Observational, prospective, multicentre study to evaluate the effects of counselling on the choice of combined hormonal contraceptives in Italy--the ECOS (Educational COunselling effectS) study.

BACKGROUND: Adequate counselling on contraceptive methods can help users choose the most appropriate method. The aim of this study was to assess the effects of structured counselling provided by gynaecologists on selection of a combined hormonal contraception method. METHODS: Women aged 18-40 years (n = 1871) who were considering the use of a combined hormonal contraception method (pill, transdermal patch or vaginal ring) underwent a structured counselling session in which gynaecologists provided comprehensive information. Pre- and post-counselling questionnaires on combined hormonal contraception choice were completed by participants. RESULTS: After counselling, many women (38 %) selected a combined hormonal contraception method that was different from the originally intended one. Preferences for the transdermal patch approximately doubled (from 3.2 % pre-counselling to 7 %; p < 0.0001) and those for the vaginal ring increased four-fold (from 5.2 to 21.2 %; p < 0.0001), while preference for the pill remained unchanged (from 64.5 % [pre-] to 64.1 % [post-counselling]). The proportion of undecided women decreased from 18 to 2.1 % (p < 0.0001). The main reasons for choosing a method were related to ease of use (all methods), and preferences for administration frequency (daily, weekly or monthly). The number of patients requiring post-counselling contact with the physician's office was low (5.1-6.9 %), as was the incidence of adverse events (1.8-3.1 %). CONCLUSIONS: Counselling has a significant impact on women's choice of combined hormonal contraception and encourages them to consider alternative methods to combined oral contraceptives. Moreover, it also enables women to use their chosen method with confidence. TRIAL REGISTRATION: NCT01181778 , Trial registration date: August 12, 2010.

Premature ovarian insufficiency in young girls: repercussions on uterine volume and bone mineral density.

STUDY OBJECTIVE: To evaluate biological differences among young subjects with premature ovarian insufficiency (POI) commencing at different stages of life. DESIGN: Retrospective observational study. SETTING: Careggi University Hospital Participants: One hundred sixty-two females aged between 15 and 29 years with premature ovarian insufficiency. METHODS: Data were collected as a retrospective chart review of baseline evaluation at diagnosis of premature ovarian insufficiency (POI). About 162 participants were divided into four groups based on gynecological age. Two primary outcome variables (uterine development and bone mineral density (BMD)) were analyzed in terms of differences among groups and in a multivariate logistic regression analysis. RESULTS: Uterine development was clearly jeopardized when estrogen insufficiency started at a very young age. Total body BMD showed significant differences among the four groups studied, clearly corresponding to the duration of ovarian function. Data were discussed in relation to the choice of hormone replacement therapy regimens.

Fetal striatal grafting slows motor and cognitive decline of Huntington's disease.

OBJECTIVE: To assess the clinical effect of caudate-putaminal transplantation of fetal striatal tissue in Huntington's disease (HD). METHODS: We carried out a follow-up study on 10 HD transplanted patients and 16 HD not-transplanted patients. All patients were evaluated with the Unified HD Rating Scale (UHDRS) whose change in motor, cognitive, behavioural and functional capacity total scores were considered as outcome measures. Grafted patients also received morphological and molecular neuroimaging. RESULTS: Patients were followed-up from disease onset for a total of 309.3 person-years (minimum 5.3, median 11.2 years, maximum 21.6 years). UHDRS scores have been available since 2004 (median time of 5.7 years since onset, minimum zero, maximum 17.2 years). Median post-transplantation follow-up was 4.3 years, minimum 2.8, maximum 5.1 years. Adjusted post-transplantation motor score deterioration rate was reduced compared to the pretransplantation period, and to that of not-transplanted patients by 0.9 unit/years (95% CI 0.2 to 1.6). Cognitive score deterioration was reduced of 2.7 unit/years (95% CI 0.1 to 5.3). For grafted patients the 2-year post-transplantation [(18)F]fluorodeoxyglucose positron emission tomography (PET) showed striatal/cortical metabolic increase compared to the presurgical evaluation; 4-year post-transplantation PET values were slightly decreased, but remained higher than preoperatively. [(123)I]iodobenzamide single photon emission CT demonstrated an increase in striatal D2-receptor density during postgrafting follow-up. CONCLUSIONS: Grafted patients experienced a milder clinical course with less pronounced motor/cognitive decline and associated brain metabolism improvement. Life-time follow-up may ultimately clarify whether transplantation permanently modifies the natural course of the disease, allowing longer sojourn time at less severe clinical stage, and improvement of overall survival.

Young Human Cholinergic Neurons Respond to Physiological Regulators and Improve Cognitive Symptoms in an Animal Model of Alzheimer's Disease.

The degeneration of cholinergic neurons of the nucleus basalis of Meynert (NBM) in the basal forebrain (BF) is associated to the cognitive decline of Alzheimer's disease (AD) patients. To date no resolutive therapies exist. Cell-based replacement therapy is a strategy currently under consideration, although the mechanisms underlying the generation of stem cell-derived NBM cholinergic neurons able of functional integration remain to be clarified. Since fetal brain is an optimal source of neuronal cells committed towards a specific phenotype, this study is aimed at isolating cholinergic neurons from the human fetal NBM (hfNBMs) in order to study their phenotypic, maturational and functional properties. Extensive characterization confirmed the cholinergic identity of hfNBMs, including positivity for specific markers (such as choline acetyltransferase) and acetylcholine (Ach) release. Electrophysiological measurements provided the functional validation of hfNBM cells, which exhibited the activation of peculiar sodium (INa) and potassium (IK) currents, as well as the presence of functional cholinergic receptors. Accordingly, hfNBMs express both nicotinic and muscarinic receptors, which were activated by Ach. The hfNBMs cholinergic phenotype was regulated by the nerve growth factor (NGF), through the activation of the high-affinity NGF receptor TrkA, as well as by 17-ß-estradiol through a peculiar recruitment of its own receptors. When intravenously administered in NBM-lesioned rats, hfNBMs determined a significant improvement in memory functions. Histological examination of brain sections showed that hfNBMs (labeled with PKH26 fluorescent dye prior to administration) reached the damaged brain areas. The study provides a useful model to study the ontogenetic mechanisms regulating the development and maintenance of the human brain cholinergic system and to assess new lines of research, including disease modeling, drug discovery and cell-based therapy for AD.

Fetal cells in a transcervical cell sample collected at 5 weeks of gestation.

Transcervical cell (TCC) sampling is being investigated as a promising method for obtaining fetal cells for prenatal genetic diagnosis. The present case report describes the identification of fetal cells by both fluorescent in situ hybridisation (FISH) and quantitative fluorescent polymerase chain reaction (QF-PCR) analyses in a TCC sample collected by intrauterine lavage at 5 + 0 weeks. This finding underscores the possible relevance of TCC sampling for extremely early prenatal genetic diagnosis.

Donor-Specific Anti-HLA Antibodies in Huntington's Disease Recipients of Human Fetal Striatal Grafts.

Fetal grafting in a human diseased brain was thought to be less immunogenic than other solid organ transplants, hence the minor impact on the efficacy of the transplant. How much prophylactic immune protection is required for neural allotransplantation is also debated. High-sensitive anti-HLA antibody screening in this field has never been reported. Sixteen patients with Huntington's disease underwent human fetal striatal transplantation in the frame of an open-label observational trial, which is being carried out at Florence University. All patients had both brain hemispheres grafted in two separate robotic-stereotactic procedures. The trial started in February 2006 with the first graft to the first patient (R1). R16 was given his second graft on March 2011. All patients received triple immunosuppressive treatment. Pre- and posttransplant sera were analyzed for the presence of anti-HLA antibodies using the multiplexed microsphere-based suspension array Luminex xMAP technology. Median follow-up was 38.5 months (range 13-85). Six patients developed anti-HLA antibodies, which turned out to be donor specific. Alloimmunization occurred in a time window of 0-49 months after the first neurosurgical procedure. The immunogenic determinants were non-self-epitopes from mismatched HLA antigens. These determinants were both public epitopes shared by two or more HLA molecules and private epitopes unique to individual HLA molecules. One patient had non-donor-specific anti-HLA antibodies in her pretransplant serum sample, possibly due to previous sensitization events. Although the clinical significance of donor-specific antibodies is far from being established, particularly in the setting of neuronal transplantation, these findings underline the need of careful pre- and posttransplant immunogenetic evaluation of patients with intracerebral grafts.

Use of the quantitative fluorescent-PCR assay in the study of fetal DNA from micromanipulated transcervical samples.

AIM: The purpose of this study was to evaluate the validity of the combined use of micromanipulation and quantitative fluorescent (QF)-PCR assay for the identification of fetal elements in transcervical cell (TCC) samples collected in early pregnancy. METHODS: TCC samples were obtained by intrauterine lavage (IUL) in 113 pregnant women who were between 7 and 12 weeks pregnant before termination of pregnancy. All IUL samples were screened under an inverted microscope, at which time the isolation of fetal cells by micromanipulation was attempted. QF-PCR assay, using 9 small tandem repeat (STR) markers for chromosomes 13, 18, 21, X, and Y, was performed in all specimens to identify fetal cells in TCC samples and the corresponding placental tissue and blood specimens. TCC samples from male fetuses in which either the micromanipulation or QF-PCR analysis were unsuccessful, were studied with fluorescent in situ hybridization (FISH), using probes for X and Y chromosomes. RESULTS: Isolation of supposed fetal material from IUL samples was carried out by means of micromanipulation in 93 cases (82.3%), where discernible chorionic villous filaments or cell clumps of probable trophoblastic origin were present. The QF-PCR analysis was performed in all 93 IUL samples and paternal peaks could be documented in 88 cases (94.6%) thus confirming the presence of fetal cells. Thirteen cases negative to micromanipulation and derived from male fetuses and four male cases not informative with QF-PCR analysis, after micromanipulation, were then tested with FISH assay using probes for sexual chromosomes. In six samples, rare (2-3%) male fetal cells were detected. Considering the combined results obtained from QF-PCR and FISH assays, the overall fetal sexing was correct in 83.2% of cases (94 of 113). CONCLUSION: This study provides evidence that fetal cells are present in a high proportion of IUL samples. Micromanipulation appears to be an extremely efficient method for the isolation of trophoblastic elements. This study also confirms the potential of IUL as a possible alternative to the traditional prenatal diagnostic procedures for the recovery of fetal cells in precocious stage of gestation, and validates the combination of the isolation of such fetal elements by means of micromanipulation and analysis with the QF-PCR assay for the identification of the most frequent prenatal chromosomal aneuploidies.

Premature ovarian failure and fragile X premutation: a study on 45 women.

OBJECTIVE: The aim of this study was to test for the presence of the fragile X (FRAXA) premutation a group of women with early menopause. STUDY DESIGN: 45 women with idiopathic premature ovarian failure (POF), five with a familial and 40 with a sporadic form, were screened for the presence of FRAXA premutation. A control group of 28 women >45 years, with one or more children and no signs of POF, was also studied. RESULTS: We found three cases of fragile X premutations in women all belonging to the group with sporadic POF. CONCLUSION: Our results seems to confirm previous observations on the non random association between POF and FRAXA premutation.

Human striatal neuroblasts develop and build a striatal-like structure into the brain of Huntington's disease patients after transplantation.

Rebuilding brain structure and neural circuitries by transplantation of fetal tissue is a strategy to repair the damaged nervous system and is currently being investigated using striatal primordium in Huntington's disease (HD) patients. Four HD patients underwent bilateral transplantation with human fetal striatal tissues (9-12 week gestation). Small blocks of whole ganglionic eminencies were processed to obtain cell suspension and then stereotactically grafted in the caudate head and in the putamen. Follow-up period ranged between 18 and 34 months (mean, 24.7 months). Surgery was uneventful. Starting from the fourth month after grafting, neo-generation of metabolically active tissue with striatal-like MRI features was observed in 6 out of 8 grafts. The increase in D2 receptor binding suggested striatal differentiation of the neo-generated tissue in 3 patients. New tissue, connecting the developing grafts with the frontal cortex and, in one case, with the ventral striatum, was also observed. The new tissue growth halted after the ninth month post transplantation. All patients showed stabilization or improvement in some neurological indices. No clinical and imaging signs, suggestive of graft uncontrolled growth, were seen. This study provides the first evidence in humans that neuroblasts of a striatal primordium can develop and move into the brain after neurotransplantation. Primordium development resulted in the building of a new structure with the same imaging features as the corresponding mature structure, combined with short- and long-distance targeted migration of neuroblasts. The results of this study support both the reconstructive potential of fetal tissue and the remarkably retained plasticity of adult brain. Further studies are necessary to assess the clinical efficacy of the human fetal striatal transplantation.

The presence of trophoblastic cells in intrauterine lavage samples: lack of correlation with maternal and obstetric characteristics.

OBJECTIVES: To investigate the correlation between maternal, obstetric and sample characteristics and the quality (i.e. yield of trophoblastic cells) of intrauterine lavage (IUL) samples. METHODS: We collected 202 IUL samples from women scheduled for first trimester termination of pregnancy (TOP). Trophoblastic cells were isolated from IUL samples and used for DNA analysis by a multiplex quantitative fluorescent polymerase chain reaction (QF-PCR) assay. A multivariate logistic regression analysis was performed, and a p<0.05 was considered statistically significant. RESULTS: The presence of trophoblastic cells in IUL samples was documented in 151/202 cases (74.7%). Blood contamination of IULs was the only characteristic to positively correlate with the presence of trophoblasts (p=0.039; OR: 1.99; 95% CI: 1.03-3.82). CONCLUSIONS: The correlation between the presence of contaminating blood and trophoblastic cells would indirectly confirm the hypothesis that IUL might act as a mini-CVS. The high yield rate of trophoblasts irrespective of maternal characteristics and past obstetric history would support the clinical use of this sampling technique, provided that its safety is clearly defined.

Development of human striatal anlagen after transplantation in a patient with Huntington's disease.

Replacement of damaged neuronal population by fetal tissue transplantation represents a potential treatment for neurodegenerative diseases. Consistent success has been achieved with fetal striatal transplantation in Huntington's disease animal models and patients. We report the neo-generation of metabolically active tissue with striatum-like imaging features after transplantation of striatal primordia in a patient with Huntington's disease. This study represents the first "in vivo" demonstration that a human striatal anlagen, transplanted into the adult human brain, is able to progress in its development and to generate a new anatomical structure in the host, without evidence of neoplasia or teratoma.

Comparison of two techniques for transcervical cell sampling performed in the same study population.

OBJECTIVES: The aim of this study was to evaluate and compare the presence of fetal cells in transcervical cell (TCC) samples collected in the first trimester of pregnancy by two different procedures [mucus collection and intrauterine lavage (IUL)], performed consecutively in the same subjects scheduled for elective termination of pregnancy (TOP). METHODS: A total of 126 mucus/IUL sample pairs were retrieved from pregnant women immediately before TOP at a gestational age ranging from 7 to 12 weeks; at termination, samples of placental tissue were collected in all cases. All mucus samples were analysed by a polymerase chain reaction (PCR) assay and, in a subset of experiments involving 56 specimens, also by fluorescence in situ hybridization (FISH) procedure. IULs were divided in two aliquots, one for PCR analysis and one for the preparation of FISH slides. All placental tissue samples obtained at termination were analysed by FISH for fetal sexing. The PCR assay for fetal sex determination was performed by using, in a multiplex reaction, primers for SRY (Y chromosome sex-determining region, 738 bp) and HUMARA (human androgen receptor on the X chromosome, 280 bp) genes. The FISH analysis was carried out using direct-labelled commercial probes for X chromosome alpha-satellite (DXZ1, Xp11.1-q11.1, spectrum green) and Y chromosome alpha-satellite (DYZ3, Yp11.1-q11.1, spectrum orange) regions. RESULTS: In samples from known male pregnancies (n = 67), full concordance between IUL and mucus results could be found in 11 cases (16.4%); in 41 cases, Y chromosome material was detected by FISH (n = 2), by PCR (n = 5) or both (n = 34) in IUL samples, but not in the corresponding mucus samples. Y chromosome material was not documented in 10 mucus/IUL sample pairs. In 5 cases, the FISH (n = 2), the PCR (n = 1) or both (n = 2) failed to detect Y chromosome material in IULs, which was detected, however, by PCR in the corresponding mucus samples. Overall, correct sex prediction was achieved in 55/67 IULs (82%) and in 16/67 (23.9%) mucus samples from male pregnancies. In samples from known female pregnancies (n = 56), full concordance between results of IUL/mucus pairs and those on placental samples could be found in 53 cases (94.6%); in 3 cases, Y chromosome material was documented by PCR in mucus samples, but not in the corresponding IULs. Correct sex prediction was therefore achieved in 56/56 IULs (100%) and in 53/56 (94.6%) mucus samples from female pregnancies. CONCLUSION: This study provides evidence that, among TCC sampling techniques, IUL, but not mucus collection, can yield fetal cells in a constant and reliable fashion, which is a basic prerequisite for possible clinical usage. This suggestion had already emerged from some previous investigations but, owing to the study design, differences in study populations can no longer be used to explain the very different and sometimes-conflicting results reported in earlier studies.

Fetal cells in cervical mucus in the first trimester of pregnancy.

OBJECTIVES: The aim of this study was to first evaluate the presence of fetal cells in cervical mucus samples collected in the first trimester of pregnancy and then to compare different laboratory methods for the detection of these cells. METHODS: Mucus samples were collected by using a cytobrush before termination of pregnancy (TOP) from 143 pregnant women between 7 and 12 weeks of gestation. None of the women had undergone an invasive diagnostic procedure prior to cervical mucus sampling. Samples of placental tissue were collected from each patient at TOP. Slides from each sample were first observed under an inverted microscope to detect possible sperm contamination. In the first part of our experiments, 40 mucus samples were treated with a mucolytic solution containing N-acetylcysteine (AC) and were analysed by a polymerase chain reaction (PCR) assay. The second series, consisting of 71 mucus samples, was treated with a mucolytic solution containing dithiothreitol (DTT): all 71 samples were analysed by a PCR-based assay, and an aliquot for fluorescent in situ hybridisation (FISH) analysis was also obtained from 48 out of 71 samples. In the third part of our experiments, performed on 32 mucus samples, mucus trapped on the cytobrush was directly spread on two slides for FISH analysis without any mucolytic treatment. All placental tissue samples obtained at termination were analysed by FISH for fetal sexing. RESULTS: Overall, the use of PCR-based or FISH analyses on 143 mucus samples resulted in correct sex prediction in 92/143 (64.3%) samples [20/66 (30.3%) cases from known male pregnancies and 72/77 (93.5%) cases from known female pregnancies]. In the AC group, Y-derived sequences were found in 7/23 samples (30.4%) from known male pregnancies and in 1/17 cases from known female pregnancies, with an overall correct sex prediction in 23/40 cases (57.5%). In the DTT group, Y-derived sequences could be amplified in 10/30 samples (33.3%) from known male pregnancies and in 4/41 cases from known female pregnancies, with an overall correct sex prediction in 47/71 cases (66.2%). In the DTT samples analysed by FISH, nuclei bearing XY signals were detected in 5/26 (19.2%) cases from known male pregnancies and in none from female pregnancies, the rate of correct sex prediction being 56.2% (27/48). On untreated mucus samples analysed by FISH, nuclei with XY signals were documented in 3/13 (23%) samples from male conceptuses and in none from known female pregnancies, with an overall correct sex prediction in 22/32 cases (68.7%). CONCLUSION: Fetal cells were not detected in a constant and reliable fashion in cervical mucus samples collected in the first trimester of pregnancy. The detection rate was poorly influenced by the use of different laboratory methods. This sampling technique cannot be regarded as a promising tool towards minimally invasive prenatal diagnosis.

Strategies for the isolation and detection of fetal cells in transcervical samples.

OBJECTIVE: The aim of the study was to evaluate the detection of fetal cells from transcervical samples, collected in early pregnancy, by means of different molecular techniques. The value of the isolation of trophoblasts using an inverted microscope, also referred to as micromanipulation, is discussed. METHODS: All the 89 specimens were obtained by intrauterine lavages before termination of pregnancy (TOP), between 7 and 12 weeks of gestation. Micromanipulation was carried out in a subgroup of 57 for the isolation of fetal material. Fetal sexing was achieved by FISH (fluorescent in situ hybridisation) using fluorescently labelled probes for X and Y chromosomes and by polymerase chain reaction (PCR). Male samples were also investigated for aneuploidy of the chromosome 21. Quantitative fluorescent (QF)-PCR using two short tandem repeat (STR) markers for chromosome 21 was carried out in 26 micromanipulated samples. RESULTS: FISH analysis revealed that 45/89 placental samples derived from pregnancies with male fetuses. Correct sexing of the lavage samples from male pregnancies was achieved in 41/45 (91%) using dual-FISH technique, and in 43/45 (95.5%) with PCR. All the samples derived from male pregnancies tested for chromosome 21 were normal. From 57 samples subjected to micromanipulation, 51 (89.5%) showed discernible chorionic villous filaments or cell clumps of possible trophoblastic origin. One case of tetraploidy and two cases of monosomy were recorded. The rate of fetal cells, in the non-micromanipulated samples, was between 4% and 97% (mean 54.3%). In micromanipulated specimens, maternal contaminant cells were absent or extremely rare (1-2%). The efficiency of the QF-PCR analysis in detecting paternal peaks in all lavage samples was only 61.5%. CONCLUSION: The present study confirms the presence of fetal cells in a very high proportion of both whole and micromanipulated intrauterine lavage samples. The isolation of trophoblastic elements can be achieved in most cases by micromanipulation. FISH and PCR techniques allowed the analysis of the most common fetal aneuploidies, confirming the power of this minimally invasive method.

Detection of fetal cells in intrauterine lavage samples collected in the first trimester of pregnancy.

OBJECTIVES: The aim of the present study was first to evaluate the presence of fetal cells in transcervical cell (TCC) samples collected by intrauterine lavage in the first trimester of pregnancy, and then to compare different methods for the detection of these cells. METHODS: TCC samples were collected by intrauterine lavage before termination of pregnancy (TOP) from 81 pregnant women between 7 and 12 weeks of gestation. Samples of placental tissue were collected from each patient at TOP, whereas maternal peripheral blood samples were obtained in 57 cases. DNA extracted from 81 lavage and the corresponding placental samples was amplified by a polymerase chain reaction (PCR) assay using primers for SRY and HUMARA genes. All 81 lavage samples were also analysed by fluorescent in situ hybridisation (FISH) using direct-labelled probes for X chromosome alpha-satellite (DXZ1, Xp11.1-q11.1) and Y chromosome alpha-satellite (DYZ3, Yp11.1-q11.1) regions. In 57 cases, a quantitative fluorescent (QF) PCR assay, involving the use of two small tandem repeat (STR) markers (D21S11, D21S14.11) specific to chromosome 21 was employed to analyse DNA extracted from placental tissue, lavage and maternal blood samples. RESULTS: PCR analysis revealed that 40/81 placental samples were from male pregnancies. Correct sexing was achieved with the PCR technique in 30/40 (75%) lavage samples retrieved from pregnant women with male conceptuses and in all 41 (100%) samples collected from pregnancies with female fetuses. With the FISH analysis, nuclei bearing X and Y signals were observed in 32/40 cases (80%) from known male pregnancies, the rate of fetal cells ranging between 2% and 95%, whereas nuclei showing X and Y signals were not detected in any of the 41 lavage samples from known female pregnancies. Paternal peaks were present in 30/57 (52.6%) lavage samples tested by QF-PCR. CONCLUSION: The results suggest that fetal cells can be found, at a significant rate, in a very high proportion of intrauterine lavage samples. Therefore, this sampling technique can be regarded as a promising tool towards minimally invasive prenatal diagnosis. The FISH and PCR methods showed a similar efficiency in detecting fetal cells.