Metabolomic Investigation of ß-Thalassemia in Chorionic Villi Samples.

BACKGROUND: Beta-thalassemias are blood disorders characterized by poorly understood clinical phenotypes ranging from asymptomatic to severe anemia. Metabolic composition of the human placenta could be affected by the presence of pathological states such as ß-thalassemia. The aim of our study was to describe metabolic changes in chorionic villi samples of fetuses affected by ß-thalassemia compared to a control group by applying a metabolomics approach. METHODS: Chorionic villi samples were differentiated according to the genetic diagnosis of ß-thalassemia: control (Group 1, n = 27); heterozygous (Group 2, n = 7); homozygous (Group 3, n = 7). Gas chromatography-mass spectrometry was used to detect the metabolic composition of the samples. Subsequently, multivariate and univariate statistical analysis was performed. The discriminant metabolites were used to identify the altered pathways. RESULTS: Supervised multivariate models were devised to compare the groups. The model resulting from the comparison between Group 1 and Group 3 was the most significant. Discriminant metabolites were identified, and the most altered pathways were as follows: pentose phosphate pathway (PPP), arachidonic acid metabolism, glycolysis, and gluconeogenesis, suggesting the presence of an energetic shift toward the PPP and the presence of oxidative stress in ß-thalassemia chorionic villi samples. CONCLUSIONS: The metabolomics approach identified a specific metabolic fingerprint in chorionic villi of fetuses affected by ß-thalassemia.

Metabolic fingerprinting of chorionic villous samples in normal pregnancy and chromosomal disorders.

OBJECTIVE: Placenta-related biological samples are used in biomedical research to investigate placental development. Metabolomics represents a promising approach for studying placental metabolism in an effort to explain physiological and pathological mechanisms. The aim of this study was to investigate metabolic changes in chorionic villi during the first trimester of pregnancy in euploid and aneuploid cases. METHODS: Samples from 21 women (13 euploid and eight aneuploid) were analyzed with 1 H-nuclear magnetic resonance (NMR), gas chromatography-mass spectrometry (GC-MS), and high-performance liquid chromatography (HPLC). Multivariate statistical analysis was performed, and differences in metabolites were used to identify the altered metabolic pathways. RESULTS: A regression model to test the correlation between fetal crown-rump length (CRL) and metabolic profile of chorionic villi was performed in euploid pregnancies (R2 was 0.69 for the NMR analysis and 0.94 for the GC-MS analysis). Supervised analysis was used to compare chorionic villi of euploid and aneuploid fetuses (NMR: R2 X = 0.70, R2 Y = 0.65, Q2  = 0.30, R2 X = 0.62; GC-MS: R2 Y = 0.704, Q2  = 0.444). Polyol pathways, myo-inositol, and oxidative stress seem to have a fundamental role in euploid and aneuploid pregnancies. CONCLUSION: Polyol pathways may have a crucial role in energy production in early pregnancy. Excessive activation in aneuploid pregnancies may lead to increased oxidative stress. Metabolomics represents a promising approach to investigate placental metabolic changes.

Metabolic characterization of amniotic fluids of fetuses with enlarged nuchal translucency.

Background In prenatal diagnosis, a thickened nuchal translucency (NT) is one of the most sensitive and specific markers for several defects but it may also be found in 5% of healthy fetuses. The pathophysiological causes that lead to an increase in NT are not yet fully understood. Metabolomics represents a new promising approach, useful for studying different metabolites in biological organisms in response to environmental stressors. The aim of our study was to investigate the metabolomic profile of the amniotic fluid samples (AFS) of euploid fetuses with enlarged nuchal translucency (ENT) compared to a control group (C group). Methods This study was carried out on a group of women who underwent second-trimester amniocentesis for advanced maternal age (C group) or for NT ≥95th percentile (ENT group) found during first-trimester aneuploidy screening. AFS were analyzed with proton nuclear magnetic resonance (1H-NMR) and high-performance liquid chromatography (HPLC), and subsequent multivariate and univariate statistical analyses were conducted, followed by pathway analysis. Results In total, 67 AFS from the C group and 23 from the ENT group were analyzed. Partial least square discriminate analysis was carried out (R2X=0.784, R2Y=0.658, Q2=0.622, P<0.0001). A different metabolic profile was observed in the ENT group compared with the C group, suggesting an energetic shift to a glycolytic phenotype in an oxidative environment in the ENT group compared to the C group. Conclusion Metabolomic studies enable the identification of metabolic alterations occurring in fetuses with ENT. These findings may provide a new basis for better understanding the pathophysiological mechanisms in this prenatal phenomenon.

From Prenatal to Preimplantation Genetic Diagnosis of ß-Thalassemia. Prevention Model in 8748 Cases: 40 Years of Single Center Experience.

The incidence of ß-thalassemia in Sardinia is high and ß-39 is the most common mutation. The prevention campaign started in 1977 and was performed in a single center (Microcitemico Hospital, Cagliari, Sardinia, Italy). It was based on educational programs, population screening by hematological and molecular identification of the carriers. Prenatal and pre-implantation diagnosis was offered to couples at risk. 8564 fetal diagnosis procedures using different invasive approaches and analysis techniques were performed in the last 40 years. Trans-abdominal chorionic villous sampling was preferred due to lower complication risks and early diagnosis. Chorionic villous DNA was analyzed by PCR technique. 2138 fetuses affected by ß-thalassemia were diagnosed. Women opted for termination of the pregnancy (TOP) in 98.2% of these cases. Pre-implantation genetic diagnosis (PGD) was proposed to couples at risk to avoid TOP. A total of 184 PGD were performed. Initially, the procedure was exclusively offered to infertile couples, according to the law in force. The success rate of pregnancies increased from 11.1% to 30.8% when, crucial law changes were enacted, and PGD was offered to fertile women as well. Forty years of ß-thalassemia prevention programs in Sardinia have demonstrated the important decrease of this severe genetic disorder.

Early fetal megacystis: Is it possible to predict the prognosis in the first trimester?

OBJECTIVE: To evaluate the best management of fetal megacystis diagnosed in the first trimester and define the prognosis and the most appropriate follow-up as early as possible. METHODS: This is a retrospective study of first-trimester fetal megacystis diagnosed in pregnant women who performed a combined screening for fetal aneuploidy. Megacystis was defined as a longitudinal bladder diameter (LBD) greater than 7 mm. All fetuses were divided into two groups according to the LBD: Group A with LBD > 15 mm and Group B with LBD < 15 mm. The fetal karyotype and associated anomalies were evaluated. Ultrasound monitoring was performed every 2 weeks (a second ultrasound scan after 2 weeks from diagnosis and a third ultrasound scan 2 weeks after the second one). RESULTS: Twenty-six cases were identified between 2011 and 2016; three cases of aneuploidy were excluded from the study. Of the remaining 23 cases, 11 were included in Group A and 12 in Group B. All Group A fetuses had an adverse outcome. In Group B: five (41.7%) cases had an adverse outcome and seven (58.3%) had a spontaneous resolution of megacystis. The ultrasound findings of both the ultrasound scans, the second and the third, were 100% concordant. CONCLUSION: An ultrasound scan performed 2 weeks after the megacystis diagnosis can predict the outcome in fetuses with an LBD < 15 mm as early as the end of the first trimester. The outcome of euploid fetuses with an LBD < 15 mm was favorable in 58.3% of the cases.

Past, present and future ultrasonographic techniques for analyzing ovarian masses.

Ultrasonography is today the method of choice for distinguishing between benign and malignant adnexal pathologies. Using pattern recognition several types of tumors can be recognized according to their characteristic appearance on gray-scale imaging. Color Doppler imaging should be used only to perform a semiquantitative color score or evaluate the flow location. International Ovarian Tumor Analysis group had standardized definitions characterizing adnexal masses and suggested the use of 'simple rules' in premenopausal women. Recently, the use of 3D vascular indices has been proposed but its potential use in clinical practice is debated. Also computerized aided diagnosis algorithms showed encouraging results to be confirmed in the future.

Three-dimensional ultrasonography in the diagnosis of deep endometriosis.

STUDY QUESTION: In the use of 'tenderness-guided' transvaginal ultrasound, is the diagnostic accuracy of three-dimensional (3D) ultrasonography better than two-dimensional (2D) ultrasonography in the identification of deep endometriosis? SUMMARY ANSWER: Three-dimensional ultrasonography has a significantly higher diagnostic accuracy in the diagnosis of posterior locations of deep endometriosis without intestinal involvement, such as the uterosacral ligaments, vaginal and rectovaginal endometriosis. WHAT IS KNOWN ALREADY: The only previous study of the diagnosis of posterior compartment endometriosis reported an poor sensitivity of 3D ultrasonography for uterosacral and sigmoid colon involvement. STUDY DESIGN, SIZE, DURATION: This diagnostic test study included 202 patients scheduled for surgery because of clinical suspicion of deep pelvic endometriosis and was carried out between January 2009 and September 2012. PARTICIPANTS/MATERIALS, SETTING, METHODS: Modified transvaginal ultrasonography was performed on all of the women by a single examiner. Two locations of deep endometriosis were considered: intestinal involvement and other posterior lesions (including vaginal location, rectovaginal septum and uterosacral ligaments). Once the 2D ultrasonography had been performed, the 3D acquisition was performed and the obtained volume was stored. To avoid the risk of recall bias, the same operator evaluated the 3D volumes 6 months after the last examination using virtual navigation to provide a presumptive diagnosis of the presence and localization of deep endometriosis. In addition, to evaluate the reproducibility of 3D, two operators with different levels of expertise performed a retrospective review of 3D volumes from a random sample of 35 patients, twice, 1 week apart to also assess intraobserver agreement. The diagnostic performance of both tests was expressed as area under the receiver-operating characteristics curve (AUC), sensitivity, specificity, positive and negative predictive values, positive (LR+) and negative (LR-) likelihood ratios, with their respective 95% confidence interval (CI). Reproducibility was evaluated using kappa statistics. MAIN RESULTS AND THE ROLE OF CHANCE: Surgery revealed deep endometriosis in 129 patients. The AUCs for endometriosis of intestinal location were similar for both ultrasound techniques. The AUCs for endometriosis of other posterior locations were significantly different (0.891, 95% CI 0.839-0.943 for 3D versus 0.789, 95% CI 0.720-0.858 for 2D; P = 0.0193). For the intestinal involvement, the specificity, sensitivity, positive and negative predictive value, and LR+ and LR- were 93% (89-95%), 95% (88-98%), 89% (83-92%), 97% (93-99%), 13, and 0.06, respectively, for 2D ultrasound and 97% (93-99%), 91% (84-94%), 95% (88-98%), 95% (91-96%), 25, and 0.09, respectively, for 3D ultrasound. For other posterior locations, the specificity, sensitivity, positive and negative predictive value, and LR+ and LR- were 88% (82-93%), 71% (64-77%), 83% (75-90%), 79% (74-83%), 6.10, 0.32, respectively, for 2D ultrasound and 94% (89-97%), 87% (81-91%), 92% (86-96%), 90% (85-93%), 14.0, 0.14, respectively, for 3D ultrasound. Intraobserver agreement was substantial for both examiners (kappa 0.8754, for operator A and 0.7087, for operator B, respectively). Interobserver agreement was also substantial. LIMITATIONS, REASONS FOR CAUTION: The disadvantages of 3D ultrasound to be considered are the necessity of newer ultrasonographic equipment and that fewer sonographers completely know the 3D technique. There are also some limitations within this study. First, an expert examiner performed the real-time ultrasound and 3D volume acquisitions. Second, the same operator also performed the 3D evaluations but at least 6 months after the last acquisition to avoid a possible recall bias. WIDER IMPLICATIONS OF THE FINDINGS: The diagnostic performance obtained in the present study is superior to the accuracy reported in other studies of 3D ultrasonography, but not superior to all other published articles of 2D ultrasonography. The reported high diagnostic accuracy of 3D ultrasound could be widely generalizable because good reproducibility was demonstrated even with an operator with less expertise. STUDY FUNDING/COMPETING INTEREST(S): This study was supported in part by the Regione Autonoma della Sardegna (project code CPR-24750).

Assessing the reproducibility of the IOTA simple ultrasound rules for classifying adnexal masses as benign or malignant using stored 3D volumes.

OBJECTIVE: To analyze the reproducibility of the IOTA simple ultrasound rules for classifying adnexal masses as benign or malignant among examiners with different level of expertise using stored 3D volumes of adnexal masses. STUDY DESIGN: Five examiners, with different levels of experience and blinded to each other, evaluated 100 stored 3D volumes from adnexal masses and looked for the presence or absence of malignant or benign features according to the IOTA definitions. Multiplanar view and virtual navigation were used. All examiners had to assess the 3D volume of each adnexal mass and classify it as benign or malignant. To analyze intra-observer agreement each examiner performed the assessment twice with a two-week interval between the first and second assessments. To analyze the inter-observer agreement, the second assessment from each examiner was used. Reproducibility was assessed calculating the weighted Kappa index. RESULTS: Intra-observer reproducibility was moderate or good for all observers (Kappa index ranging from 0.59 to 0.74). Inter-observer reproducibility was moderate to good (Kappa index range: 0.46-0.67). CONCLUSIONS: The simple rules are reasonably reproducible among observers with different level of expertise when assessed in stored 3D volumes.