Investigating the "Fetal Side" in Recurrent Pregnancy Loss: Reliability of Cell-Free DNA Testing in Detecting Chromosomal Abnormalities of Miscarriage Tissue.

(1) Background: The aim of our study is to evaluate whether cell-free DNA testing can overlap the genetic testing of miscarriage tissue in women with early pregnancy loss (EPL) and length of recurrent pregnancy loss (RPL); (2) Methods: We conducted a prospective cohort study at the Pregnancy Loss Unit of the Fondazione Policlinico Universitario A. Gemelli (IRCCS), Rome, Italy between May 2021 and March 2022. We included women with EPL and length of RPL. Gestational age was >9 weeks + 2 days and <12 weeks + 0 days of gestation corresponding to a crown rump length measurement of >25 and <54 mm. Women underwent both dilation and curettage for the collection of miscarriage tissue and for blood sample collection. Chromosomal microarray analysis (CMA) on miscarriage tissues was performed by oligo-nucleotide- and single nucleotide polymorphisms (SNP)-based comparative genomic hybridization (CGH+SNP). Maternal blood samples were analyzed by Illumina VeriSeq non-invasive prenatal testing (NIPT) to evaluate the cell-free fetal DNA (cfDNA) and the corresponding fetal fraction and the presence of genetic abnormalities; (3) Results: CMA on miscarriage tissues revealed chromosome aneuploidies in 6/10 cases (60%), consisting of trisomy 21 (5 cases) and monosomy X (one case). cfDNA analysis was able to identify all cases of trisomy 21. It failed to detect monosomy X. A large 7p14.1p12.2 deletion concomitant to trisomy 21 was, in one case, detected by cfDNA analysis but it was not confirmed by CMA on miscarriage tissue. (4) Conclusions: cfDNA largely reproduces the chromosomal abnormalities underlying spontaneous miscarriages. However, diagnostic sensitivity of cfDNA analysis is lower with respect to the CMA of miscarriage tissues. In considering the limitations when obtaining biological samples from aborted fetuses suitable for CMA or standard chromosome analysis, cfDNA analysis is a useful, although not exhaustive, tool for the chromosome diagnosis of both early and recurrent pregnancy loss.

Effects of Simulated Microgravity on Wild Type and Marfan hiPSCs-Derived Embryoid Bodies.

BACKGROUND: Mechanical unloading in microgravity is thought to induce tissue degeneration by various mechanisms, including the inhibition of regenerative stem cell differentiation. In this work, we investigate the effects of microgravity simulation on early lineage commitment of hiPSCs from healthy and Marfan Syndrome (MFS; OMIM #154700) donors, using the embryoid bodies model of tissue differentiation and evaluating their ultra-structural conformation. MFS model involves an anomalous organization of the extracellular matrix for a deficit of fibrillin-1, an essential protein of connective tissue. METHODS: In vitro models require the use of embryoid bodies derived from hiPSCs. A DRPM was used to simulate microgravity conditions. RESULTS: Our data suggest an increase of the stemness of those EBs maintained in SMG condition. EBs are still capable of external migration, but are less likely to distinguish, providing a measure of the remaining progenitor or stem cell populations in the earlier stage. The microgravity response appears to vary between WT and Marfan EBs, presumably as a result of a cell structural component deficiency due to fibrillin-1 protein lack. In fact, MFS EBs show a reduced adaptive capacity to the environment of microgravity that prevented them from reacting and making rapid adjustments, while healthy EBs show stem retention, without any structural changes due to microgravity conditions. CONCLUSION: EBs formation specifically mimics stem cell differentiation into embryonic tissues, this process has also significant similarities with adult stem cell-based tissue regeneration. The use of SMG devices for the maintenance of stem cells on regenerative medicine applications is becoming increasingly more feasible. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s12195-021-00680-1.

Mutation analysis of the FBN1 gene in a cohort of patients with Marfan Syndrome: A 10-year single center experience.

BACKGROUND: Marfan Syndrome (MFS) is a chronic, life-threatening, autosomal dominant connective tissue disorder caused by mutations in the FBN1 gene, coding for fibrillin-1. All organ systems may be affected, but particularly the cardiovascular system, eyes, and skeleton. Mortality generally results from cardiovascular complications, mainly aortic dissection. Currently, the diagnosis of MFS is based on the revised Ghent nosology. Molecular analysis of the FBN1 gene reduces diagnostic uncertainty in patients with suspected MFS or MFS-related disorders (MFS-RD). To date, more than 2700 FBN1 mutations are known. METHODS: Using Next Generation Sequencing (NGS) followed by Multiplex Ligation-dependent Probe Amplification on NGS-negative samples, we screened FBN1 gene on 124 unrelated patients (101 MFS fulfilling revised Ghent criteria, 20 suspected MFS, 3 MFS-RD) enrolled from 2008 to 2018 at the Multidisciplinary Marfan Clinic, Tor Vergata Hospital, Rome. RESULTS: An FBN1 variant was identified in 107/124 (86.3%) patients, including 48 novel variants (46 pathogenic/likely pathogenic, 2 VUS). A pathogenic/likely pathogenic variant was detected in 90/101 (89.1%) MFS patients. Our approach allowed early diagnosis for 10 young patients (age 3-19 years) with suspected MFS. CONCLUSIONS: This study broadens the mutation spectrum of FBN1, providing a full update of the molecular basis of MFS in Italy.

Uncovering genetic and non-genetic biomarkers specific for exudative age-related macular degeneration: significant association of twelve variants.

Age-related Macular Degeneration (AMD) represents one of the most sight-threatening diseases in developed countries that substantially impacts the patients' lifestyle by compromising everyday activities, such as reading and driving. In this context, understanding the prevalence, burden, and population-specific risk/protective factors of AMD is essential for adequate health care planning and provision. Our work aimed to characterize exudative AMD in Italian population and to identify the susceptibility/protective factors (genetic variants, age, sex, smoking and dietary habits) which are specific for the onset of disease. Our study involved a cohort of 1976 subjects, including 976 patients affected with exudative AMD and 1000 control subjects. In particular, the sample cohort has been subjected to a large genotyping analysis of 20 genetic variants which are known to be associated with AMD among European and Asiatic populations. This analysis revealed that 8 genetic variants (CFH, ARMS2, IL-8, TIMP3, SLC16A8, RAD51B, VEGFA and COL8A1) were significantly associated with AMD susceptibility. Successively, we performed a multivariate analysis, considering both genetic and non-genetic data available for our sample cohort. The multivariate analysis showed that age, smoking, dietary habits and sex, together with the genetic variants, were significantly associated with AMD in our population. Altogether, these data represent a starting point for the set-up of adequate preventive and personalized strategies aimed to decrease the burden of disease and improve the patients' quality of life.

Pharmacogenomics of multifactorial diseases: a focus on psoriatic arthritis.

This review will outline the current pharmacogenomics knowledge about psoriatic arthritis with a special attention to the perspectives and the challenges for its implementation in the clinical practice. To date, different drugs have been developed to contrast the symptoms and the progression of psoriatic arthritis. However, patients have shown high variability of drug response in relation to their genetic makeup. In this context, the advances made in the knowledge and the potentialities of genome-drugs associations paved the path for the development of a precision medicine. In fact, these associations may be successfully combined with the environment information to provide new strategies able to prevent and improve the disease management as well as to enhance the patients quality of life.

Three-hour analysis of non-invasive foetal sex determination: application of Plexor chemistry.

BACKGROUND: The knowledge of the individual genetic "status" in the prenatal era is particularly relevant in the case of positive family history for genetic diseases, in advanced maternal age and in the general screening for foetal abnormalities. In this context, here, we report an innovative molecular assay which utilizes the cell-free foetal DNA (cffDNA) as a source for the early and fast detection of the foetal sex. The study involved 132 pregnant women in their first 3 months of pregnancy, who agreed to give a blood sample. All the collected samples were immediately subjected to the separation of the plasma, which was utilized for the extraction of the cffDNA. Successively, the extracted cffDNA was analysed by a quantitative PCR (qPCR) method based on Plexor-HY chemistry, which is able to simultaneously identify, quantify and discriminate the autosomal DNA from the sex-linked DNA. RESULTS: Overall, the Plexor-HY assay demonstrated to be sensitive and specific for the determination of low-template DNA, such as the cffDNA. In fact, the Plexor-HY assay has been successfully performed in all the samples, identifying 70 males and 62 females. As the foetal sex can be provided in 120 min just by utilizing a maternal blood sample as cffDNA source, the assay represents a very fast, safe and non-invasive prenatal method. CONCLUSIONS: The possibility of determining the foetal sex in the early prenatal life consents the application of our assay as a helpful screening test for subjects and families at risk of sex-linked disorders. Moreover, the early knowledge of the foetal sex may be of great help even for the specialist, who might promptly advise the patients concerning the foetal risk of inheriting sex-linked disorders and the clinical utility of performing an invasive prenatal diagnosis.

Two molecular assays for the rapid and inexpensive detection of GJB2 and GJB6 mutations.

The hypoacusia can be classified in two clinical forms: Syndromic (SHL) and Nonsyndromic (NSHL). In particular, the NSHL describes the 70-80% of hypoacusia cases and it is mainly due to genetic factors, which are causative of the deafness at the birth. The genetic hypoacusia presents different inheritance patterns: autosomal dominant (20%), autosomal recessive (80%), X-linked (1%), and mitochondrial (1%), respectively. To date, about 35 deafness-causative genes have been identified and most of them codify for connexin transmembrane proteins. Approximately 1:2500 children with NSHL carries mutations in the GJB2 and GJB6 (13q12) genes, which code for connexin 26 (Cx26) and connexin 30 (Cx30), respectively. In the Caucasian population, the most common mutations are 35delG, M34T and 167delT, and D13S1830. Given the frequency distribution of the four mutations in the Caucasian population and the pathogenic connection with NSHL, the development of accurate, rapid, and "low-cost" molecular assays should be strongly encouraged. To this purpose, we set up two different molecular assays (namely the Cx26 and Cx26-30 molecular assays) for the fast and inexpensive detection of 35delG, M34T, 167delT, and D13S1830 mutations. Both the molecular approaches showed to be accurate, sensitive, reproducible, and "low-cost" alternatives for the proper evaluation of the GJB2 and GJB6 genes, which are causative of NSHL. In conclusion, the Cx26 and Cx26-30 molecular assays can be applied to individual, preconception, prenatal, or postnatal screening for the causative-mutations of NSHL.

Use of N-Butyl-2-Cyanoacrylate (Glubran2(®)) in Fractures of Orbital-Maxillo-Zygomatic Complex.

INTRODUCTION: Fractures of the orbital-maxillo-zygomatic complex are among the most common fractures affecting the facial skeleton. Goal of surgical treatment is the realignment of fracture lines for a complete functional and aesthetic rehabilitation. MATERIALS AND METHODS: From January 2008 to January 2011 in the Department of Maxillofacial Surgery of Complesso Integrato Columbus of the Università Cattolica del Sacro Cuore in Rome, 25 patients, affected by comminute fractures of the anterior wall of the maxillary sinus associated with fractures of the orbital-maxillary complex were selected. The synthesis of the larger fracture fragments was performed by plates and screws (1.5 mm) while a biocompatible glue (N-Butyl-2-Cyanoacrylate-Glubran2(®)) was applied to treat the comminute fractures of the anterior wall of the maxillary sinus. RESULTS AND CONCLUSION: The aim of our article is to report our experience and a review of the literature on application of-Butyl-2-Cyanoacrylate for treatment of comminute fractures of the anterior wall of the maxillary sinus. According to the results achieved in our study the N-Butyl-2-Cyanoacrylate can be indicated to treat comminuted fractures of the anterior wall of the maxillary sinus which could not easily be treated with internal rigid fixation.

HFE gene variants and iron-induced oxygen radical generation in idiopathic pulmonary fibrosis.

In idiopathic pulmonary fibrosis (IPF), lung accumulation of excessive extracellular iron and macrophage haemosiderin may suggest disordered iron homeostasis leading to recurring microscopic injury and fibrosing damage. The current study population comprised 89 consistent IPF patients and 107 controls. 54 patients and 11 controls underwent bronchoalveolar lavage (BAL). Haemosiderin was assessed by Perls' stain, BAL fluid malondialdehyde (MDA) by high-performance liquid chromatography, BAL cell iron-dependent oxygen radical generation by fluorimetry and the frequency of hereditary haemochromatosis HFE gene variants by reverse dot blot hybridisation. Macrophage haemosiderin, BAL fluid MDA and BAL cell unstimulated iron-dependent oxygen radical generation were all significantly increased above controls (p<0.05). The frequency of C282Y, S65C and H63D HFE allelic variants was markedly higher in IPF compared with controls (40.4% versus 22.4%, OR 2.35, p=0.008) and was associated with higher iron-dependent oxygen radical generation (HFE variant 107.4±56.0, HFE wild type (wt) 59.4±36.4 and controls 16.7±11.8 fluorescence units per 10(5) BAL cells; p=0.028 HFE variant versus HFE wt, p=0.006 HFE wt versus controls). The data suggest iron dysregulation associated with HFE allelic variants may play an important role in increasing susceptibility to environmental exposures, leading to recurring injury and fibrosis in IPF.

The Use of a Superiorly Based Melolabial Interpolated Flap for Reconstruction of Anterior Oronasal Fistulas: An Easy and Practical Solution.

PURPOSE: This study aimed to propose the use of a superiorly based melolabial interpolated flap for reconstruction of anteriorly located oronasal fistulas maxillary defects. MATERIALS AND METHODS: Using a prospective study design, we evaluated indications and outcomes of the reconstructive technique using the interpolated melolabial flap in 6 patients affected by anteriorly located maxillary defects with naso-sinonasal communication. The cases differed in demographic characteristics and etiology of the defect. The outcome variables were flap vitality/failure and persistent/recurrent oronasal fistula. Both the outcomes were clinically evaluated. RESULTS: No partial or total flap failures were recorded. Two patients experienced recurrent oronasal fistula after previous attempts of correction that required second surgery repair; in both cases, the melolabial flap was available and functional for the secondary procedure. CONCLUSIONS: In selected cases, the superiorly based interpolated melolabial flap could represent a valuable choice for repairing of anteriorly located maxillary defects with oronasal fistulas.

Long term follow-up in inferior alveolar nerve transposition: our experience.

INTRODUCTION: Inferior alveolar nerve transposition (IANT) is a surgical technique used in implantoprosthetic rehabilitation of the atrophic lower jaw which has not been well embraced because of the high risk of damage to the inferior alveolar nerve (IAN). There are cases in which this method is essential to obtain good morphologic and functional rebalancing of the jaw. In this paper, the authors present their experience with IANT, analyzing the various situations in which IANT is the only surgical preprosthetic option. METHODS: Between 2003 and 2011, 35 patients underwent surgical IANT at our center. Thermal and physical sensitivity were evaluated in each patient during follow-up. The follow-up ranged from 14 to 101 months. RESULTS AND CONCLUSION: Based on our experience, absolute indications of IANT are as follows: (1) class IV, V, or VI of Cawood and Howell with extrusion of the antagonist tooth and reduced prosthetic free space; (2) class V or VI of Cawood and Howell with presence of interforaminal teeth; (3) class V or VI of Cawood and Howell if patient desires fast implantoprosthetic rehabilitation with predictable outcomes; (4) class VI of Cawood and Howell when mandibular height increase with inlay grafts is advisable.

Iliac crest flap: donor site morbidity.

INTRODUCTION: Starting from the 1980s, with the advent of microsurgery, microvascular flaps are used for the reconstruction of wide and complex bone defects of the maxillomandibular district. Compared with conventional and implant-supported prostheses, the free flaps allow aesthetic-functional rehabilitations more adapt to answer to problems that these wide disablements involve. The anatomic characteristics of the crest flap make it one of the best available flap in the maxillomandibular bone reconstruction. METHODS: The authors introduce a retrospective analysis of their own experience in the reconstruction of wide and complex bone defects of the maxillomandibular district. Specifically, the attention is focused on the use of the iliac crest flap. The surgical technique of flap preparation is discussed. Moreover, a review of results from international studies about the morbidity of the donor site is presented and compared with own experience. RESULT: As reported in the literature, the iliac crest flap donor site may encounter several complications. Among these, chronic pain, loss of regional sensibility or paresthesias, hematoma, seroma, walking troubles, unaesthetic scars, abdominal hernia, and loss of the normal bone profile of the hip. DISCUSSION: At present, the use of the iliac crest free flap in the microvascular reconstruction of the complex deficits of the maxillomandibular district represents a well-established method in the experience of the maxillofacial surgeon. Several information about results obtained in the maxillomandibular rehabilitation are available from the literature; however, little attention has been addressed to complications and morbidity of the donor site. Such aspect will be discussed in this work.

Scapula free flap for complex maxillofacial reconstruction.

INTRODUCTION: Composite tissue defects of the mandible and maxilla, after resection of head and neck malignancies, osteoradionecrosis, malformations, or traumas, cause functional and aesthetic problems. Nowadays, microvascular free flaps represent the main choice for the reconstruction of these defects. Among the various flaps proposed, the scapula flap has favorable characteristics that make it suitable for bone, soft tissue, or combined defects. MATERIALS: We report 7 cases of reconstruction of complex maxillofacial defects with subscapular system flaps. The patients treated had Romberg syndrome (1 case), malignant tumors (5 cases), and result of previous trauma (1 case).Location of deficit was the maxilla (3 cases), the mandible (2 case), the ethmoidal-maxillary region (1 case) and the upper and middle thirds of the face in the last case. METHODS: In 2 cases, a parascapular system flap was used; in 5 cases, a composite flap with latissimus dorsi muscle and scapular bone. RESULTS: Neither failure of the harvested flaps nor complications in the donor site were evidenced. A good aesthetic and functional outcome was obtained in all cases. DISCUSSION: : Many free flaps have been proposed for the reconstruction of defects in the maxillofacial region such as fibula, deep circumflex iliac artery, scapula, among the bone flaps; and forearm, rectus abdominis, and anterolateral thigh, among the soft tissue flaps. The choice of the flap to use depends on the length of the bone defect and the amount of soft tissues required. The subscapular system has the advantage of providing different flaps based on the same pedicle. The osteofasciocutaneous scapular free flap, in particular, allows wide mobility of soft tissues (parascapular flap) with respect to its bone component (scapular bone), resulting suitable for defects of large size involving both the soft tissues and the bone. CONCLUSIONS: Although the fibula flap and the deep circumflex iliac artery flap remain the first choice for bone reconstructions of the mandible and maxilla, the scapula flap has some features that make its use extremely advantageous in some circumstances. In particular, we advocate the use of the osteomuscular latissimus dorsi-scapula flap for reconstruction of large-volume defects involving the bone and soft tissues, whereas fasciocutaneous parascapular flaps represent a valid alternative to forearm flap and anterolateral thigh flap in the reconstruction of soft tissue defects.

Microvascular reconstruction of the mandible in irradiated patients.

This work focuses on the use of revascularized free flaps for the reconstruction of the major defects of the mandible after the removal of advanced-stage tumors in irradiated patients. It uses three representative cases to study the problems of complex patients and the possible reconstructive options. The cases, all three young patients (two females and one male), had undergone a mandibulectomy and adjuvant radiotherapy for malignant neoplasms. In each case, secondary reconstruction of the mandible and soft tissue was necessary and was performed using microvascular free flaps. An osteomyocutaneous iliac crest free flap was used in two cases, whereas a double flap (fibula free flap + rectus abdominis free flap) was used in the other case. In all three cases, after the microvascular reconstruction, an orthognathic procedure was performed to obtain the correct maxillomandibular relationship. The advantages and disadvantages of the various techniques used are discussed.