Safety of Procalcitonin Guided Early Discontinuation of Antibiotic Therapy among Children Receiving Cancer Chemotherapy and Having Low-Risk Febrile Neutropenia: A Randomized Feasibility Trial (ProFenC Study).

In low-risk febrile neutropenia (LR-FN), the safety of early discontinuation of empiric antibiotics without marrow recovery is not well established. This study aimed to evaluate the safety of procalcitonin (PCT) guided early discontinuation of antibiotics in LR-FN. In this trial, children with LR-FN with an afebrile period of at least 24 h, sterile blood culture, and negative/normalized PCT were randomized at 72 h of starting antibiotics into two groups: intervention arm and standard arm. The antibiotics were stopped in the intervention arm regardless of absolute neutrophil count (ANC), while in the standard arm, antibiotics were continued for at least 7 days or until recovery of ANC (>500/mm3). The primary objective was to determine the treatment failure rates, and the secondary objective was to compare the duration of antibiotics and all-cause mortality between the two arms. A total of 46 children with LR-FN were randomized to either the intervention arm (n = 23) or the standard arm (n = 23). Treatment failure was observed in 2/23 (8.7%) of patients in the intervention arm compared to 1/23 (4.3%) in the standard arm [RR: 2 (95% CI: 0.19-20.6); p = 0.55]. The median duration of antibiotics in the intervention arm and standard arm were 3 days vs 7 days (P= <0.001). There was no mortality in this study. PCT-guided early discontinuation of empirical antibiotics in LR-FN is feasible. There was no significant difference observed in treatment failure between the early discontinuation of antibiotics vs standard therapy. The total duration of antibiotic exposure was significantly lesser in the discontinuation arm. Further, larger multicenter studies are needed to confirm the finding of this study.

Serum anti-Müllerian hormone: A potential biomarker for polycystic ovary syndrome.

BACKGROUND OBJECTIVES: Polycystic ovary syndrome (PCOS) is characterized by chronic ovulatory dysfunction, hyperandrogenism and polycystic ovary morphology (PCOM). Although hyperandrogenism is one of the major features of PCOS, it is rarely observed in southeast Asia. Recently, however, there has been growing evidence on association of anti-Müllerian hormone (AMH) with PCOS. The objective of this study was to investigate the diagnostic potentials of AMH in PCOS individuals. METHODS: This case-control study included a total of 131 women with PCOS and 49 healthy controls who were enrolled after the exclusion of secondary causes of PCOS. Serum AMH was measured using an ultra-sensitive AMH ELISA kit in addition to other diagnostic biomarkers. Statistical analyses was carried out using the Student's t test, Wilcoxon rank-sum test, receiver operating characteristic (ROC) curve analysis, Spearman's rank correlation test and multivariable binary logistic regression analysis. RESULTS: The median AMH values were 8.5 ng/ml and 2.5 ng/ml in the study group and controls, respectively ( P <0.001). The normal cutoff value of 4.1 ng/ml for AMH was derived from ROC curve analysis. With a 4.1 ng/ml cut-off value, high levels of AMH was found in about 84 per cent of PCOS cases. However, no significant difference in AMH level was noted between age groups (<20 vs . ≥20 yr), body mass index (BMI) (<25 vs . ≥25 kg/m 2 ) and PCOM types. The area under the ROC curve (AUC) for AMH yielded diagnostic range values. In total PCOS cases, AUC was 0.93 (95% CI: 0.88 and 0.96), and in phenotype A PCOS cases, AUC was 0.96 (95% CI: 0.91 and 0.98). The correlation test also showed no association with BMI, the FG score, PCOM, free androgen index, androstenedione, dehydroepiandrosterone sulphate and luteinizing hormone. However, a weak correlation was observed with testosterone in total PCOS cases and with DHT as well as age in phenotype A PCOS cases. The prediction model for PCOS using multivariable binary logistic regression analysis showed AMH as the best marker. INTERPRETATION CONCLUSIONS: The results of this study suggest that AMH can be considered as the most promising biomarker in PCOS women, particularly with phenotype A and phenotype D.

Whole Exome Sequencing Reveals Rare Variants in Genes Associated with Metabolic Disorders in Women with PCOS.

Background: Polycystic ovary syndrome (PCOS) is a complex genetic trait, the pathogenesis of which is governed by an interplay of genetic and epigenetic factors. However, the aetiology of PCOS is not fully understood. Aims: The objective of this study was to investigate the genetic causes of PCOS by identifying rare variants in genes implicated in its pathophysiology. Settings and Design: This was a hospital-based observational study. Materials and Methods: We used whole-exome sequencing for 52 PCOS women to identify the rare variants in genes related to PCOS pathogenesis. Subsequently, we analysed these variants using in silico prediction software to determine their functional effects. We then assessed the relationship between these variants and the clinical outcomes of the patients. Statistical Analysis Used: Student's t-test and Fisher's exact test were used to compare clinical parameters and frequency differences amongst PCOS patients with and without variants. Results: A total of four rare exonic variants in obesity- and hyperinsulinaemia-related genes including UCP1 (p.Thr227Ile), UCP2 (p.Arg88Cys), IRS1 (p.Ser892Gly) and GHRL (p.Leu72Met) were identified in eight patients. Significant differences were observed between the patients carrying variants and those without variants. PCOS patients with identified variants exhibited significantly higher average body mass index and fasting insulin levels of PCOS subjects with identified variants compared to those without variants (P < 0.05). Additionally, there were significant differences in the variant frequencies of four variants when compared to the population database (P < 0.05). Conclusion: This study shows a prevalence of rare variants in obesity and hyperinsulinaemia-related genes in a cohort of PCOS women, thereby underscoring the impact of the identified rare variants on the development of obesity and associated metabolic derangements in PCOS women.

An Investigation of Steroid Biosynthesis Pathway Genes in Women with Polycystic Ovary Syndrome.

Background: Polycystic ovary syndrome (PCOS) is a common endocrinopathy whose heterogeneous genetic basis results in a variable clinical presentation. One of the main clinical features of PCOS is hyperandrogenism which occurs due to dysregulation of ovarian and adrenal steroidogenesis. Aims: This study aimed to investigate potentially pathogenic variants in steroidogenic genes associated with PCOS. Settings and Design: This was a hospital-based observational study. Materials and Methods: We recruited 51 women who presented with PCOS. Fasting blood samples were drawn from the participants and their whole-exome sequencing analysis was carried out to look for pathogenic variants involved in steroidogenic pathways. The variants were predicted for their probable deleterious effects on proteins through in silico prediction tools. We evaluated the variants with respect to the hormonal characteristics and clinical outcomes of the patients. Statistical Analysis Used: All variables were analysed using GraphPad Prism 8. Kruskal-Wallis t-test and Fisher's exact test were used to compare clinical parameters and frequency differences among PCOS patients with and without variants. Results: The data presented here reveal eight heterozygous exonic variants, namely CYP21A2 (p.Ala392Thr, p.Gln319Ter and p.I143N), steroidogenic acute regulatory (p.Arg53 Leu), AKR1C3 (p.Phe205Val), P450 oxidoreductase (p.Val334Ile and p.Val251Met) and HSD17B6 (p.Gly40Ser), of which three were pathogenic, and four variants of uncertain significance in 8 out of 51 patients (15.68%). The identified variants were predicted to cause protein destabilisation, thus likely contributing to the pathogenesis of PCOS. Some of the variants showed significant differences between PCOS patients and population database (P < 0.05). Conclusion: The results of this study add to the mutational spectrum of steroidogenic genes and their association with PCOS.

Transcriptome Analysis of Insulin Signaling-Associated Transcription Factors in C. elegans Reveal Their Genome-Wide Target Genes Specificity and Complexity.

Insulin/IGF-1-like signaling (IIS) plays a crucial, conserved role in development, growth, reproduction, stress tolerance, and longevity. In Caenorhabditis elegans, the enhanced longevity under reduced insulin signaling (rIIS) is primarily regulated by the transcription factors (TFs) DAF-16/FOXO, SKN-1/Nrf-1, and HSF1/HSF-1. The specific and coordinated regulation of gene expression by these TFs under rIIS has not been comprehensively elucidated. Here, using RNA-sequencing analysis, we report a systematic study of the complexity of TF-dependent target gene interactions during rIIS under analogous genetic and experimental conditions. We found that DAF-16 regulates only a fraction of the C. elegans transcriptome but controls a large set of genes under rIIS; SKN-1 and HSF-1 show the opposite trend. Both of the latter TFs function as activators and repressors to a similar extent, while DAF-16 is predominantly an activator. For expression of the genes commonly regulated by TFs under rIIS conditions, DAF-16 is the principal determining factor, dominating over the other two TFs, irrespective of whether they activate or repress these genes. The functional annotations and regulatory networks presented in this study provide novel insights into the complexity of the gene regulatory networks downstream of the IIS pathway that controls diverse phenotypes, including longevity.

Identification of genomic imbalances (CNVs as well as LOH) in sertoli cell only syndrome cases through cytoscan microarray.

Sertoli cell only syndrome (SCOS) is characterized by complete absence of germ cells in seminiferous tubules of testis. SCOS is multifactorial but genetic factors play a major role in pathogenesis of the disorder with idiopathic origin. Genetic factors majorly include sex chromosomal aneuploidy and Yq Microdeletion. But a large number of cases are still idiopathic. The study aimed to evaluate the genomic imbalances (CNVs and LOH) in idiopathic SCOS patients. The study is based on 28 apparent idiopathic SCOS cases and 10 controls. Molecular cytogenetic techniques viz., FISH, STS-Multiplex PCR and Affymetrix cytoscan microarray (750 K) were used. The microarray screened whole genomic imbalances in DNA from peripheral blood of 25 cases (excluded Klinefelter syndrome patients) and testicular FNAC sample of 2 cases. High FSH and low Inhibin B were observed in cases as compared to control controls groups. Four cases of sex chromosomal abnormality (i.e., three non-mosaic 47, XXY males and one non-mosaic 46, XX male) as well as four cases of Yq microdeletion (i.e., three cases with AZFc deletion and one case with complete AZFa, b and c deletion) were identified. Microarray detected unbalanced translocation of two segments of Y-chromosome i.e., Yp11.31-p11.2 (~4.o mb region, involving SRY) and Yp11.2 (~2.5 mb region) on X-chromosome in XX male. Also, loss of segment on same X-chromosome involving PAR1 region was identified. We have identified both autosomal and sex chromosomal CNVs (recurrent as well as private) involving candidate genes like SYCE1, ZFPM2, SRPK1, DAZ1, BPY2, HSFY1, VCY1 etc. All these CNVs are possibly associated with SCOS pathogenesis. CNVs identified in cases were already reported as pathogenic variant in clinical database DECIPHER. Microarray also detected many LOH (all autosomal, >3.0 mb size) that covered genes with spermatogenesis related function. The mechanism of action of LOH in pathogenesis of SCOS still remains unravelled. CNVs and LOH related to spermatogenesis identified from two different sample types (blood vs. testicular tissue) were discordant. This study should be extended for larger cohort of patients.

Sex ratio trajectory in mouse.

Skewing of the sex ratio towards males occurs in humans. The possible explanation for excess male births could be a preference for Y-bearing sperm at fertilization and/or selective elimination of female embryos during pregnancy. In this study, we have tested the sex ratio in the preimplantation embryo (2-3 cells stage/closest possible primary sex ratio), the post-implantation embryo (day E7.5), and at birth (secondary sex ratio) on a homogenous (genetic, environmental, and dietary) population of mice to ascertain the biological reason i.e., male preference at fertilization or female elimination during pregnancy or both. Primary sex ratio on early preimplantation embryos (2-3 cells stage) was studied on 598 embryos and secondary sex ratio (at birth) on 721 pups using PCR-based sexing (both X & Y chromosome-specific) besides sex ratio of 80 post-implantation embryos (day E7.5). We have also investigated whether the fat content (high & low) of the diet affects the sex ratio. We observed a skewed sex ratio (more female) in preimplantation embryos (0.436; 95 % CI 0.39, 0.48), and post-implantation embryos (0.462; 95 % CI 0.35, 0.57) but reverse skewing (more male) at birth (0.539; 95 % CI 0.5, 0.58). We also observed that high-fat diet promoted male sex ratio at birth (0.657; 95 % CI 0.57, 0.74) whereas a low-fat diet had the opposite effect (0.46; 95 % CI 0.36, 0.56) but no effect at fertilization (2-3 cells stage embryos). This indicates selective elimination of female embryo and fetus throughout pregnancy in mice, more so with a high-fat diet.

Human telomerase RNA component (hTERC) gene expression and chromosome 7 ploidy correlate positively with histological grade of cervical intraepithelial neoplasia.

OBJECTIVE: Cervical cancer screening by primary human papilloma virus detection and cytology is fraught with low specificity and variable sensitivity, respectively. Cytology-histology correlation remains modest. Biomarkers associated with early genetic events in cervical squamous carcinogenesis and detectable in cytology material are likely to be relevant. Human telomerase RNA component (hTERC) gene overexpression and aneuploidy are promising candidates in view of their reported early and consistent association with cervical squamous oncogenesis. METHODS: We analysed hTERC gene expression and chromosome 7 ploidy by fluorescent in-situ hybridisation (FISH) in 50 women with cytological precursor squamous intraepithelial lesions and available histology outcomes. Results were expressed as percentages of cells showing ≥3 signals, mean signals/nucleus, and maximum amplitude across various cytology and histology categories. Proportions of positive cases were calculated from threshold values derived from 6 controls. Distribution of above indices with respect to ≥cervical intraepithelial neoplasia 2 (CIN2) was explored. RESULTS: For both genetic aberrations, there was significant positive correlation (for all indices) between the proportion of positive cases and worsening cytological and histological outcomes (P < .05), with significant intergroup differences (P < .05). High-grade lesions (≥CIN2) had significantly higher results compared to

22(R)-hydroxycholesterol for dopaminergic neuronal specification of MSCs and amelioration of Parkinsonian symptoms in rats.

Oxysterols play vital roles in the human body, ranging from cell cycle regulation and progression to dopaminergic neurogenesis. While naïve human mesenchymal stem cells (hMSCs) have been explored to have neurogenic effect, there is still a grey area to explore their regenerative potential after in vitro differentiation. Hence, in the current study, we have investigated the neurogenic effect of 22(R)-hydroxycholesterol (22-HC) on hMSCs obtained from bone marrow, adipose tissue and dental pulp. Morphological and morphometric analysis revealed physical differentiation of stem cells into neuronal cells. Detailed characterization of differentiated cells affirmed generation of neuronal cells in culture. The percentage of generation of non-DA cells in the culture confirmed selective neurogenic potential of 22-HC. We substantiated the efficacy of these cells in neuro-regeneration by transplanting them into Parkinson's disease Wistar rat model. MSCs from dental pulp had maximal regenerative effect (with 80.20 ± 1.5% in vitro differentiation efficiency) upon transplantation, as shown by various behavioural examinations and immunohistochemical tests. Subsequential analysis revealed that 22-HC yields a higher percentage of functional DA neurons and has differential effect on various tissue-specific primary human MSCs. 22-HC may be used for treating Parkinson's disease in future with stem cells.

Intra-individual Genomic Variation Analysis in Tissues (Blood vs. Testis) Through SNP Microarray: A Case Report of Two Patients with Idiopathic Sertoli Cell Only Syndrome (SCOS).

BACKGROUND: Sertoli cell only syndrome (SCOS) or germ cell aplasia is characterized by the existence of only sertoli cells in the seminiferous tubule without any germ cells. SCOS is a multifactorial disorder but genetic factors play a major role in pathogenesis of idiopathic SCOS. CASE PRESENTATION: Two cases of idiopathic SCOS had been reported with no non-genetic factor in their medical history that could play a role in aetiology of SCOS. Also, two normal fertile males were recruited as controls in this study. For evaluation of genomic imbalance, karyotyping (G-banding), FISH, STS-PCR and SNP microarray were carried out. SNP microarray was carried out in DNA of peripheral blood for cases as well as controls. However, for cases, SNP microarray was conducted in DNA of testicular Fine needle aspiration cytology (FNAC). CONCLUSION: No chromosome abnormality and Yq microdeletion was found in cases as well as in controls. Microarray detected many CNVs and LOH that cover genes with spermatogenesis related function and PAR CNVs in both cases. Differential genomic variations were found in blood and testis for cases. Therefore, the evaluation of pathogenesis of idiopathic SCOS might be dependent on both tissue samples. The evaluation of genomic imbalances at both tissue levels should be done for a large cohort of patients.

Cytokine profile in multiple myeloma.

BACKGROUND: Cytokines play a crucial role in the growth, survival and dissemination of malignant plasma cells in patients of multiple myeloma (MM). We estimated concentrations of five key cytokines: Vascular Endothelial Growth Factor (VEGF), Interleukin-6 (IL-6), Tumor Necrosis Factor- alpha (TNF- α), B-cell activating factor (BAFF), and Receptor Activator of Nuclear Factor-κB ligand (RANKL) in newly diagnosed and relapsed/refractory MM (RRMM). METHODS: The study groups include 68 newly diagnosed and 21 relapsed/refractory (RR) MM patients. 32 out of 68 newly diagnosed MM patients were evaluated for serum cytokine concentrations after their treatment. For survival analysis, the various parameters were studied in relation to both progression free survival (PFS) and overall survival (OS). RESULTS: The median serum levels of VEGF, IL-6, BAFF and RANKL were higher in RRMM compared with newly diagnosed patients. However, the difference was significant for BAFF levels (p = 0.04). The median serum levels of VEGF, IL-6, TNF-α, BAFF and RANKL were significantly higher in newly diagnosed and RRMM patients, compared to controls. We also observed lower plasma levels of VEGF (p=<0.0001) and BAFF (p=<0.0001) in BM compartment compared to the levels in serum from peripheral blood of newly diagnosed patients. Significant reduction in the median levels of IL-6, TNF-α, BAFF and RANKL was seen after 4-6 cycles of induction treatment in responders but not in non-responders. On survival analysis, RRMM patients had inferior median OS and PFS compared to that in newly diagnosed MM patients and found to be significantly associated with low haemoglobin representing the more aggressive disease biology in recurrent myeloma. The mean levels of IL-6 were significantly different in patients who died as compared to patients who were alive. CONCLUSIONS: The present study demonstrates that the serum levels of VEGF, IL-6, TNF, BAFF and RANKL are significantly elevated and decrease significantly after treatment. The concentrations of circulating cytokines will reflect those of the bone marrow and could be used for subsequent analyses.

Cellular mesenchymal epithelial transition (C-MET) gene copy number variation in gastric adenocarcinoma: A pilot search for new marker for targeted therapy in HER-2/neu resistance.

Increasing HER-2/neu resistance in gastric carcinoma has encouraged search for new biomarkers for targeted therapy. Cellular mesenchymal epithelial transition (C-MET) is one such tyrosine kinase inhibitor proposed for personalized salvage treatment. We determined frequency of C-MET gene copy number variation (CNV) by Fluorescent in-situ hybridization (FISH) in gastric adenocarcinoma (GAC) and sought its correlation with conventional clinicopathologic parameters. Dual-coloured FISH was done on 32 GAC cases. C-MET gene and centromere 7 signals were counted under fluorescent microscope and ratio was calculated for each case. Correlation between C-MET CNV and conventional clinic-pathologic parameters was done by Fischer exact test. CNV was identified in the form of amplification and polysomy (3.1% each) and associated with poorer prognostic parameters. Our pilot study highlights limited subset of patients that may benefit from anti-C-MET-targeted therapy and thus could be a novel biomarker for targeted intervention in GAC.

Association of raised levels of IL-4 and anti-TPO with hyperprolactinemia.

BACKGROUND AND OBJECTIVE: The modulatory role of prolactin in autoimmune regulation is well established. Hyperprolactinemia is often associated with autoimmune disease like systemic lupus erythematosus and autoimmune thyroid diseases. The objective was to compare levels of direct and indirect autoimmune factors in different categories of hyperprolactinemia cases and predict the direction of association between hyperprolactinemia and autoimmune factors, if any. METHODS: A total of 102 hyperprolactinemia cases (>100 ng/mL serum prolactin level) were included along with 24 controls. Among 102 hyperprolactinemia cases, there were 36 idiopathic cases, 19 pituitary adenoma cases, 36 drug-induced cases, and 11 cases associated with other secondary/systemic diseases (chronic renal failure, chronic hepatic failure, etc). MEASUREMENTS: Direct autoimmune markers, IL-2, IFN-γ, IL-4, and IL-5, were measured in serum by ELISA. Indirect autoimmune markers, anti-TPO, anti-tg, anti-CCP, VDRL, platelet count, and aPTT, were measured as per laboratory-defined protocol. RESULTS: Serum levels of IL-4 and anti-TPO were significantly high in idiopathic hyperprolactinemia cases. Serum IL-4 levels were also significantly high in pituitary adenoma cases, drug-induced cases, and in cases with other secondary causes of hyperprolactinemia. Serum anti-TPO levels were also significantly high in drug-induced hyperprolactinemia cases. CONCLUSION: No significant difference in autoimmune factors is observed between macroprolactinemia and true hyperprolactinemia. Serum IL-4 and anti-TPO were high in all categories of hyperprolactinemia. This suggests a possible association of hyperprolactinemia with autoimmune conditions (high IL-4 and anti-TPO), mostly subclinical. Thus, hyperprolactinemia case with serum prolactin level >100 ng/mL may require long-term follow-up for the development of autoimmune disease in future.

Prevalence and reproductive manifestations of macroprolactinemia.

PURPOSE: Macroprolactinemia is characterized by predominance of macroprolactin molecules in circulation and generally has extra-pituitary origin. Macroprolactin is viewed as biologically inactive, therefore asymptomatic, and thus may not require any treatment or prolonged follow-up. In addition, data on prevalence of macroprolactinemia and its clinical manifestation are also rare. Therefore, the present study was aimed to find out prevalence of macroprolactinemia and its association, if any, with reproductive manifestations. MATERIAL AND METHODS: Macroprolactin was measured in 102 hyperprolactinemia cases (>100 ng/ml prolactin level), 135 physiological hyperprolactinemia cases (50 pregnant and 85 lactating females; >100 ng/ml prolactin level) and 24 controls. Poly ethylene glycol (PEG) precipitation method was carried out to screen macroprolactin. Prolactin recovery of <25% was considered overt macroprolactinemia. Detailed clinical data was recorded which included complete medical history, physical examination and hormone measurements besides CT/MRI for pituitary abnormalities. RESULTS: Prevalence of macroprolactinemia was 21.57% (22/102) in hyperprolactinemia (prolactin >100 ng/ml). There was no case of macroprolactinemia in physiological hyperprolactinemia, or healthy control females. Reproductive manifestations were present in 72.73% (16/22) macroprolactinemia cases, out of which macroprolactinemia was the sole cause of associated reproductive manifestations in 68.7% (11/16) cases. Reversal of reproductive dysfunction/s was observed in five cases with appropriate treatment for high macroprolactin. CONCLUSION: Macroprolactinemia prevalence was found to be 21.5%, out of which 72.73% cases had associated reproductive dysfunctions.

Genomics: Tool to predict and prevent male infertility.

A large number of human diseases arise as a result of genetic abnormalities. With the advent of improved molecular biology techniques, the genetic etiology of male infertility is increasing. The common genetic factors responsible for male infertility are chromosomal abnormalities, Yq microdeletion and cystic fibrosis. These are responsible for approximately 30 percent cases of male infertility. About 40 percent cases of male infertility are categorized as idiopathic. These cases may be associated with genetic and genomic abnormalities. During last few years more and more genes are implicated in male infertility leading to decline in prevalence of idiopathic etiology. In this review we will summarize up to date published works on genetic etiologies of male infertility including our own works. We also briefly describe reproductive technologies used to overcome male infertility, dangers of transmitting genetic disorders to offspring and ways to prevent transmission of genetic disorders during assisted reproduction. At the end we will provide our points on how genomic information can be utilized for prediction and prevention of male infertility in coming years.

SNP Microarray in FISH Negative Clinically Suspected 22q11.2 Microdeletion Syndrome.

The present study evaluated the role of SNP microarray in 101 cases of clinically suspected FISH negative (noninformative/normal) 22q11.2 microdeletion syndrome. SNP microarray was carried out using 300 K HumanCytoSNP-12 BeadChip array or CytoScan 750 K array. SNP microarray identified 8 cases of 22q11.2 microdeletions and/or microduplications in addition to cases of chromosomal abnormalities and other pathogenic/likely pathogenic CNVs. Clinically suspected specific deletions (22q11.2) were detectable in approximately 8% of cases by SNP microarray, mostly from FISH noninformative cases. This study also identified several LOH/AOH loci with known and well-defined UPD (uniparental disomy) disorders. In conclusion, this study suggests more strict clinical criteria for FISH analysis. However, if clinical criteria are few or doubtful, in particular newborn/neonate in intensive care, SNP microarray should be the first screening test to be ordered. FISH is ideal test for detecting mosaicism, screening family members, and prenatal diagnosis in proven families.

High Serum Estradiol and Heavy Metals Responsible for Human Spermiation Defect-A Pilot Study.

INTRODUCTION: Spermiation is a process of releasing sperm into the lumen of seminiferous tubules. Failure in releasing sperm into the lumen is designated as spermiation defect. Spermiation defect cases present as oligo-azoospermia or azoospermia despite normal gonadotropins and testicular histology/cytology. Human spermiation defect never got attention to investigate infertility practice. Most of the information on spermiation defect, so far is from animal experiments. We assume some cases of non-obstructive azoospermia with normal gonadotropins and testicular histology/cytology could be due to spermiation defect. AIM: The aim of the study was to find out the underlying aetiology in cases of human spermiation defect. MATERIALS AND METHODS: A total of 13 cases of spermiation defect and 20 fertile men as control constituted study material. Cases were studied for chromosomal abnormalities by conventional karyotyping, sex chromosome mosaicism by interphase XY FISH, Yq microdeletion by STS PCR, sertoli cell quality (function) and quantity (numbers) by serum Anti-Mullerian Hormone (AMH) and inhibin B besides other hormones like Follicular Stimulating Hormone (FSH), prolactin, testosterone and estradiol. Vitamin A concentration in serum was also measured. Presence of heavy metal was investigated by elemental electron microscopy in seminal cells (eight cases) & by spectrometry in serum as well as seminal plasma. RESULTS: Chromosomal and Yq microdeletion study failed to detect any abnormalities. AMH, inhibin B and vitamin A were also normal. Estradiol level was high in 6 out of 13 cases (46%) while platinum in seminal cells was high in 4 cases (50%). High (four times or more) serum level of lead and nickel was observed in 11 (85%) and 6 (46%) cases, respectively. CONCLUSION: High serum concentration of heavy metals like lead & nickel or high platinum accumulation in seminal cells or high serum estradiol alone or in combinations may be underlying aetiologic factors in human spermiation defect.

Williams-Beuren Syndrome: Experience of 43 Patients and a Report of an Atypical Case from a Tertiary Care Center in India.

Williams-Beuren syndrome (WBS) or Williams syndrome (OMIM 194050) is a multisystem disorder manifested by neurodevelopmental delay and is caused by a hemizygous deletion of ∼ 1.5-1.8 Mb in the 7q11.23 region. Clinical features include cardiovascular anomalies (mainly supravalvular aortic stenosis), peripheral pulmonary stenosis, distinctive facies, intellectual disability (usually mild), unique personality characteristics, and growth and endocrine abnormalities. Clinical diagnostic criteria are available for WBS; however, the mainstay of diagnosis is the detection of the contiguous gene deletion. Although FISH remains the most widely used laboratory test, the diagnosis can also be established by means of qPCR, MLPA, microsatellite marker analysis, and chromosomal microarray (CMA). We evaluated the utility of MLPA to detect deletion/duplication in the 7q11.23 region in 43 patients suspected to have WBS using MLPA kits for microdeletion syndromes. A hemizygous deletion in the 7q11.23 region was found in 41 (95.3%) patients using MLPA. One patient had an atypical deletion detected by CMA. During the initial period of this study, the results of 12 patients tested by MLPA were also confirmed by FISH. Compared to FISH and CMA, MLPA is a cheaper, high-throughput, less labor-intensive and less time-consuming technique for the diagnosis of WBS. Although CMA is expensive and labor-intensive, its effectiveness is demonstrated to detect an atypical deletion and to delineate the breakpoints.

Fluorescence in situ hybridization (FISH) using non-commercial probes in the diagnosis of clinically suspected microdeletion syndromes.

BACKGROUND & OBJECTIVES: Microdeletion syndromes are characterized by small (<5 Mb) chromosomal deletions in which one or more genes are involved. These are frequently associated with multiple congenital anomalies. The phenotype is the result of haploinsufficiency of genes in the critical interval. Fluorescence in situ hybridization (FISH) technique is commonly used for precise genetic diagnosis of microdeletion syndromes. This study was conducted to assess the role of FISH in the diagnosis of suspected microdeletion syndrome. METHODS: FISH was carried out on 301 clinically suspected microdeletion syndrome cases for the confirmation of clinical diagnosis using non-commercial probes. Of these, 177 cases were referred for 22q11.2 microdeletion, 42 cases were referred for William syndrome, 38 cases were referred for Prader Willi/Angelman and 44 cases were referred for other suspected microdeletion syndromes. RESULTS: FISH was confirmatory in 23 cases only (7.6%). There were 17 cases of 22q11.2 microdeletion, four cases of Prader Willi syndrome and two cases of William syndrome. INTERPRETATION & CONCLUSION: We conclude that FISH should not be the method of choice for clinically suspected microdeletion syndromes. We propose to follow strict clinical criteria for FISH testing or preferably to follow better methods (genotype first approach). Whole genome screening may be used as first line of test and FISH may be used for confirmation of screening result, screening of family members and prenatal diagnosis.