Metabolomics in Prenatal Medicine: A Review.

Pregnancy is a complicated and insidious state with various aspects to consider, including the well-being of the mother and child. Developing better non-invasive tests that cover a broader range of disorders with lower false-positive rates is a fundamental necessity in the prenatal medicine field, and, in this sense, the application of metabolomics could be extremely useful. Metabolomics measures and analyses the products of cellular biochemistry. As a biomarker discovery tool, the integrated holistic approach of metabolomics can yield new diagnostic or therapeutic approaches. In this review, we identify and summarize prenatal metabolomics studies and identify themes and controversies. We conducted a comprehensive search of PubMed and Google Scholar for all publications through January 2020 using combinations of the following keywords: nuclear magnetic resonance, mass spectrometry, metabolic profiling, prenatal diagnosis, pregnancy, chromosomal or aneuploidy, pre-eclampsia, fetal growth restriction, pre-term labor, and congenital defect. Metabolite detection with high throughput systems aided by advanced bioinformatics and network analysis allowed for the identification of new potential prenatal biomarkers and therapeutic targets. We took into consideration the scientific papers issued between the years 2000-2020, thus observing that the larger number of them were mainly published in the last 10 years. Initial small metabolomics studies in perinatology suggest that previously unidentified biochemical pathways and predictive biomarkers may be clinically useful. Although the scientific community is considering metabolomics with increasing attention for the study of prenatal medicine as well, more in-depth studies would be useful in order to advance toward the clinic world as the obtained results appear to be still preliminary. Employing metabolomics approaches to understand fetal and perinatal pathophysiology requires further research with larger sample sizes and rigorous testing of pilot studies using various omics and traditional hypothesis-driven experimental approaches.

Recommendations for neonatologists and pediatricians working in first level birthing centers on the first communication of genetic disease and malformation syndrome diagnosis: consensus issued by 6 Italian scientific societies and 4 parents' associations.

BACKGROUND: Genetic diseases are chronic conditions with relevant impact on the lives of patients and their families. In USA and Europe it is estimated a prevalence of 60 million affected subjects, 75% of whom are in developmental age. A significant number of newborns are admitted in the Neonatal Intensive Care Units (NICU) for reasons different from prematurity, although the prevalence of those with genetic diseases is unknown. It is, then, common for the neonatologist to start a diagnostic process on suspicion of a genetic disease or malformation syndrome, or to make and communicate these diagnoses. Many surveys showed that the degree of parental satisfaction with the methods of communication of diagnosis is low. Poor communication may have short and long-term negative effects on health and psychological and social development of the child and his family. We draw up recommendations on this issue, shared by 6 Italian Scientific Societies and 4 Parents' Associations, aimed at making the neonatologist's task easier at the difficult time of communication to parents of a genetic disease/malformation syndrome diagnosis for their child. METHODS: We used the method of the consensus paper. A multidisciplinary panel of experts was first established, based on the clinical and scientific sharing of the thematic area of present recommendations. They were suggested by the Boards of the six Scientific Societies that joined the initiative: Italian Societies of Pediatrics, Neonatology, Human Genetics, Perinatal Medicine, Obstetric and Gynecological Ultrasound and Biophysical Methodologies, and Pediatric Genetic Diseases and Congenital Disabilities. To obtain a deeper and global vision of the communication process, and to reach a better clinical management of patients and their families, representatives of four Parents' Associations were also recruited: Italian Association of Down People, Cornelia de Lange National Volunteer Association, Italian Federation of Rare Diseases, and Williams Syndrome People Association. They worked from September 2019 to November 2020 to achieve a consensus on the recommendations for the communication of a new diagnosis of genetic disease. RESULTS: The consensus of experts drafted a final document defining the recommendations, for the neonatologist and/or the pediatrician working in a fist level birthing center, on the first communication of genetic disease or malformation syndrome diagnosis. Although there is no universal communication technique to make the informative process effective, we tried to identify a few relevant strategic principles that the neonatologist/pediatrician may use in the relationship with the family. We also summarized basic principles and significant aspects relating to the modalities of interaction with families in a table, in order to create an easy tool for the neonatologist to be applied in the daily care practice. We finally obtained an intersociety document, now published on the websites of the Scientific Societies involved. CONCLUSIONS: The neonatologist/pediatrician is often the first to observe complex syndromic pictures, not always identified before birth, although today more frequently prenatally diagnosed. It is necessary for him to know the aspects of genetic diseases related to communication and bioethics, as well as the biological and clinical ones, which together outline the cornerstones of the multidisciplinary care of these patients. This consensus provide practical recommendations on how to make the first communication of a genetic disease /malformation syndrome diagnosis. The proposed goal is to make easier the informative process, and to implement the best practices in the relationship with the family. A better doctor-patient/family interaction may improve health outcomes of the child and his family, as well as reduce legal disputes with parents and the phenomenon of defensive medicine.

Prenatal screening diagnosis and management in the era of coronavirus: the Sardinian experience.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), a new coronavirus, was first identified in December 2019 in Wuhan, China and spread rapidly, affecting many other countries. The disease is now referred to as coronavirus disease 2019 (COVID-19).The Italian government declared a state of emergency on 31st January 2020 and on 11th March World Health Organization (WHO) officially declared the COVID-19 outbreak a global pandemic. Although the COVID-19 incidence remained considerably lower in Sardinia than in the North Italy regions, which were the most affected, the field of prenatal screening and diagnosis was modified because of the emerging pandemic. Data on COVID-19 during pregnancy are so far limited. Since the beginning of the emergency, our Ob/Gyn Department at Microcitemico Hospital, Cagliari offered to pregnant patients all procedures considered essential by the Italian Ministry of Health. To evaluate the influence of the COVID-19 pandemic on the activities of our center, we compared the number of procedures performed from 10th March to 18th May 2020 with those of 2019. Despite the continuous local birth rate decline, during the 10-week pandemic period, we registered a 20% increment of 1st trimester combined screening and a slight rise of the number of invasive prenatal procedures with a further increase in chorionic villi sampling compared to amniocentesis. Noninvasive prenatal testing remained unvariated. The request for multifetal pregnancy reduction as a part of the growing tendency of voluntary termination of pregnancy in Sardinia increased. The COVID-19 pandemic provides many scientific opportunities for clinical research and study of psychological and ethical issues in pregnant women.

The decline of amniocentesis and the increase of chorionic villus sampling in modern perinatal medicine.

Objective The aims of this study were to determine the rate of change by type of diagnosis by transabdominal chorionic villus sampling (TA-CVS) vs. amniocentesis for aneuploidy and to describe a successful and intensive international training program for TA-CVS in ongoing pregnancies. Methods We conducted a retrospective cohort study of all deliveries from 2010 to 2018 in Sardinia. All invasive diagnostic procedures are conducted at a single regional perinatal referral center. Descriptive statistics were used to compare data across groups, and inter-correlations between variables were investigated by Pearson's correlation coefficient. We subsequently describe the international trainee experiences in TA-CVS over a 35-year period. Results A total of 101,025 deliveries occurred over 9 years. The number of deliveries (13,413-9143, P < 0.0001) and total invasive diagnostic procedures (1506-858 per year, P = 0.019) declined over this period. The percentage of deliveries undergoing invasive diagnostic procedures remained steady (mean: 12.2%). In 2010, TA-CVS made up 32.3% of all invasive diagnostic procedures, while amniocentesis made up 67.7%. By 2018, TA-CVS made up 61.3% of the invasive diagnostic procedures, and amniocentesis, only 38.7%. The rate of TA-CVS increased over 9 years, while the rate of amniocentesis declined. A total of 236 trainees from 39 different countries and 5 different continents rotated through this site. The average length of stay was 2.4 weeks. Conclusion We demonstrate an increasing prevalence of TA-CVS vs. amniocentesis in the current era of prenatal testing and underscore the importance of continuing to train specialists skilled in TA-CVS. Our global operative experience is feasible and sustainable and will have a lasting impact on physicians conducting invasive fetal procedures.

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.

Pregnancy outcome and long-term follow-up of fetuses with isolated increased NT: a retrospective cohort study.

OBJECTIVE: To assess the prevalence of associated findings, diagnosed either prior to or after birth, among fetuses with increased nuchal translucency (NT) and normal karyotype and to investigate the prevalence of children under therapy at school age, among cases confirmed to be isolated both prior to and after birth. METHODS: Retrospective cohort study of all singleton pregnancies were referred for the 1st trimester screening for Down syndrome between January 2004 and December 2006. Cases with NT >95th percentiles who undertook fetal karyotype were analyzed. We evaluated the prevalence of the following: (i) abnormal fetal karyotype, (ii) associated structural abnormalities, (iii) genetic syndromes, and (iv) children needing treatment (i.e. medications or psychopedagogical intervention) at school age. RESULTS: There were 19 106 women screened for Down syndrome. Among them, 846 (4.4%) had a NT >95th centile, of whom 541 (64.0%) undertook fetal karyotyping, 422 (78.0%) had normal karyotype, two (0.5%) opted for termination of pregnancy, and four (1.0%) ended in miscarriages. Among the 384 cases thought to be "normal" prior to birth, 14 (3.6%) were lost at follow-up and 10 (2.7%) had an associated abnormality diagnosed postnatally. Among the cases confirmed to be isolated, 10/270 (3.7%) were under treatment at the time of telephonic interview with parents. CONCLUSIONS: Parents should be informed that fetuses with increased NT and normal karyotype need detailed ultrasound scans during pregnancy and detailed pediatric evaluation at birth; if these are normal, the risk from adverse outcome at school age is the same as for general population.

Single center experience in selective feticide in high-order multiple pregnancy: clinical and ethical issues.

OBJECTIVE: This paper describes the 20-year experience with selective feticide (SF) of high-order multiple quadruplet and higher pregnancies in a single center. METHODS: The paper describes protocols, procedures, management, outcomes, and ethical issues. RESULTS: SF was performed in 49 pregnancies with 244 fetuses, with median gestational age of 12+2 weeks. The initial number was nine (one case), eight (one case), seven (three cases), six (11 cases), five (eight cases), and four (27 cases). Nuchal translucency was utilized prior to the procedure starting in 1996. The technique was transabdominal ultrasound-guided and intrathoracic injection of potassium chloride. One pregnancy (with seven fetuses) was reduced to three, 42 to two, and four (starting with four fetuses) to singletons. There were ten pregnancy losses (20.4%). A decreasing trend in losses was evident over the 20-year time period: 7/23 (30.4%) from 1994 to 2004 down to 3/26 (11.5%) for 2004-2014. No chromosomal abnormalities were present in any of the survivors. The ethical issues focus on the justification of SF in high-order multifetal pregnancies. CONCLUSION: In this series, pregnancy loss decreased with operator experience. Excellent outcomes can be achieved with the ethically justified use of feticide in high-order multiple pregnancies.

How to perform transabdominal chorionic villus sampling: a practical guideline.

The spread of both first trimester screening for chromosomal abnormalities and the possibility to check for single gene disorders at DNA-analysis has increased the request for chorionic villus sampling (CVS) in the first trimester. In order to perform placental biopsy, two routes are possible: the transcervical (TC) and the transabdominal (TA). In early days, the trancervical technique was the most diffused, but since its introduction into clinical practice, the TA technique has become the approach of choice in detriment of the TC technique. In our institution, we have a 30-year experience in TA-CVS with more than 26 000 procedures performed. Considering the expertise and the volume of procedures undertaken at our unit, we suggest a practical guideline for novel operators in TA-CVS.

Brain stem/brain stem occipital bone ratio and the four-line view in nuchal translucency images of fetuses with open spina bifida.

Objective: Brain stem depth/brain stem occipital bone distance (BS/BSOB ratio) and the four-line view, in images obtained for nuchal translucency (NT) screening in fetuses with open spina bifida (OSB).Methods: Single center, retrospective study based on the assessment of NT screening images of fetuses with OSB. A ratio between the BS depth and the BSOB distance was calculated (BS/BSOB ratio) and the four-line view observed, and the sensitivity for a BS/BSOB ratio superior/equal to 1, and for the lack of detection of the four-line view were calculated.Results: There were 17 cases of prenatal diagnosis OSB. In six cases, the suspicion on OSB was raised during NT screening, in six cases, the diagnosis was made before 20 weeks and in five cases during anomaly scan. The BS/BSOB ratio was superior/equal to 1 in all 17 cases, and three lines, were visualized in 15/17 images of the OSB cases, being the sensitivity 100% (95% CI, 81 to 100%) and 88% (95% CI, 65 to 96%).Conclusion: Assessment of BS/BSOB ratio and four-line view in NT images is feasible detecting affected by OSB with high sensitivity. The presence of associated anomalies or of an enlarged NT enhances the early detection.