carried in two groups (0-2 versus 3-4 polymorphic alleles). The presence of 3 or more polymorphic alleles was associated with significantly lower E2 levels on the day of hCG administration and a significantly lower rate of good quality embryos. CONCLUSION: There is an association between ESR1 polymorphisms and some ART parameters such as the level of E2 on the day of hCG administration and the quality of the embryos. These results underline the importance of ESR1 as a candidate gene for the prediction of ovarian response to IVF/ICSI protocols. Future research work concerning several more genes is necessary for a better evaluation of patients before entering an IVF/ICSI program.

Detection of RUNX2 gene expression in cumulus cells in women undergoing controlled ovarian stimulation.

BACKGROUND: RUNX2 is a transcription factor, whose expression has been recently identified in the mouse ovary. Regulation of RUNX2 expression and its function in the human ovary have not been determined yet. The aim of the present study is the investigation of the possible correlation between RUNX2 gene expression in cumulus cells and controlled ovarian stimulation and pregnancy outcomes after ART treatment. METHODS: A total of 41 patients undergoing ICSI treatment for male factor infertility were enrolled into a specific ART program, during which cumulus cells were collected. The expression of RUNX2 gene in cumulus cells was examined by real-time PCR. RESULTS: Concerning RUNX2 gene expression, 12 out of 41 women were detected with RUNX2 expression, with ratios ranging from 0.84 to 1.00, while 28 out of 41 women had no expression (ratio = 0). Only 1 woman presented a weak RUNX2 gene expression (ratio = 0.52). From 8 women that proceeded to pregnancy, 7 of them did not express RUNX2 gene in cumulus cells, while one was the woman with weak gene expression that also achieved pregnancy. The group of women without RUNX2 expression presented higher number of follicles (p = 0.013), higher number of retrieved oocytes (p = 0.016), higher basal LH serum levels (p = 0.016) and higher peak estradiol levels (p = 0.013), while the number of fertilized oocytes differed marginally between the two groups (p = 0.089). Moreover, RUNX2 expression was negatively associated with LH levels (OR = 0.22, p = 0.021) and E2 levels (OR = 0.25, p = 0.026). CONCLUSIONS: Consequently, based on the preliminary findings of the present pilot study a potential inhibitory mechanism of RUNX2 gene is observed in the ovary when high mRNA levels are detected, suggesting that RUNX2 could possibly be used as a candidate genetic marker in the monitoring of the outcome of an ART treatment.

LH receptor gene expression in cumulus cells in women entering an ART program.

PURPOSE: Luteinizing hormone (LH) exerts its actions through its receptor (LHR), which is mainly expressed in theca cells and to a lesser extent in oocytes, granulosa and cumulus cells. The aim of the present study was the investigation of a possible correlation between LHR gene and LHR splice variants expression in cumulus cells and ovarian response as well as ART outcome. METHODS: Forty patients undergoing ICSI treatment for male factor infertility underwent a long luteal GnRH-agonist downregulation protocol with a fixed 5-day rLH pre-treatment prior to rFSH stimulation and samples of cumulus cells were collected on the day of egg collection. RNA extraction and cDNA preparation was followed by LHR gene expression investigation through real-time PCR. Furthermore, cumulus cells were investigated for the detection of LHR splice variants using reverse transcription PCR. RESULTS: Concerning LHR expression in cumulus cells, a statistically significant negative association was observed with the duration of ovarian stimulation (odds ratio = 0.23, p = 0.012). Interestingly, 6 over 7 women who fell pregnant expressed at least two specific types of LHR splice variants (735 bp, 621 bp), while only 1 out of 19 women that did not express any splice variant achieved a pregnancy. CONCLUSIONS: Consequently, the present study provide a step towards a new role of LHR gene expression profiling as a biomarker in the prediction of ovarian response at least in terms of duration of stimulation and also a tentative role of LHR splice variants expression in the prediction of pregnancy success.

Unusual association between increased bone resorption and presence of paroxysmal nocturnal hemoglobinuria phenotype in multiple myeloma.

Paroxysmal nocturnal hemoglobinuria (PNH) clones deficient in glycosylphosphatidylinositol-anchored molecules, including CD55 and CD59, have been previously described in patients with multiple myeloma (MM). The aim of this study was to investigate the possible association between existence of the PNH phenotype and myeloma bone disease. Forty-three patients with newly diagnosed MM were the subjects of the study. Radiographic evaluation of the skeleton was performed in all patients at diagnosis. The following biochemical markers were measured: bone resorption markers (tartrate-resistant acid phosphatase isoform 5b [TRACP-5b]and N-terminal cross-linking telopeptide of type-I collagen [NTX]), bone formation markers (bone alkaline phosphatase [bALP] and osteocalcin [OC]), osteoprotegerin (OPG), soluble receptor activator of nuclear factor KB ligand (sRANKL), and interleukin 6 (IL-6). Detection of CD55- and/or CD59-deficient red cell populations was performed after diagnosis. Patients with MM had elevated mean baseline NTX, TRACP-5b, sRANKL, and IL-6 levels compared with controls, whereas the mean values of bALP, OC, and OPG were significantly decreased. Four patients had no osteolytic lesions, whereas 8 patients had 1 to 3 lytic lesions, and 31 patients had more than 3 lytic lesions and/or pathologic fractures in the skeletal survey. CD55- and/or CD59-deficient red cell populations were observed in 56% of patients with MM. There was a strong correlation between the presence of PNH-like erythrocytes and increased bone resorption, as measured by NTX, TRACP-5b, and sRANKL/OPG ratio (P < .03, P < .02, and P < .02, respectively). There was also a significant correlation between PNH phenotype and severe bone disease (P < .02). These results suggest that there is a possible link between PNH phenotype and increased osteoclastic activity in MM owing to a potential effect of myeloma microenvironment on a preexisting PNH clone. Further studies are required for clarifying this phenomenon and investigating possible mechanisms of this unusual association.

Red cells with paroxysmal nocturnal hemoglobinuria-phenotype in patients with acute leukemia.

CD55 and CD59 are complement regulatory proteins that are linked to the cell membrane via a glycosyl-phosphatidylinositol anchor. They are reduced mainly in paroxysmal nocturnal hemoglobinuria (PNH) and in other hematological disorders. However, there are very few reports in the literature concerning their expression in patients with acute leukemias (AL). We studied the CD55 and CD59 expression in 88 newly diagnosed patients with AL [65 with acute non-lymphoblastic leukemia (ANLL) and 23 with acute lymphoblastic leukemia (ALL)] using the sephacryl gel test, the Ham and sucrose lysis tests and we compared the results with patients' clinical data and disease course. Eight patients with PNH were also studied as controls. Red cell populations deficient in both CD55 and CD59 were detected in 23% of ANLL patients (especially of M(0), M(2) and M(6) FAB subtypes), 13% of ALL and in all PNH patients. CD55-deficient erythrocytes were found in 6 ANLL patients while the expression of CD59 was decreased in only 3 patients with ANLL. No ALL patient had an isolated deficiency of these antigens. There was no correlation between the existence of CD55 and/or CD59 deficiency and the percentage of bone marrow infiltration, karyotype or response to treatment. However no patient with M(3), M(5), M(7) subtype of ANLL and mature B- or T-cell ALL showed a reduced expression of both antigens. The deficient populations showed no alteration after chemotherapy treatment or during disease course. This study provides evidence about the lower expression of CD55 and CD59 in some AL patients and the correlation with their clinical data. The possible mechanisms and the significance of this phenotype are discussed.

Leukemogenic risk of hydroxyurea therapy as a single agent in polycythemia vera and essential thrombocythemia: N- and K-ras mutations and microsatellite instability in chromosomes 5 and 7 in 69 patients.

Polycythemia vera (PV) and essential thrombocythemia (ET) are chronic myeloproliferative diseases that carry intrinsically the potential for leukemic transformation. The aims of this study were (1) to detect involvement of N- and K-ras mutations in codons 12 and 13 in the pathogenesis of the chronic and blastic phases of PV and ET, (2) to study the occurrence of microsatellite instability (MSI) in chromosomes 5 and 7 during the chronic phase and blastic transformation of the disease, and (3) to examine the incidence of leukemia in patients treated with hydroxyurea (HU). Samples of PV and ET patients were analyzed with a polymerase chain reaction. No N- or K-ras mutations were detected. A positive score for MSI in chromosome 7 was found in 1 patient with PV during leukemic transformation. Three of 69 patients developed acute myelogenous leukemia, 2 with PV and 1 with ET. As of this report, the overall incidence of leukemic transformation is 5.7% (2/35 patients) in PV and 3.3% (1/30 patients) in ET patients treated with HU. These results indicate that (1) MSI is a genetic marker that can be detected, even in a small group of patients, at the blastic phase of the disease and (2) no increased leukemogenicity was noted in this group of patients treated with HU.

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

Paroxysmal nocturnal hemoglobinuria (PNH) is an acquired clonal disorder characterized by a decrease or absence of glycosylphosphatidylinositol (GPI)-anchored molecules such as CD55 and CD59 from the surface of affected cells, resulting in intravascular hemolysis, cytopenia, and venous thrombosis. A PNH-like phenotype has been detected in various hematological disorders, mainly in aplastic anemia and myelodysplastic syndromes, but also in lymphoproliferative syndromes (LPSs). To the best of our knowledge, CD55- or CD59-deficient red cells have not been detected in plasma cell dyscrasias (PCDs). The aim of this study was the detection of CD55- and/or CD59-deficient red cell populations in patients with PCD. Seventy-seven patients were evaluated; 62 with multiple myeloma (MM), 7 with Waldenstrom macroglobulinemia (WM), 6 with monoclonal gammopathy of undetermined significance (MGUS), and 2 with heavy chain disease (HCD). The sephacryl gel microtyping system was applied; Ham and sucrose lysis tests were also performed on all samples with CD55- or CD59-negative populations. Red cells deficient in both molecules were detected in 10 (12.9%) of 77 patients with PCD: 2 (28.6%) of 7 with WM, 1 (16.6%) of 6 with MGUS, 6 (9.6%) of 62 with MM, and 1 of 2 patients with HCD. Isolated CD55 deficiency was found in 28.5% of all PCD patients, whereas isolated CD59 deficiency was not observed in any patients. These findings illustrate the existence of the PNH phenotype in the red cells of patients with PCD; further investigation is needed into the mechanisms and significance of this phenotype.