Organochlorine pollutants' levels in hair, amniotic fluid and serum samples of pregnant women in Greece. A cohort study.

Persistent organic pollutants are synthetic chemicals highly resistant to degradation with strong tendency to bioaccumulation. Assessment of human exposure to these compounds is crucial for public health protection, especially during vulnerable periods. The aim of the present cohort study was to evaluate the level of contamination to PCBs, o,p'- and p,p'-DDE, o,p' and p,p'-DDD, o,p' and p,p'-DDT and HCB in pregnant women. Hair, amniotic fluid and serum samples were collected and analyzed by HS-SPME-GCMS. The most detected analytes in amniotic fluids were p,p'-DDE, p,p'-DDD, o,p'-DDE and PCB101, in serum p,p'-DDE, HCB and PCB101 and in hair p,p'-DDE, HCB and PCB101. The levels of HCB and PCB101 in amniotic fluids were positively correlated with those in hair. Higher levels of DDDs and DDTs in hair samples and PCB28 in amniotic fluids were observed in smoker pregnant women. Gestation age was inversely proportional with the detected levels of PCB101 in all tested samples.

mTOR downstream effectors, 4EBP1 and eIF4E, are overexpressed and associated with HPV status in precancerous lesions and carcinomas of the uterine cervix.

The present study aims to investigate the expression levels of two critical mammalian target of rapamycin (mTOR) downstream effectors, 4E binding protein 1 (4EBP1) and eukaryotic initiation factor 4E (eIF4E) proteins, in precancerous squamous intraepithelial lesions and cancer of the uterine cervix, and their association with human papilloma virus (HPV) infection status. Uterine cervical biopsies from 73 patients were obtained, including 40 fresh-frozen samples and 42 archival formalin-fixed, paraffin-embedded tissue specimens. Whole protein extracts were analyzed for the expression of 4EBP1 and eIF4E proteins using western blotting. In addition, distribution of 4EBP1 and eIF4E protein expression and 4EBP1 phosphorylation (P-4EBP1) were analyzed by immunohistochemistry in archival tissues and correlated with the degree of dysplasia. The presence of high-risk HPV (HR-HPV) types was assessed by polymerase chain reaction. Using western blot analysis, high expression levels of 4EBP1 and eIF4E were observed in all uterine cervical carcinomas, which significantly correlated with the degree of dysplasia. By immunohistochemistry, overexpression of 4EBP1 and eIF4E was detected in 20 of 21 (95%) and 17 of 21 (81%) samples, respectively, in patients with high-grade dysplasia and carcinomas, compared with 1 of 20 (5%) and 2 of 20 (10%) samples, respectively, in patients with low-grade lesions or normal histology. All 4EBP1-positive cases tested were also positive for P-4EBP1. Furthermore, overexpression of 4EBP1 and eIF4E significantly correlated with the presence of HR-HPV oncogenic types. The present study demonstrated that critical effectors of mTOR signaling, which control protein synthesis initiation, are overexpressed in cervical high-grade dysplasia and cancer, and their levels correlate with oncogenic HPV types. These findings may provide novel targets for investigational therapeutic approaches in patients with cancer of the uterine cervix.

IGF2BP1 expression in human mesenchymal stem cells significantly affects their proliferation and is under the epigenetic control of TET1/2 demethylases.

Mesenchymal stem cells (MSCs) are a population of cells harboring in many tissues with the ability to differentiate toward many different lineages. Unraveling the molecular profile of MSCs is of great importance due to the fact that these cells are very often used in preclinical and clinical studies. We have previously reported the expression of insulin-like growth factor 2 mRNA binding protein 1 (IGF2BP1) an oncofetal mRNA-binding protein-in different stem cell types such as bone marrow (BM)-MSC and umbilical cord blood (UCB)-hematopoietic stem cells. Here, we demonstrate that MSCs of adipose tissue, BM, and UC origin have a differential pattern of IGF2BP1 and ten-eleven-translocate 1/2 (TET1/2) expression that could correlate with their proliferation potential. Upon IGF2BP1 interference, a significant reduction of cell proliferation is observed, accompanied by reduced expression of c-MYC and GLI1 and increased p21. We also present, for the first time, evidence that IGF2BP1 is epigenetically regulated by TET1 and TET2 demethylases. Specifically, we show that TET1 directly binds to the promoter of IGF2BP1 gene and affects the hydroxymethylation status of its promoter. These results indicate that IGF2BP1 and TET1/2 contribute to the stemness of MSCs, at least regarding their proliferative potential.

Sox2 suppression by miR-21 governs human mesenchymal stem cell properties.

MicroRNAs (miRNAs) have recently been shown to act as regulatory signals for maintaining stemness and for determining the fate of adult and fetal stem cells, such as human mesenchymal stem cells (hMSCs). hMSCs constitute a population of multipotent stem cells that can be expanded easily in culture and are able to differentiate into many lineages. We have isolated two subpopulations of fetal mesenchymal stem cells (MSCs) from amniotic fluid (AF) known as spindle-shaped (SS) and round-shaped (RS) cells and characterized them on the basis of their phenotypes, pluripotency, proliferation rates, and differentiation potentials. In this study, we analyzed the miRNA profile of MSCs derived from AF, bone marrow (BM), and umbilical cord blood (UCB). We initially identified 67 different miRNAs that were expressed in all three types of MSCs but at different levels, depending on the source. A more detailed analysis revealed that miR-21 was expressed at higher levels in RS-AF-MSCs and BM-MSCs compared with SS-AF-MSCs. We further demonstrated for the first time a direct interaction between miR-21 and the pluripotency marker Sox2. The induction of miR-21 strongly inhibited Sox2 expression in SS-AF-MSCs, resulting in reduced clonogenic and proliferative potential and cell cycle arrest. Strikingly, the opposite effect was observed upon miR-21 inhibition in RS-AF-MSCs and BM-MSCs, which led to an enhanced proliferation rate. Finally, miR-21 induction accelerated osteogenesis and impaired adipogenesis and chondrogenesis in SS-AF-MSCs. Therefore, these findings suggest that miR-21 might specifically function by regulating Sox2 expression in human MSCs and might also act as a key molecule determining MSC proliferation and differentiation.

The validation of international consultation on incontinence questionnaires in the Greek language.

AIMS: The objective of this study was to validate four specific International Consultation on Incontinence Questionnaires (ICIQ) modules in the Greek language: (i) the ICIQ-FLUTS long form (ICIQ-FLUTS-LF), (ii) the ICIQ-FLUTS, (iii) the ICIQ-FLUTS-SEX, and (iv) the ICIQ-Vaginal Symptoms Questionnaire (ICIQ-VS), originally validated in English. METHODS: The English questionnaires were initially translated into Greek, then back-translated into English and final modifications were made after testing the questionnaires on a sample of patients. To validate the translated questionnaires, the following tests were undertaken: Content/face validity, internal consistency (reliability) and stability (test-retest reliability). RESULTS: A total of 122 women participated in the study. Eighty-nine presented with pelvic organ prolapse (POP) and/or urinary incontinence (UI) symptoms and 33 attended an outpatient gynecological clinic without POP/UI symptoms. All modules showed excellent content/face validity (missing values 0-2.5%). Cronbach's alpha test for internal consistency showed satisfactory to excellent reliability (0.876 for ICIQ-FLUTS-LF, 0.85 for ICIQ-FLUTS, and 0.83 for ICIQ-VS), with the exception ICIQ-FLUTS-SEX which was 0.69. The test-retest reliability showed moderate to near-perfect agreement (weighted kappa value 0.52-0.99). CONCLUSIONS: The Greek versions of the ICIQ-FLUTS-LF, ICIQ-FLUTS, and ICIQ-VS questionnaires were successfully validated. Our data showed that the ICIQ FLUTS-SEX questionnaire, as it stands in its current English version, cannot be reliably used to assess sex symptoms in the Greek female population.

VEGF directly suppresses activation of T cells from ovarian cancer patients and healthy individuals via VEGF receptor Type 2.

The role of vascular endothelial growth factor (VEGF) in tumor angiogenesis is well characterized; nevertheless, it is also a key element in promoting tumor evasion of the immune system by downregulating dendritic cell maturation and thus T cell activation. We sought to investigate the possible direct effect of VEGF on T cell activation and through which type of VEGF receptor (VEGFR) it exerts this effect. Circulating T cells from healthy donors and ovarian cancer patients were expanded in cultures with anti-CD3 and IL-2 with or without VEGF for 14 days, and the number of T cells was assessed. Cultured T cells were also tested for their cytotoxic activity in a standard 4-hr (51) Cr-release assay, and the expression of VEGFRs 1, 2 and 3 was assayed by flow cytometry, immunocytochemistry and Western blotting. To assess the ability of activated T cells to secrete VEGF, levels in culture supernatants were measured by enzyme linked immunosorbent assay. The addition of VEGF in cultures significantly reduced T cell proliferation in a dose-dependent manner. Protein expression studies demonstrated that CD3(+) T cells express VEGFR-2 on their surface upon activation. Experiments with anti-VEGFR-2 antibodies showed that the direct suppressive effect of VEGF on T cell proliferation is mediated by VEGFR-2. We also showed that VEGF significantly reduced the cytotoxic activity of T cells and that activated T cells secrete VEGF in the culture environment. Overall, our study shows that T cells secret VEGF and expresses VEGFR-2 upon activation. VEGF directly suppresses T cell activation via VEGF receptor type 2.

Therapeutic potential of a distinct population of human amniotic fluid mesenchymal stem cells and their secreted molecules in mice with acute hepatic failure.

BACKGROUND: There is increasing interest in the therapeutic potential of human mesenchymal stem cells (hMSCs), especially in diseases such as acute hepatic failure (AHF) that are predominantly caused by a variety of drugs and viruses. In previous studies, a distinct population termed human spindle-shaped MSCs were isolated and expanded from second trimester amniotic fluid (AF-MSCs) and characterised based on their phenotype, pluripotency and differentiation potential. METHODS: AF-MSCs, hepatic progenitor-like (HPL) cells and hepatocyte-like (HL) cells derived from AF-MSCs were transplanted into CCl4-injured NOD/SCID mice with the AHF phenotype in order to evaluate their therapeutic potential. Conditioned medium (CM) derived from AF-MSCs or HPL cells was then delivered intrahepatically in order to determine whether the engraftment of the cells or their secreted molecules are the most important agents for liver repair. RESULTS: Both HPL cells and AF-MSCs were incorporated into CCl(4)-injured livers; HPL cell transplantation had a greater therapeutic effect. In contrast, HL cells failed to engraft and contribute to recovery. In addition, HPL-CM was found to be more efficient than CM derived from AF-MSCs in treatment of the liver. Proteome profile analysis of HPL-CM indicated the presence of anti-inflammatory factors such as interleukins IL-10, IL-1ra, IL-13 and IL-27 which may induce liver recovery. Blocking studies of IL-10 secretion from HPL cells confirmed the therapeutic significance of this cytokine in the AHF mouse model. CONCLUSIONS: Human spindle-shaped AF-MSCs or HPL cells might be valuable tools to induce liver repair and support liver function by cell transplantation. More importantly, the factors they release may also play an important role in cell treatment in diseases of the liver.

Importance of maternal and cord blood viremia in pregnant women with chronic hepatitis B virus infection.

BACKGROUND/AIM: The spontaneous preterm birth (SPB) rates in a group of HBeAg-negative chronic HBV infected pregnant women without several known risk factors for preterm delivery as well as the mother to infant HBV transmission rates was evaluated. Moreover the role of maternal data during perinatal period as well as the role of HBsAg and/or HBV-DNA presence in cord blood in respect to preterm labour and vertical transmission of the infection was examined. METHODS: 138 consecutive chronic HBV infected pregnant women were haematologically, serologically and virologically evaluated during the perinatal period. 102 women were finally evaluated and fifteen of them (14.7%) exhibited SPB. Overall, 44 infants who had completed the proposed vaccination schedule were evaluated at month 12 of their life. RESULTS: A significant association between SPB and HBV-DNA presence in cord blood was observed (p=0.007). HBV-DNA positivity in cord blood was significantly associated with maternal HBV-DNA levels (p=0.002). The relative risk of HBV-DNA presence in cord blood was 6.43 times higher among women with serum HBV-DNA ≥ 10.000 copies/ml and lymphocyte count<1500 compared to those with all the other combinations of both parameters (p=0.001). All infants evaluated at month 12 were HBsAg-negative and exhibited undetectable HBV-DNA levels. CONCLUSION: The presence of HBV-DNA in cord blood is significantly associated with SPB in chronic HBV infected pregnant women. Maternal or cord blood viremia does not pose an additional risk factor for vertical transmission of HBV infection, in passive-active immunoprotected infants from HBeAg-negative chronic HBV infected mothers.

Presence of HBV-DNA in cord blood is associated with spontaneous preterm birth in pregnant women with HBeAg-negative chronic hepatitis B virus infection.

Spontaneous preterm birth is the leading cause of perinatal morbidity and mortality. In this study the spontaneous preterm birth rates in a group of hepatitis B e antigen (HBeAg)-negative chronic hepatitis B virus (HBV)-infected pregnant women without known risk factors for preterm delivery as well as the role of maternal laboratory data and hepatitis B surface antigen/HBV deoxyribonucleic acid (HBV-DNA) in cord blood in respect to preterm labour were evaluated. 138 consecutive HBeAg-negative chronic HBV-infected pregnant women were evaluated during the perinatal period. Serum HBV-DNA was determined by using the Cobas Amplicor HBV Test in both maternal and cord blood samples. 102 women were finally evaluated (36 were excluded) and 15 of them (14.7%) had spontaneous preterm birth. A significant association between spontaneous preterm birth and HBV-DNA in cord blood was observed (p = 0.007). HBV-DNA positivity in cord blood was significantly associated with maternal HBV-DNA levels (p = 0.002). The relative risk of HBV-DNA in cord blood was 6.43 times higher among women with serum HBV-DNA ≥10,000 copies/ml and lymphocyte count <1,500 compared to those with all the other combinations of both parameters (p = 0.001). In conclusion, the presence of HBV-DNA in cord blood is significantly associated with spontaneous preterm birth in chronic HBV-infected pregnant women. Women with HBV-DNA ≥10,000 copies/ml and lymphocyte count <1,500 during the perinatal period have a higher probability of HBV-DNA in their cord blood.

Effects of donor age, gender, and in vitro cellular aging on the phenotypic, functional, and molecular characteristics of mouse bone marrow-derived mesenchymal stem cells.

Mesenchymal stem cells (MSCs) are a very important adult stem cell population with a multitude of potential applications in regenerative medicine. The thorough characterization of the bone marrow MSC (BM-MSC) population derived from the BALB/c species was essential, considering the significance of the murine model amongst animal models. In the present study, we examined the effect of gender, age, and in vitro culture on the basic properties (proliferation, differentiation, and immunosuppressive potential) of BM-MSCs. We found a decline in the progenitor frequencies from the BM of adult mice, lower MSC frequencies in all female donors, and an increase in the BM-MSC proliferation rate upon in vitro propagation. We also examined BM-MSCs for the expression of the 3 major embryonic stem cell transcription factors, Oct3/4, Sox-2, and Nanog, as well as 2 mRNA binding proteins, coding region determinant binding protein/insulin-like growth factor 2 mRNA binding protein 1 (Crd-bp/Imp1) and Deleted in azoospermia-like (Dazl), which are expressed in primitive stem cells, umbilical cord blood-hematopoietic stem cells and amniotic fluid stem cells, respectively. Further, it has been reported that these 2 genes are critical for embryonic development. In this study, therefore, we report, for the first time, the expression of Crd-bp/Imp1 and Dazl in BM-MSCs. Dazl, Oct3/4, and Sox2 were detected in relatively low levels in contrast to Crd-bp/Imp1, its major target c-Myc, as well as Nanog, which were expressed redundantly, irrespective of sex, donor age, or in vitro passaging. These findings could further support the extrinsic theory of aging of the MSC population and the potential implication of embryonic genes in adult stem cell physiology.

In vitro and in vivo properties of distinct populations of amniotic fluid mesenchymal progenitor cells.

Human mesenchymal progenitor cells (MPCs) are considered to be of great promise for use in tissue repair and regenerative medicine. MPCs represent multipotent adherent cells, able to give rise to multiple mesenchymal lineages such as osteoblasts, adipocytes or chondrocytes. Recently, we identified and characterized human second trimester amniotic fluid (AF) as a novel source of MPCs. Herein, we found that early colonies of AF-MPCs consisted of two morphologically distinct adherent cell types, termed as spindle-shaped (SS) and round-shaped (RS). A detailed analysis of these two populations showed that SS-AF-MPCs expressed CD90 antigen in a higher level and exhibited a greater proliferation and differentiation potential. To characterize better the molecular identity of these two populations, we have generated a comparative proteomic map of SS-AF-MPCs and RS-AF-MPCs, identifying 25 differentially expressed proteins and 10 proteins uniquely expressed in RS-AF-MPCs. Furthermore, SS-AF-MPCs exhibited significantly higher migration ability on extracellular matrices, such as fibronectin and laminin in vitro, compared to RS-AF-MPCs and thus we further evaluated SS-AF-MPCs for potential use as therapeutic tools in vivo. Therefore, we tested whether GFP-lentiviral transduced SS-AF-MPCs retained their stem cell identity, proliferation and differentiation potential. GFP-SS-AF-MPCs were then successfully delivered into immunosuppressed mice, distributed in different tissues and survived longterm in vivo. In summary, these results demonstrated that AF-MPCs consisted of at least two different MPC populations. In addition, SS-AF-MPCs, isolated based on their colony morphology and CD90 expression, represented the only MPC population that can be expanded easily in culture and used as an efficient tool for future in vivo therapeutic applications.

A risk-adapted strategy of adjuvant paclitaxel/carboplatin in early-stage ovarian cancer: time-dependent effect of 4 versus 6 cycles on outcome.

OBJECTIVE: We investigated the efficacy of risk-adapted adjuvant paclitaxel/carboplatin chemotherapy in early-stage ovarian carcinoma. METHODS: Fifty-three patients were treated according to the risk of relapse: patients with stages IA or IB or with grade 1 (low risk) received 4 cycles of paclitaxel and carboplatin; patients with IC/IIA and grade 2 or 3 (high risk) received 6 cycles of chemotherapy. The outcome was compared with that of 95 patients who were all treated with 4 cycles. RESULTS: Median follow-up was 88, 113 and 42 months for the whole cohort, non-risk-adapted and risk-adapted treatment, respectively. Five-year relapse-free and disease-specific survival was 86 and 93% for the whole population, 96 and 97% for low-risk and 81 and 91% for high-risk patients. Risk classification was the only significant prognostic factor for relapse-free (p = 0.011) and disease-specific survival (p = 0.039). Among high-risk patients, the administration of 6 cycles was associated with a significantly lower relapse rate after censoring events, which occurred beyond 2 years (3 vs. 18%; p = 0.013), but this difference was diminished at 5 years (23 vs. 25%; p = 0.797). CONCLUSIONS: Six cycles of chemotherapy reduced the risk of relapse within 2 years, but the benefit from two additional cycles beyond this time is questionable.

The role of calprotectin in obstetrics and gynecology.

The purpose of this article is to create the first complete review concerning the role of calprotectin, a calcium- and zinc-binding protein of the S100/calgranulins family, in obstetrics and gynecology. A Medline search was conducted between 6 and 8 June 2009 using the term calprotectin and its synonyms combined with the following ones: calprotectin, obstetrics and gynecology, breast cancer, ovarian cancer, endometrial cancer, cervical cancer, menstrual cycle, pregnancy, fetal implantation, labor, intra-amniotic inflammation, preeclampsia, HELLP syndrome, Rh(-) incompatibility. We found 46 studies which referred to obstetrics and gynecology. We excluded 11 studies which referred to obstetrics and gynecology but did not include enough information about calprotectin, and another two which referred to calprotectin but were not related to subjects of obstetrics and gynecology. Thus, we ended up with 33 studies which contained sufficient information to extract data for this review. All the articles were written in English. It was found that calprotectin is associated with many physiologic and pathologic processes in obstetrics and gynecology, such as: breast cancer, ovarian cancer, endometrial cancer, cervical cancer, cervical and vaginal physiology, menstrual cycle, pregnancy and labor. The role of calprotectin in these conditions is significant. In conclusion, the role of calprotectin seems to be important in several issues of obstetrics and gynecology. For example, calprotectin could be used as a diagnostic, prognostic or metastatic marker in several types of cancer, as a marker of inflammation and as a pharmaceutical target in many conditions. Further studies must be conducted to elucidate this role.

Quantitative analysis of heparanase gene expression in normal cervical, cervical intraepithelial neoplastic, and cervical carcinoma tissues.

Heparanase is an endoglycosidase that specifically cleaves heparan sulfate side chains of heparan sulfate proteoglycans, the major proteoglycans in the extracellular matrix and cell surfaces. Traditionally, heparanase activity was implicated in cellular invasion associated with angiogenesis, inflammation, and cancer metastasis. More recently, heparanase up-regulation was documented in an increasing number of primary human tumors. Iotan this study, we sought to investigate the expression of heparanase messenger RNA (mRNA) in normal cervical tissue and intraepithelial cervical lesion and its clinicopathologic importance in invasive cervical cancer. Gene expression of heparanase was assessed by quantitative real-time reverse transcriptase polymerase chain reaction in 28 normal cervical, 26 intraepithelial neoplastic, and 48 cervical cancer tissue samples. Heparanase mRNA expression was different between the 3 groups and lower in normal cervical specimens in relationship with intraepithelial cervical lesions and invasive cervical cancer tissue samples (P = 0.048). Gradually increasing expression of heparanase was evident as the cells progressed from low-grade to high-grade squamous intraepithelial lesions (P = 0.002). In invasive cervical cancer cases, there was a direct correlation between heparanase expression and tumor size (P = 0.002). In cases treated with radical hysterectomy and pelvic lymphadenectomy, the heparanase mRNA expression was significantly higher in tumors exhibiting lymph vascular space invasion (P = 0.044) and in cases with big tumor size (P = 0.005). In our study, we did not find any significant correlation between disease-free and overall survival rates and expression of heparanase (P = 0.396 and P = 0.712, respectively). The results of this study suggest that the gene expression of heparanase in cervical cancer enhances growth, invasion, and angiogenesis of the tumor and may have therapeutic applications.

Paraneoplastic humorally mediated hypercalcemia induced by parathyroid hormone-related protein in gynecologic malignancies: a systematic review.

Humoral hypercalcemia of malignancy (HHM) is a metabolic phenomenon that is mediated by the paraneoplastic secretion of parathyroid hormone-related peptide (PTHrP). Gynecologic malignant neoplasms complicated by HHM have been reported for organs such as the uterus, cervix, ovary, vulva and the vagina. The purpose of our study was to perform a review of the published cases in the literature and, further, to identify parameters with effect on outcome. Among 34 women with gynecologic neoplasms, 22 suffered from ovarian and 6 from uterine malignancies, while 3 had vulvar and another 3 cervical cancer. Furthermore, clear cell carcinoma was the predominant histology associated with PTH-rP expression. A significant correlation was found between serum calcium and PTH-rP levels. Treatment of hypercalcemia was successful in all cases; pamidronate was utilized in 8 patients. Ovarian cancer patients with severe hypercalcemia and high PTH-rP serum levels had shorter survival compared to their counterparts with mild hypercalcemia or moderately elevated PTH-rP serum levels, but the differences were not statistically significant.

Corifollitropin alfa, a long-acting follicle-stimulating hormone agonist for the treatment of infertility.

Corifollitropin alfa is being developed by Schering-Plough Corp as an injectable, long-acting follicle-stimulating hormone (FSH) agonist for the treatment of infertility. A single dose of corifollitropin alfa could initiate and sustain multifollicular growth in patients undergoing controlled ovarian stimulation, such as during in vitro fertilization or intracytoplasmic sperm injection. The agent comprises an alpha-subunit, which is identical to that of FSH, and a beta-subunit, which is produced by the fusion of the C-terminal peptide from the beta-subunit of chorionic gonadotropin to the beta-subunit of FSH. Corifollitropin alfa has a longer half-life compared with FSH and thus requires less frequent dosing. The drug was well tolerated and does not appear to be associated with any serious adverse events or the formation of antibodies. The initial results from a large, phase III, double-blind clinical trial indicated that the ongoing pregnancy rate achieved with corifollitropin alfa treatment was high and similar to the rate established with daily treatment of recombinant FSH. The number of oocytes retrieved following the administration of corifollitropin alfa was slightly higher compared with the number observed with daily recombinant FSH treatment. Thus, corifollitropin alfa has the potential to serve as a viable fertility agent and to gain a place in the infertility market.

Molecular and proteomic characterization of human mesenchymal stem cells derived from amniotic fluid: comparison to bone marrow mesenchymal stem cells.

Human mesenchymal stem cells (hMSCs) constitute a population of multipotent adherent cells able to give rise to multiple mesenchymal lineages such as osteoblasts, adipocytes, or chondrocytes. So far, the most common source of MSCs has been the bone marrow (BM); however BM-MSC harvesting and processing exhibits major drawbacks and limitations. Thus, identification and characterization of alternative sources of MSCs are of great importance. In the present study, we isolated and expanded fetal MSCs from second-trimester amniotic fluid (AF). We documented that these cells are of embryonic origin, can differentiate under appropriate conditions into cell types derived from all three germ layers, and express the pluripotency marker Oct-4, the human Nanog protein, and the stage-specific embryonic antigen-4 (SSEA-4). Furthermore, we systematically tested the immunophenotype of cultured MSCs by flow cytometry analysis using a wide variety of markers. Direct comparison of this phenotype to the one derived from cultured BM-MSCs demonstrated that cultured MSCs from both sources exhibit similar expression patterns. Using the two-dimensional gel electrophoresis and matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF-MS) approach, we have generated for the first time the protein map of cultured AF-MSCs by identifying 261 proteins, and we compared it directly to that of cultured BM-MSCs. The functional pattern of the identified proteins from both sources was similar. However, cultured AF-MSCs displayed a number of unique proteins related to proliferation and primitive phenotype, which may confer to the distinct features of the two types. Considering the easy access to this new cell source and the yield of expanded MSCs for stem cell research, AF may provide an excellent source of MSCs both for basic research and for potential therapeutic applications.

Fully automated FISH examination of amniotic fluid cells.

OBJECTIVE: Fluorescence in situ hybridization (FISH) analysis has become a valuable adjunct in cytogenetics, providing a rapid screen for common chromosome abnormalities that is particularly helpful in prenatal diagnosis. FISH analysis using standard microscopy is expensive and labor intensive, requiring both a high skill level and subjective signal interpretation. A reliable fully automated system for FISH analysis could improve laboratory efficiency and potentially reduce errors and costs. METHODS: The efficacy of an automated system was compared to standard manual FISH analysis. Two sets of slides were generated from each of 152 amniotic fluid samples. Following hybridization with a standard panel of five chromosome FISH probes, one set of slides was evaluated using manual microscopy. The other set was evaluated using an automated microscopy system. RESULTS: A diagnostic outcome was obtained for all 152 samples using manual microscopy and for 146 of 152 (96%) samples using automated microscopy. Three cases of aneuploidy were detected. For those samples for which a diagnostic outcome was determined by both manual and automated microscopy, 100% concordance was observed. All FISH analysis results were confirmed by karyotype. CONCLUSION: These data suggest that an automated microscopy system is capable of providing accurate and rapid enumeration of FISH signals in amniocytes.

Estradiol and progesterone supplementation during luteal phase improved the receptivity of the endometrium in a patient with a history of diethylstilboestrol exposure in-utero.

BACKGROUND: Diethylstilboestrol (DES) exposure in-utero has been shown to have negative effects on pregnancy. DES-exposed women are at increased risk of early spontaneous pregnancy loss, ectopic gestation and infertility. DESIGN: A 34-year old woman with a 6-year history of primary infertility is presented. The patient underwent in vitro fertilization (IVF) treatment without success. To improve the quality of the endometrium following IVF treatment, E2 and progesterone supplementation was added to the usual therapeutic regimen. The pregnancy progressed uneventfully and a normal female was born. CONCLUSIONS: This case indicates that the administration of E2 and progesterone in DES-exposed women might improve endometrium receptivity and consequently pregnancy outcome.

Oocyte maturation in assisted reproductive techniques.

Human oocyte maturation is a long process during which nuclear maturation occurs resulting in germinal vesicle breakdown (transition from prophase I to metaphase II) and extrusion of the first polar body. During oocyte maturation, in parallel with nuclear maturation, a number of events take place in the oocyte cytoplasm that assist fertilization and early embryonic development. So far several attempts have been made to mature human oocytes in vitro. The main patient group to which in vitro maturation (IVM) has been applied is polycystic ovarian syndrome. In a concise review we present the techniques used for the IVM of oocytes and the role of hormones and growth factors in IVM and subsequent fertilization and early embryonic development.