Pietro Cavalli

Results from the International Consensus Conference on Myo-inositol and d-chiro-inositol in Obstetrics and Gynecology: the link between metabolic syndrome and PCOS.

In recent years, interest has been focused to the study of the two major inositol stereoisomers: myo-inositol (MI) and d-chiro-inositol (DCI), because of their involvement, as second messengers of insulin, in several insulin-dependent processes, such as metabolic syndrome and polycystic ovary syndrome. Although these molecules have different functions, very often their roles have been confused, while the meaning of several observations still needs to be interpreted under a more rigorous physiological framework. With the aim of clarifying this issue, the 2013 International Consensus Conference on MI and DCI in Obstetrics and Gynecology identified opinion leaders in all fields related to this area of research. They examined seminal experimental papers and randomized clinical trials reporting the role and the use of inositol(s) in clinical practice. The main topics were the relation between inositol(s) and metabolic syndrome, polycystic ovary syndrome (with a focus on both metabolic and reproductive aspects), congenital anomalies, gestational diabetes. Clinical trials demonstrated that inositol(s) supplementation could fruitfully affect different pathophysiological aspects of disorders pertaining Obstetrics and Gynecology. The treatment of PCOS women as well as the prevention of GDM seem those clinical conditions which take more advantages from MI supplementation, when used at a dose of 2g twice/day. The clinical experience with MI is largely superior to the one with DCI. However, the existence of tissue-specific ratios, namely in the ovary, has prompted researchers to recently develop a treatment based on both molecules in the proportion of 40 (MI) to 1 (DCI).

Results from the International Consensus Conference on myo-inositol and D-chiro-inositol in Obstetrics and Gynecology – assisted reproduction technology

Abstract A substantial body of research on mammalian gametogenesis and human reproduction has recently investigated the effect of myo-inositol (MyoIns) on oocyte and sperm cell quality, due to its possible application to medically assisted reproduction. With a growing number of both clinical and basic research papers, the meaning of several observations now needs to be interpreted under a solid and rigorous physiological framework. The 2013 Florence International Consensus Conference on Myo- and D-chiro-inositol in obstetrics and gynecology has answered a number of research questions concerning the use of the two stereoisomers in assisted reproductive technologies. Available clinical trials and studies on the physiological and pharmacological effects of these molecules have been surveyed. Specifically, the physiological involvement of MyoIns in oocyte maturation and sperm cell functions has been discussed, providing an answer to the following questions: (1) Are inositols physiologically involved in oocyte maturation? (2) Are inositols involved in the physiology of spermatozoa function? (3) Is treatment with inositols helpful within assisted reproduction technology cycles? (4) Are there any differences in clinical efficacy between MyoIns and D-chiro-inositol? The conclusions of this Conference, drawn depending on expert panel opinions and shared with all the participants, are summarized in this review paper.

Effects of Inositol supplementation in a cohort of mothers at risk of producing an NTD pregnancy

Neural tube defects (NTDs), most commonly spina bifida and anencephaly, can be prevented with periconceptional intake of folic acid in about 70% of cases. Recurrence of NTDs despite supplementation of high dose of folic acid further suggests that a proportion of NTD cases might be resistant to folic acid. Moreover, heterogeneity of NTDs has been suggested in animal studies, indicating that only some sub-type of NTDs should be considered sensitive to folate intake. Inositol isomers (particularly myo- and chiro-inositol) can prevent folate-resistant NTDs in the curly-tail mutant mouse, suggesting that some cases of human NTDs might benefit from inositol supplementation. In humans, lower inositol blood concentration was found in pregnant women carrying NTD fetuses, whereas a periconceptional combination therapy with folic acid associated with inositol has been linked to normal live births, despite high NTD recurrence risk. Fifteen pregnancies from 12 Caucasian women from different parts of Italy with at least one previous NTD-affected pregnancy underwent periconceptional combined myo-inositol and folic acid supplementation. Maternal serum α-feto-protein levels were found in the normal range, and normal results on ultrasound examination were found in all the pregnancies that followed. No collateral effects or intense uterine contractions were demonstrated in this pilot study in any of the pregnancies after inositol supplementation, and seventeen babies were born without any type of NTD.

Wolf–Hirschhorn syndrome-associated chromosome changes are not mediated by olfactory receptor gene clusters nor by inversion polymorphism on 4p16

The basic genomic defect in Wolf–Hirschhorn syndrome (WHS), including isolated 4p deletions and various unbalanced de novo 4p;autosomal translocations and above all t(4p;8p), is heterogeneous. Olfactory receptor gene clusters (ORs) on 4p were demonstrated to mediate a group of WHS-associated t(4p;8p)dn translocations. The breakpoint of a 4-Mb isolated deletion was also recently reported to fall within the most distal OR. However, it is still unknown whether ORs mediate all 4p-autosomal translocations, or whether they are involved in the origin of isolated 4p deletions. Another unanswered question is whether a parental inversion polymorphism on 4p16 can act as predisposing factor in the origin of WHS-associated rearrangements. We investigated the involvement of the ORs in the origin of 73 WHS-associated rearrangements. No hotspots for rearrangements were detected. Breakpoints on 4p occurred within the proximal or the distal olfactory receptor gene cluster in 8 of 73 rearrangements (11%). These were five t(4p;8p) translocations, one t(4p;7p) translocation and two isolated terminal deletions. ORs were not involved in one additional t(4p;8p) translocation, in a total of nine different 4p;autosomal translocations and in the majority of isolated deletions. The presence of a parental inversion polymorphism on 4p was investigated in 30 families in which the 4p rearrangements, all de novo, were tested for parental origin (7 were maternal and 23 paternal). It was detected only in the mothers of 3 t(4p;8p) cases. We conclude that WHS-associated chromosome changes are not usually mediated by low copy repeats. The 4p16.3 inversion polymorphism is not a risk factor for their origin.

Design and validation of a pericentromeric BAC clone set aimed at improving diagnosis and phenotype prediction of supernumerary marker chromosomes

BackgroundSmall supernumerary marker chromosomes (sSMCs) are additional, structurally abnormal chromosomes, generally smaller than chromosome 20 of the same metaphase spread. Due to their small size, they are difficult to characterize by conventional cytogenetics alone. In regard to their clinical effects, sSMCs are a heterogeneous group: in particular, sSMCs containing pericentromeric euchromatin are likely to be associated with abnormal outcomes, although exceptions have been reported. To improve characterization of the genetic content of sSMCs, several approaches might be applied based on different molecular and molecular-cytogenetic assays, e.g., fluorescent in situ hybridization (FISH), array-based comparative genomic hybridization (array CGH), and multiplex ligation-dependent probe amplification (MLPA).To provide a complementary tool for the characterization of sSMCs, we constructed and validated a new, FISH-based, pericentromeric Bacterial Artificial Chromosome (BAC) clone set that with a high resolution spans the most proximal euchromatic sequences of all human chromosome arms, excluding the acrocentric short arms.ResultsBy FISH analysis, we assayed 561 pericentromeric BAC probes and excluded 75 that showed a wrong chromosomal localization. The remaining 486 probes were used to establish 43 BAC-based pericentromeric panels. Each panel consists of a core, which with a high resolution covers the most proximal euchromatic ~0.7 Mb (on average) of each chromosome arm and generally bridges the heterochromatin/euchromatin junction, as well as clones located proximally and distally to the core. The pericentromeric clone set was subsequently validated by the characterization of 19 sSMCs. Using the core probes, we could rapidly distinguish between heterochromatic (1/19) and euchromatic (11/19) sSMCs, and estimate the euchromatic DNA content, which ranged from approximately 0.13 to more than 10 Mb. The characterization was not completed for seven sSMCs due to a lack of information about the covered region in the reference sequence (1/19) or sample insufficiency (6/19).ConclusionsOur results demonstrate that this pericentromeric clone set is useful as an alternative tool for sSMC characterization, primarily in cases of very small SMCs that contain either heterochromatin exclusively or a tiny amount of euchromatic sequence, and also in cases of low-level or cryptic mosaicism. The resulting data will foster knowledge of human proximal euchromatic regions involved in chromosomal imbalances, thereby improving genotype–phenotype correlations.

Array CGH in routine prenatal diagnosis practice

The past 20 years witnessed an explosion in genetic research and genetic tests, leading to improved knowledge about diagnosis of genetic conditions. However, many genetic tests are marketed on the basis of limited scientific information, leading to the risk of misuse and overuse and prospective physical and/or psychological consequences to the public. The practice of medicine requires that every new test should be evaluated in terms of performance, quality control, effectiveness, and usefulness. Starting from 2000, the ACCE evaluation process was developed by the Centres for Disease Control and Prevention as a model system to assess safety and effectiveness of DNA-based genetic tests. ACCE takes its name from the terms analytical validity, clinical validity, clinical utility, and associated ethical, legal and social implications. After reading the article by Fiorentino et al. suggesting that aCGH might be introduced into routine prenatal diagnosis as a first line diagnostic test, we came up with the above considerations. Moreover, even if the use of aCGH is associated with an increased detection rate of chromosomal abnormalities both in post-natal and in prenatal diagnosis, little is known about the standardization and the optimal resolution of arrays, the cost-effectiveness of those tests, the uptake of genetic counselling, the psychological and emotional response of the couple, and the clinical management of uncertainties linked to the large number of copy number variants (CNVs) with no major phenotypic effects found in many healthy individuals. Not to mention forensic problems that might arise, for example, after the choice to terminate or to continue a pregnancy on the basis of uncertain or incomplete diagnostic data. Most medical decisions are based on a Bayesian process, which combines clinical findings and laboratory tests to make a diagnosis, where genetic tests should be considered as a means, not an end goal. It has been claimed that laboratory tests are decision-support tools, not clinical decisionreplacement tools. Undoubtedly, aCGH may improve the diagnostic process, but only if used within a comprehensive medical approach. For all those reasons, it is still wise, in our opinion, to use aCGH in prenatal diagnosis according to ultrasound fetal abnormalities and genotype-phenotype correlation in unexpected chromosomal findings in the fetus (e.g. supernumerary marker chromosomes, de novo chromosomal rearrangements). With these considerations in mind, we will discuss the data presented by Fiorentino et al. particularly the clinically significant aCGH results, not detected by conventional karyotyping and listed in Table 3. Indications for prenatal diagnosis in 5/9 (55%) cases reported in Table 3 were abnormal ultrasound findings that, in the presence of a normal karyotype, most laboratories will further study by aCGH in any case, according to plain common sense and good medical practice. In 3/9 cases (33%) the deletion/duplication size was well above the estimated size of 10Mb, which conventional karyotyping at the 400-band level should detect reliably. As a suggestion, it would be of interest to compare molecular cytogenetic analysis with good quality conventional karyotyping, because a resolution of less than 400 bands is considered to be inappropriate for routine chromosome analysis. Two cases (case 1 and case 4) had inherited 17p12 deletion/ duplication. It would be interesting to assess the clinical utility of those findings in prenatal diagnosis. According to those observations, we suggest that the claimed clinical sensitivity of aCGH used as a first-line prenatal choice should be corrected in the above mentioned paper, and a figure of 0.29% (3/1027) instead of 0.9% (9/1027) would probably better represent the improvement over current protocols based on conventional karyotyping and aCGH as second line analysis in high risk pregnancies. Besides this small increase in sensitivity, other issues need to be taken into account, including incomplete penetrance and variable clinical expressivity of CNVs, late-onset CNVassociated diseases, variants of unknown significance, CNVs containing an imprinted gene, recessive conditions unmasked by inherited deletions, CNVs falling in gene-desert regions and much more. Case 9 (Table 3) is emblematic of the difficulty to translate a laboratory result into a clinically relevant information. In fact, the inherited duplication found at 22q11.21, leading to termination of pregnancy in that case, is often associated with an unpredictable phenotype, depending on the penetrance of that specific CNV.

The future of prenatal diagnosis: karyotype, microarray or both? Technical and ethical considerations.

Evaluation of: Wapner RJ, Martin CL, Levy B et al. Chromosomal microarray versus karyotyping for prenatal diagnosis. N. Engl. J. Med. 367(23), 2175–2184 (2012). Prenatal diagnosis is now offered to high-risk pregnancies, including advanced maternal age, ultrasound anomalies and positive Down’s syndrome screening, and karyotype on cultured fetal material is the test of choice to screen these pregnancies. However, microscope analysis can only detect gross chromosome abnormalities, highlighting the need for more sensitive techniques. It has recently been established that the higher resolution of microarray-based platforms can increase the diagnostic yield, offering more information to couples, and it is being discussed as a replacement to the standard karyotype. Conversely, the very high sensitivity of microarray-based analysis allows us to detect small microdeletions/microduplications (copy number variations) with unknown functional role and difficult genotype/phenotype correlation. In addition, the new copy number variation syndromes are often associated with variable outcomes, ranging from normal to severely affected individuals. This means that the microarray-based analysis introduced routinely in prenatal diagnosis needs to answer the question: are laboratory staff, clinical geneticists and counselors really experienced enough to manage these new scenarios?

Acrania-Anencephaly Associated with Hypospadias. Prenatal Ultrasound and MRI Diagnosis and Molecular Folate Metabolism Pathway Analysis

Acrania may occur as a single isolated malformation or associated with extracranial defects. Hypospadias is one of the most common congenital abnormalities of the genitalia frequently missed on prenatal sonograms. Second trimester two- and three-dimensional ultrasound and MRI diagnosis with necropsy and folate metabolism pathway analysis. The mechanisms leading to closure of both neural and urethral tubes, are far from being demonstrated, and molecular studies of this very rare association are lacking although it might be based on a common genetic mechanism, leading to a disturbed development pathway at the molecular level.

Anencephaly-exencephaly sequence and congenital diaphragmatic hernia in a fetus with 46, XX karyotype: Early prenatal diagnosis, necropsy, and maternal folate pathway genetic analysis.

Neural tube defects (NTDs) are a group of serious birth defects that affect the developing nervous system and include acrania, anencephaly, and encephalocele. Acrania may manifest as a partial or complete absence of the cranial vault and of cerebral hemispheres (forebrain) with abnormal brain development. Anencephaly instead is generally defined as the congenital absence of skull, scalp, and forebrain. Acrania occurs more infrequently than anencephaly and may be diagnosed prenatally during the first trimester [1, 2]. It may occur as a single, isolated malformation or with co-existing anomalies including extracranial defects such as rachischisis, hypoplastic ears, cleft lip and palate, omphalocele, limb defects, cardiac and kidney

First trimester diagnosis of iniencephaly associated with fetal malformations and trisomy 18: Report of a new case and gene analysis on folate metabolism in parents

ABSTRACT  Iniencephaly is a rare congenital malformation consisting of a complex alteration of the embryonic development occurring around the third post‐fertilization week and characterized by a hyper‐retroflexion of the cephalic pole. We report a case of iniencephaly associated with acrania‐encephalocele, spina bifida and abnormal ductus venosus in a fetus with trisomy 18 diagnosed at 12 weeks gestation in a 41‐year‐old woman. A co‐occurrence between aneuploidy and iniencephaly was documented and polymorphisms on folate metabolism‐related genes were investigated in the parents to assess possible etiologic factors and recurrence risk for neural tube defects (NTD). An homozygous state for the MTRR polymorphism was diagnosed in the mother, identifying a clinical risk for NTD. Once iniencephaly or any other NTD are suspected, genetic analysis, second level ultrasound and fetal karyotype are recommended. Autopsy should also be performed in all cases of early ultrasound‐based diagnosis of fetal malformations.