Good laboratory practice for PGT-M: Turkish Society of Reproductive Medicine guidelines.

This guideline was prepared by the Turkish Society of Reproductive Medicine to define the conditions and requirements for an outsourced preimplantation genetic testing (PGT) programme in line with the experience and needs of practitioners. This guideline is intended to be a reference document for assisted reproductive technology centres, genetic diagnosis centres, non-governmental organizations working on reproductive health, legal experts, consultants working on laboratory accreditation, academicians specializing in ethical issues, and policy makers. The Consortium aims to provide recommendations addressing the challenges of genetic testing, especially PGT for monogenic diseases (PGT-M) due to the high rate of consanguineous marriage in Turkey. For this purpose, this summary document specifically includes challenges and recommendations regarding PGT-M practice, and aims to identify and aid in prevention of errors leading to misdiagnosis. The recommendations can be modified to fit other locations.

A novel SLC35D1 variant causing milder phenotype of Schneckenbecken dysplasia in a large pedigree.

SLC35D1 gene encodes UDP-glucuronic acid/UDP-n-acetylgalactosamine dual transporter protein and transports organic or inorganic molecules across cellular membranes. SLC35D1 gene pathogenic variants causes Schneckenbecken dysplasia (SHNKND) which is a rare lethal autosomal recessive disorder characterized by the snail-like pelvis, flattening of vertebral bodies, short and broad long bones with a dumbbell-like appearance, thoracic hypoplasia. Only six cases with homozygous SLC35D1 variants have been reported to date, and all of these cases were lost in the perinatal period. Here we report different family members with a novel SLC35D1 variant who presented a milder phenotype of SHNKND. The affected patients have common clinical features such as short stature, mild mesomelia, shortening of the lower extremity, genu valgum, and narrow thorax. Exome sequencing of the proband revealed a homozygous missense variant of SLC35D1 gene, c.401 T > C (p. Met134Thr). The affected siblings, their two cousins, and their paternal uncle with a similar phenotype were also homozygous for the variant. This is the first case report of a family with a novel likely pathogenic variant (p. Met134Thr) and mild phenotypic features. It has the largest family with different ages of patients (ages ranged 4-31 years old) reported to date. The present report supports the evidence that the p. Met134Thr variant is responsible for a milder phenotype than previously reported cases with SLC35D1 pathogenic variants.

ASH1L (a histone methyltransferase protein) is a novel candidate globin gene regulator revealed by genetic study of an English family with beta-thalassaemia unlinked to the beta-globin locus.

In 1993, we described an English family with beta-thalassaemia that was not linked to the beta-globin locus. Whole genome sequence analyses revealed potential causative mutations in 15 different genes, of which 4 were consistently and uniquely associated with the phenotype in all 7 affected family members, also confirmed by genetic linkage analysis. Of the 4 genes, which are present in a centromeric region of chromosome 1, ASH1L was proposed as causative through functional mRNA knock-down and chromatin-immunoprecipitation studies in human erythroid progenitor cells. Our data suggest a putative role for ASH1L (Trithorax protein) in the regulation of globin genes.

Low Dose of Ti3 C2 MXene Quantum Dots Mitigate SARS-CoV-2 Infection.

MXene QDs (MQDs) have been effectively used in several fields of biomedical research. Considering the role of hyperactivation of immune system in infectious diseases, especially in COVID-19, MQDs stand as a potential candidate as a nanotherapeutic against viral infections. However, the efficacy of MQDs against SARS-CoV-2 infection has not been tested yet. In this study, Ti3 C2 MQDs are synthesized and their potential in mitigating SARS-CoV-2 infection is investigated.  Physicochemical characterization suggests that MQDs are enriched with abundance of bioactive functional groups such as oxygen, hydrogen, fluorine, and chlorine groups as well as surface titanium oxides. The efficacy of MQDs is tested in VeroE6 cells infected with SARS-CoV-2. These data demonstrate that the treatment with MQDs is able to mitigate multiplication of virus particles, only at very low doses such as 0,15 µg mL-1 . Furthermore, to understand the mechanisms of MQD-mediated anti-COVID properties, global proteomics analysis are performed and determined differentially expressed proteins between MQD-treated and untreated cells. Data reveal that MQDs interfere with the viral life cycle through different mechanisms including the Ca2 + signaling pathway, IFN-α response, virus internalization, replication, and translation. These findings suggest that MQDs can be employed to develop future immunoengineering-based nanotherapeutics strategies against SARS-CoV-2 and other viral infections.

An Incidental Detection of a Cryptic Complex Chromosome Rearrangement Found During NGS Based PGT-SR: A Case Report.

Background: Complex chromosome rearrangements (CCRs) involve more than 2 chromosomal breakpoints and cause the exchanges of chromosomal segments between two or more chromosomes. The carriers of CCRs have normal phenotypes, but they have a higher risk of reproductive failure. Case Presentation: This paper presents a couple with a history of two affected children, one spontaneous abortion, three in vitro fertilization (IVF) failures, and one healthy boy who were referred to our laboratory for preimplantation genetic testing (PGT). The wife had been evaluated as a carrier of 46,XX,t (2;6)(p21;p25); therefore, four IVF treatment cycles supported with PGT for this translocation had been performed in different IVF centers until the couple consulted our laboratory. Only one of these four IVF attempts had resulted in a healthy boy and this IVF study had been performed with fluorescence in situ hybridization (FISH)-based preimplantation genetic testing for structural chromosomal rearrangements (PGT-SR). The fifth IVF study with next-generation sequencing (NGS)-based PGT was performed by our laboratory and no healthy embryo was found in evaluated 6 embryos. During our NGS-based PGT, the cryptic involvement of 12p was firstly detected. FISH with chromosome 2,6, and 12 specific probes revealed that the mother was a carrier of a balanced 3-way translocation of 46,XX,t(2;6;12)(p21;p25;p13). Conclusion: NGS based PGT-SR method is an accurate method for detecting the copy number variations and is helpful to find out the cryptic CCRs.

Can PCNA and LIM15 gene expression levels predict sperm retrieval success in men with non-obstructive azoospermia?

INTRODUCTION AND OBJECTIVES: It is necessary to be able to predict sperm retrieval before microdissection testicular sperm extraction (mTESE) in azoospermic men. This study established the importance of proliferating cell nuclear antigen (PCNA) and LIM15 gene expression levels in predicting the success of sperm retrieval by mTESE. MATERIALS AND METHODS: One hundred and forty-three men who were diagnosed with non-obstructive azoospermia (NOA) were included in the study. Patients' age, total testosterone and follicle stimulating hormone values, testicular volume and testicular histology were recorded by prospectively. PCNA and LIM15 gene expression levels were determined by real-time PCR in the materials from both ejaculate and testicular specimens. RESULTS: Testis volume and histology were the most important factors in predicting the sperm retrieval rate (SRR). The PCNA and LIM15 gene expression levels measured in testicular tissues and the LIM15 gene expression levels measured in ejaculate significantly correlated with the SRR in mTESE (p=0.038, p=0.022, and p=0.004, respectively). Although the PCNA gene expression level measured in ejaculate was higher in men with successful sperm retrieval, the difference was not statistically significant (p=0.061). According to the multivariate logistic regression analysis, testicular volume and LIM15 gene expression level in ejaculate were independent predictive parameters for sperm retrieval. CONCLUSION: The data showed that LIM15 gene expression level in ejaculate is a useful molecular marker to predict the SRR before mTESE.

Whole Mitochondrial Genome Analysis in Turkish Patients with Mitochondrial Diseases

Background: Mitochondrial diseases are a clinically heterogeneous group of rare hereditary disorders that are defined by a genetic defect predominantly affecting mitochondrial oxidative phosphorylation. Mitochondrial diseases are caused by mutations of genes encoded by either nuclear DNA or mitochondrial DNA. Hundreds of different mitochondrial DNA point mutations and large-scale mitochondrial DNA rearrangements have been shown to cause mitochondrial diseases including Kearns­Sayre syndrome, Leber's hereditary optic neuropathy, Leigh syndrome, myoclonic epilepsy with ragged-red fibers, mitochondrial encephalopathy lactic acidosis stroke. Aims: To investigate new variants that could be associated with mitochondrial diseases and to determine the effect of mitochondrial DNA mutations on the clinical spectrum. Study Design: Cross-sectional study. Methods: We screened whole mitochondrial DNA genome using next-generation sequencing in 16 patients who are considered to have mitochondrial disease. CentoGene and Mikrogen Genetic Diseases Diagnostic Center's database were used to investigate sequence variants. Detected variants were evaluated in bioinformatic databases to determine pathogenicity and were classified as class 1 (pathogenic), class 2 (likely pathogenic), and class 3 (variant of uncertain significance) according to CentoGene-ACMG database. Results: As a result of the study, 2 patients were diagnosed with Leigh syndrome as previously reported class 1 mutations in MT-ATP6 and MT-ND5 genes. Four variants were identified for the first time in literature and 2 variants, previously reported but with uncertain pathogenic effect, are thought to be associated with mitochondrial disease. Conclusion: Mitochondrial DNA screening should be among the primary clinical tests in patients with suspected mitochondrial disease to rule out DNA-associated mutations.

Mandibuloacral dysplasia and LMNA A529V mutation in Turkish patients with severe skeletal changes and absent breast development.

Mandibuloacral dysplasia (MAD) is an autosomal recessive disorder characterized by acroosteolysis (resorption of terminal phalanges), skin changes (hyperpigmentation), clavicular hypoplasia, craniofascial anomalies, a hook nose and prominent eyes, delayed closures of the cranial sutures, lipodystrophy, alopecia, and skeletal anomalies. MAD patients are classified according to lipodystrophy patterns: type A and type B. The vast majority of MAD cases are caused by LMNA gene mutations. MAD patients with type A lipodystrophy (MADA) have been reported to have LMNA R527H, A529V, or A529T mutations. In this report, we describe two MADA patients with progressive skeletal changes, absent breast development, and cataract in addition to the classical MAD phenotype. Both patients were found to be homozygous for the Ala529Val mutation of the LMNA gene. Our female patient is the oldest MADA patient (59 years old) who has ever been reported with the LMNA mutation and also the LMNA Ala529Val mutation. This study is the second report on MADA patients with a homozygous Ala529Val mutation.

Effects of pentoxifylline and platelet activating factor on sperm DNA damage.

OBJECTIVE: Pentoxifylline and platelet-activating factor (PAF) have been used to increase sperm motility in embryology laboratories. In the present study, we aimed to investigate whether these agents pose sperm DNA damage using DNA sperm chromatin dispersion (SCD) assay. STUDY DESIGN: Following application of pentoxifylline and PAF, sperm samples of 50 individuals with different sperm parameters were compared to baseline in terms of DNA damage using SCD assay. Furthermore, the relationship between DNA damage and sperm parameters in predicting DNA damage was assessed. RESULTS AND CONCLUSIONS: Significant increase in DNA damage was observed following application of PAF and pentoxifylline. Furthermore, DNA damage was significantly increased with application of pentoxifylline compared to PAF. Sperm motility was observed to be a statistically significant indicator in predicting alterations in DNA damage in baseline and subsequent to application of PAF and pentoxifylline independent of sperm concentration and morphology. Increased DNA damage was observed in both groups following application of pentoxifylline and PAF. Furthermore, the increase in DNA damage was higher in samples treated with pentoxifylline compared to samples treated with PAF. Thus, PAF seems to be more innocent in choosing viable sperm cells and in achieving sperm motility in the in vitro fertilization laboratory.

Oogonial Precursor Cell-Derived Autologous Mitochondria Injection to Improve Outcomes in Women With Multiple IVF Failures Due to Low Oocyte Quality: A Clinical Translation.

BACKGROUND: Mitochondrial dysfunction has been suggested as a major cause of age-induced decline in oocyte quality. In the past, donor oocyte cytoplasmic transfer showed some success but was abandoned due to the concerns with heteroplasmy. Recent studies indicated presence of oogonial precursor cells (OPCs) in the human ovary, which could be an autologous source of "healthy mitochondria." We sought to investigate the clinical efficacy of OPC-derived autologous mitochondrial injection (AMI) to improve oocyte quality in women with multiple in vitro fertilization (IVF) failures. METHODS: The OPCs were isolated from laparoscopically obtained ovarian cortical pieces by cell sorting using a monoclonal anti-vasa homolog (anti-DDX) antibody. They were then disrupted and mitochondria were isolated. Reconstituted mitochondria were injected into each oocyte during intracytoplasmic sperm injection. Paired comparisons were made between the first failed cycles and the post-AMI cycles. RESULTS: Of the 15 women undergoing ovarian stimulation, 2 were canceled and 3 decided to pool oocytes for later AMI. In remaining 10 (mean age 34.7 ± 4.1), AMI significantly improved fertilization rates (49.7 ± 31.3 vs 78.3 ± 18.9; P = .03) with a trend for better embryo grades (2.3 ± 0.3 vs 3.1 ± 0.7; P = .08). Four of 10 women conceived after single frozen embryo transfer and 3 after confirmation of diploidy via array comparative genomic hybridization (aCGH) (clinical pregnancy/embryo transfer = 4/10). CONCLUSION: These data show encouraging results for AMI in comparison to previous failed IVF cycles.

Recombinant chromosome with partial 14 q trisomy due to maternal pericentric inversion.

BACKGROUND: 14q duplications caused by parental pericentric inversion of chromosome 14 are rarely reported and no clear genotype-phenotype correlation has been determined yet. CASE PRESENTATION: Here we reported a 7 years old female patient with recombinant chromosome characterized by 14 q duplication and originated from maternal pericentric inversion of chromosome 14. Principal clinical findings of the child include developmental delay, microcephaly, hypertelorism, low set ears, clinodactyly of fifth fingers, hypotonia, telecanthus and cardiac malformation. CONCLUSIONS: Her final karyotype was 46,XX,rec(14)dup(14q)inv(14)(p11.2q24)mat,arr14q24.1-qter(64,800,000-108,350,000 bp)x3.

HBS1L-MYB intergenic variants modulate fetal hemoglobin via long-range MYB enhancers.

Genetic studies have identified common variants within the intergenic region (HBS1L-MYB) between GTP-binding elongation factor HBS1L and myeloblastosis oncogene MYB on chromosome 6q that are associated with elevated fetal hemoglobin (HbF) levels and alterations of other clinically important human erythroid traits. It is unclear how these noncoding sequence variants affect multiple erythrocyte characteristics. Here, we determined that several HBS1L-MYB intergenic variants affect regulatory elements that are occupied by key erythroid transcription factors within this region. These elements interact with MYB, a critical regulator of erythroid development and HbF levels. We found that several HBS1L-MYB intergenic variants reduce transcription factor binding, affecting long-range interactions with MYB and MYB expression levels. These data provide a functional explanation for the genetic association of HBS1L-MYB intergenic polymorphisms with human erythroid traits and HbF levels. Our results further designate MYB as a target for therapeutic induction of HbF to ameliorate sickle cell and ß-thalassemia disease severity.