Low-dose letrozole - an effective option for women with symptomatic adenomyosis awaiting IVF: a pilot randomized controlled trial.

RESEARCH QUESTION: Can low-dose letrozole reduce dysmenorrhoea, menorrhagia and sonographic features in symptomatic women with adenomyosis awaiting IVF? DESIGN: This was a longitudinal randomized prospective pilot study to explore the effectiveness of low-dose letrozole and compare it with a gonadotropin releasing hormone (GnRH) agonist in reducing dysmenorrhoea, menorrhagia and sonographic features in symptomatic women with adenomyosis awaiting IVF. The women were treated for 3 months, either with the GnRH agonist goserelin 3.6 mg/month (n = 77) or the aromatase inhibitor letrozole 2.5 mg three times weekly (n = 79). Dysmenorrhoea and menorrhagia were evaluated at randomization and followed up monthly using a visual analogue score (VAS) and pictorial blood loss assessment chart (PBAC), respectively. A quantitative scoring method was used to assess the improvement of sonographic features after 3 months of treatment. RESULTS: Both groups reported a marked improvement in symptoms after 3 months of treatment. In both the letrozole and GnRH agonist groups, VAS and PBAC scores decreased significantly over the 3 months (letrozole: P = 0.0001 and P = 0.0001 for VAS and PBAC, respectively; GnRH agonist: P = 0.0001 and P = 0.0001 for VAS and PBAC, respectively). Participants on letrozole had regular menstruation cycles, while most of the women who received the GnRH agonist were amenorrhoeic, with only four women reporting mild bleeding. Haemoglobin concentrations also improved after both treatments (letrozole P = 0.0001, GnRH agonist P = 0.0001). A quantitative assessment of sonographic features showed significant improvements following both treatments (diffuse adenomyosis of the myometrium: letrozole P = 0.015, GnRH agonist P = 0.039; diffuse adenomyosis of the junctional zone: letrozole P = 0.025, GnRH agonist P = 0.001). Women with adenomyoma also responded well to both therapies (letrozole P = 0.049, GnRH agonist P = 0.024), whereas the letrozole group responded comparatively better in focal adenomyosis when the outer myometrium was involved (letrozole P < 0.001, GnRH agonist P = 0.26). No noticeable side effects were observed in women receiving letrozole therapy. Additionally, letrozole therapy was found to be more cost-effective than GnRH agonist treatment. CONCLUSIONS: Low-dose letrozole treatment is a low-cost alternative to a GnRH agonist, with comparable effects in improving the symptoms and sonographic features of adenomyosis in women awaiting IVF.

Two chair test: a substitute of 6 min walk test appear cardiopulmonary reserve specific.

BACKGROUND: A simple and efficient exercise test possible in a small space is welcome to supplement 6 min walk test (6MWT) that demands a 100 feet corridor to perform. METHODS: The proposed two chair test (2CT) makes a person to sit and move five times between two chairs placed face to face at 5 feet apart and note the changes in pulse-rate (PR) and arterial oxygen saturation (SpO2) at every 10 s for 2 min after that. Comparison of the post-exercise measurements (PR and SpO2) with a repeat performance in same patients was done for reproducibility and doing the same after 6MWT and 2CT in another set of patients was meant for for acceptability. The statistical analysis was made on moment to moment change, mean maximal difference and mean cumulative difference for the measurements using p value, z-score, r value and principal component analysis (PCA). FINDINGS: A total of 40 and 60 volunteers were included for testing reproducibility and acceptability. On both the sets, the difference in most of comparisons between the measured variable (PR and SpO2) showed the p values remaining insignificant (>0.05), and z-score being <1 SD of the corresponding other and the correlation coefficients (r) remaining excellent (>0.9). Furthermore, the PCA shows complete overlapping. The post-exercise changes did not corelate the walking distance in 6MWT. INTERPRETATION: The proposed 2CT demands small space and appears reproducible and comparable with 6MWT in terms of its post-exercise impact on PR and SpO2. This novel test also appears more of cardiopulmonary reserve specific.

Pregnancy and Live Birth Rates Are Comparable in Young Infertile Women Presenting with Severe Endometriosis and Tubal Infertility.

The aim of this study is to evaluate the effect of severe endometriosis in younger patients compared to tubal infertility on pregnancy and live birth rate undergoing in vitro fertilization (IVF). This prospective observational study included 294 women with severe endometriosis and 358 women with tubal factor as control who underwent IVF. Follicular fluid samples were collected during oocyte retrieval, and cytokines and angiogenic factors were estimated. The groups were sub-stratified based on age. Number of metaphase II oocytes, grade I/II embryos, pregnancy rate, miscarriage rate per pregnancy, and live birth rate were compared. Significantly elevated levels of cytokines and angiogenic molecules were observed in younger endometriosis patients when compared to tubal group (p < 0.001). Number of MII oocytes (p < 0.003) and grade I/II embryos (p < 0.001) were observed to be significantly lower in these women when compared with matched controls. Despite higher levels of inflammatory cytokines, angiogenic molecules, fewer MII oocytes, and grade I/II embryos, the younger endometriosis patients had similar pregnancy (OR 0.81; 95% CI 0.54-1.22; p = 0.31) and live birth rate (OR 0.78; 95% CI 0.5-1.2; p = 0.26) when compared with matched controls. In contrast, endometriosis patients of age ≥ 35 years had significantly less likelihood of live birth (OR 0.47; 95% CI 0.25-0.9; p = 0.02) and pregnancy rate (OR 0.46; 95% CI 0.22-0.95; p = 0.03), respectively, when compared with the matched controls. It appears that women with severe endometriosis have even chance of successful pregnancy if diagnosed at early age and sought for assisted reproductive technology to reduce its adverse effect on reproductive outcome.

Metabolomic alterations in invasive ductal carcinoma of breast: A comprehensive metabolomic study using tissue and serum samples.

Invasive ductal carcinoma (IDC) is the most common type of breast cancer and the leading cause of breast cancer related mortality. In the present study, metabolomic profiles of 72 tissue samples and 146 serum samples were analysed using targeted liquid chromatography multiple reaction monitoring mass spectrometry (LC-MRM/MS) and untargeted gas chromatography mass spectrometry (GC-MS) approaches. Combination of univariate and multivariate statistical treatment identified significant alterations of 42 and 32 metabolites in tissue and serum samples of IDC, respectively when compared to control. Some of the metabolite changes from tissue were also reflected in serum, indicating a bi-directional interaction of metabolites in IDC. Additionally, 8 tissue metabolites and 9 serum metabolites showed progressive change from control to benign to IDC suggesting their possible role in malignant transformation. We have identified a panel of three metabolites viz. tryptophan, tyrosine, and creatine in tissue and serum, which could be useful in screening of IDC subjects from both control and benign. The metabolomic alterations in IDC showed perturbations in purine and pyrimidine metabolism, amino sugar metabolism, amino acid metabolism, fatty acid biosynthesis etc. Comprehensively, this study provides valuable insights into metabolic adaptations of IDC, which can help to identify diagnostic markers as well as potential therapeutic targets.

Multipronged quantitative proteomics reveals serum proteome alterations in breast cancer intrinsic subtypes.

Being molecularly heterogeneous, breast cancer tends to be a complicated oncological disease with high incidence rates throughout the world. The primary aim of this study was to identify the set of serum proteins with discriminatory capabilities towards the four major subtypes of breast cancer. We employed multipronged quantitative proteomic approaches like 2D-DIGE, iTRAQ and SWATH-MS and identified 307 differentially regulated proteins. Luminal A subtype consisted of 24, Luminal B subtype 38, HER2 Enriched subtype 17 and Triple negative breast cancer subtype 10 differentially regulated subtype specific proteins. These specific proteins were further subjected to bioinformatic tools which revealed the involvement in platelet degranulation, fibrinolysis, lipid metabolism, immune response, complement activation, blood coagulation, glycolysis and cancer signaling pathways in the subtypes of the breast cancer. The significant discrimination efficiency of the models generated through multivariate statistical analysis was decent to distinguish each of the four subtypes from controls. Further, some of the statistically significant differentially regulated proteins were verified and validated by immunoblotting and mass spectrometry based selected reaction monitoring (SRM) approach. Our Multipronged proteomics approaches revealed panel of serum proteins specifically altered for individual subtypes of breast cancer. The mass spectrometry data are available via ProteomeXchange with identifier PXD006441. BIOLOGICAL SIGNIFICANCE: Worldwide, breast cancer continues to be one of the leading causes of cancer related deaths in women and it encompasses four major molecular subtypes. As breast cancer treatment majorly depends on identification of specific subtype, it is important to diagnosis the disease at subtype level. Our results using multipronged quantitative proteomics identified 307 differentially regulated proteins in which 24 were specific for Luminal A, 38 for Luminal B, 17 for HER2 enriched and 10 proteins were specific for TN subtype. Bioinformatic analysis of these proteins revealed certain biological processes and pathways altered at subtype level and validation experiments of some of these proteins using immunoblotting and SRM assays are consistent with discovery data. This is the first comprehensive proteomic study on serum proteome alterations at subtype level which will not only help to distinguish subtype of breast cancer but also contribute to a better understanding of the molecular characteristic of breast cancer at individual subtype level.

Serum metabolomics of Indian women with polycystic ovary syndrome using 1H NMR coupled with a pattern recognition approach.

Polycystic ovary syndrome (PCOS) is one of the most commonly occurring metabolic and endocrinological disorders affecting women of reproductive age. Metabolomics is an emerging field that holds promise in understanding disease pathophysiology. Recently, a few metabolomics based studies have been attempted in PCOS patients; however, none of them have included patients from the Indian population. The main objective of this study was to investigate the serum metabolomic profile of Indian women with PCOS and compare them with controls. Proton nuclear magnetic resonance (1H NMR) was used to first identify the differentially expressed metabolites among women with PCOS from the Eastern region of India during the discovery phase and further validated in a separate cohort of PCOS and control subjects. Multivariate analysis of the binned spectra indicated 16 dysregulated bins in the sera of these women with PCOS. Out of these 16 bins, 13 identified bins corresponded to 12 metabolites including 8 amino acids and 4 energy metabolites. Amongst the amino acids, alanine, valine, leucine and threonine and amongst the energy metabolites, lactate and acetate were observed to be significantly up-regulated in women with PCOS when compared with controls. The remaining 4 amino acids, l-glutamine, proline, glutamate and histidine were down-regulated along with 2 energy metabolites: glucose and 3-hydroxybutyric acid. Our findings showed dysregulations in the expression of different metabolites in the serum of women with PCOS suggesting the involvement of multiple pathways including amino acid metabolism, carbohydrate/lipid metabolism, purine and pyrimidine metabolism and protein synthesis.

Urinary proteome alterations in HER2 enriched breast cancer revealed by multipronged quantitative proteomics.

Globally, breast cancer is the second most common cancer among women. Although biomarker discoveries through various proteomic approaches of tissue and serum samples have been studied in breast cancer, urinary proteome alterations in breast cancer are least studied. Urine being a noninvasive biofluid and a significant source of proteins, it has the potential in early diagnosis of breast cancer. This study used complementary quantitative gel-based and gel-free proteomic approaches to find a panel of urinary protein markers that could discriminate HER2 enriched (HE) subtype breast cancer from the healthy controls. A total of 183 differentially expressed proteins were identified using three complementary approaches, namely 2D-DIGE, iTRAQ, and sequential window acquisition of all theoretical mass spectra. The differentially expressed proteins were subjected to various bioinformatics analyses for deciphering the biological context of these proteins using protein analysis through evolutionary relationships, database for annotation, visualization and integrated discovery, and STRING. Multivariate statistical analysis was undertaken to identify the set of most significant proteins, which could discriminate HE breast cancer from healthy controls. Immunoblotting and MRM-based validation in a separate cohort testified a panel of 21 proteins such as zinc-alpha2-glycoprotein, A2GL, retinol-binding protein 4, annexin A1, SAP3, SRC8, gelsolin, kininogen 1, CO9, clusterin, ceruloplasmin, and α1-antitrypsin could be a panel of candidate markers that could discriminate HE breast cancer from healthy controls.

Repeated implantation failure versus repeated implantation success: discrimination at a metabolomic level.

STUDY QUESTION: Is there any difference at the serum metabolic level between women with recurrent implantation failure (RIF) and women with recurrent implantation success (RIS) when undergoing in vitro fertilization (IVF)? SUMMARY ANSWER: Eight metabolites, including valine, adipic acid, l-lysine, creatine, ornithine, glycerol, d-glucose and urea, were found to be significantly up-regulated in women with RIF when compared with women with RIS. WHAT IS KNOWN ALREADY: Despite transfer of three high-grade embryos per cycle, RIF following three or more consecutive IVF attempts occurs in a group of infertile women. Conversely, there is a group of women who undergo successful implantation each cycle, yet have a poor obstetric history. STUDY DESIGN, SIZE, DURATION: This study was conducted over a period of 10 years (January 2004-October 2014). Groups of 28 women with RIF (age ≤40 years and BMI ≤28) and 24 women with RIS (age and BMI matched) were selected from couples with primary infertility reporting at the Institute of Reproductive Medicine, Kolkata, India. Women recruited in the RIF group had history of implantation failure in at least three consecutive IVF attempts, in which three embryos of high-grade quality were transferred in each cycle. PARTICIPANTS/MATERIALS, SETTING, METHODS: Blood samples were collected from both the groups during the implantation window following overnight fasting for at least 10 h (7-10 days post ovulation). Samples were analyzed using a 700 MHz NMR spectrometer and acquired spectra were subjected to chemometric and statistical analysis. Serum levels of endothelial nitric oxide synthase (eNOS) were measured using an enzyme immunoassay technique. MAIN RESULTS AND THE ROLE OF CHANCE: Valine, adipic acid, l-lysine, creatine, ornithine, glycerol, d-glucose and urea were found to be significantly down-regulated in women with RIS when compared with those with RIF, with fold change values of 0.81, 0.82, 0.79, 0.80, 0.78, 0.68, 0.76 and 0.74, respectively. Further, serum eNOS was found to be significantly lower in women with RIF when compared with RIS (P < 0.05), indicating possible impairment in nitric oxide production. Metabolites, mostly related to energy metabolism, lipid metabolism and the arginine metabolic pathway were found to be considerably altered and are likely to be associated with the RIF phenomenon. However, the interplay between these molecules in RIF is complex and holds merit for further exploration. LIMITATIONS, REASONS FOR CAUTION: In-depth studies of the arginine metabolic pathway in endometrial tissues seem necessary to validate our findings. A limitation of the present study is that the metabolic level changes, eNOS and nitric oxide levels have not been investigated in the endometrial tissues of the two groups of women. It would be interesting to investigate whether there exists a direct link between metabolic dysregulation and genetic factors that affects implantation in RIF women. WIDER IMPLICATIONS OF THE FINDINGS: We speculate that tissue metabolomics can provide an improved understanding of the metabolic dysfunction associated with RIF. The identification of serum metabolic marker(s) in women with RIS may help with strategies of early therapeutic intervention, which may improve the chances of implantation significantly in women otherwise susceptible to IVF failure. STUDY FUNDING/COMPETING INTERESTS: One of the authors, S.R.C. acknowledges the Council of Scientific and Industrial Research (CSIR), Government of India [No: 9/81(1228)/14, EMR-I] for financial support.

Quantitative tissue proteomic investigation of invasive ductal carcinoma of breast with luminal B HER2 positive and HER2 enriched subtypes towards potential diagnostic and therapeutic biomarkers.

Worldwide, breast cancer is one of the frequently diagnosed cancers in women with high mortality if not diagnosed at early stage. Although biomarker discoveries through various proteomic approaches have been studied in breast cancer, a limited number of studies have explored the invasive ductal carcinoma with Luminal B HER2 positive (LB) and HER2 enriched (HE) subtypes. The present study employed the complementary quantitative proteomic approaches to find a panel of markers that could discriminate LB and HE subtypes as well as early (ES) and late stages (LS) of these subtypes. A total of 67 and 68 differentially expressed proteins were identified by DIGE for the subtype and stage wise categories, respectively. Multivariate statistical analysis was employed to identify the set of most significant proteins, which could discriminate between these two subtypes and also early and late stages under study. Immunoblotting and MRM based validation in a separate cohort of samples confirmed that panel of biosignatures for LB are APOA1, GELS, HS90B, EF1A1, NHRF1 and PRDX3 and for HE are PRDX1, CATD, CALR, ATPB and CH60. For the diagnosis of early and late stages the potential markers are TPM4, CATD, PRDX3, ANXA3, HSPB1 and CALR, TRFE, GELS, CH60, CAPG, NHRF1, 1433G, GRP78 respectively.

Metabolomics and its integration with systems biology: PSI 2014 conference panel discussion report.

Metabolomics, being a relatively new field, is facing multiple challenges related to data acquisition and interpretation, reproducibility across analytical platforms, integration with other omics approaches and translation into theragnostic biomarkers. There is an immediate need to overcome these challenges in order to make metabolomics more useful and reliable in terms of improving our current understanding of disease biology and help in developing predictive biomarkers. Researchers interested in metabolomics gathered for a panel discussion on 'Metabolomics and its integration with systems biology' during the 6th Annual Meeting of Proteomics Society-India and International Conference on "Proteomics from Discovery to Function" held at the Indian Institute of Technology, Bombay from December 7-9, 2014. The panel discussed various challenges related to metabolomics and also proposed several effective solutions for optimum implementation of metabolomics in clinical practice. The key areas of panel discussion were improvement in metabolite databases with comprehensive spectral libraries, need for extensive bioinformatics tools for integrative approaches and serious considerations for clinical validation of the biomarkers for the successful implementation of metabolomics in clinics. BIOLOGICAL SIGNIFICANCE: Information drafted in this report is significant for researchers working in metabolomics field to overcome the challenges and successful implementation of metabolomics in clinical practice. This article is part of a special issue titled: Proteomics in India.

Regenerative nanomedicine: current perspectives and future directions.

Nanotechnology has considerably accelerated the growth of regenerative medicine in recent years. Application of nanotechnology in regenerative medicine has revolutionized the designing of grafts and scaffolds which has resulted in new grafts/scaffold systems having significantly enhanced cellular and tissue regenerative properties. Since the cell-cell and cell-matrix interaction in biological systems takes place at the nanoscale level, the application of nanotechnology gives an edge in modifying the cellular function and/or matrix function in a more desired way to mimic the native tissue/organ. In this review, we focus on the nanotechnology-based recent advances and trends in regenerative medicine and discussed under individual organ systems including bone, cartilage, nerve, skin, teeth, myocardium, liver and eye. Recent studies that are related to the design of various types of nanostructured scaffolds and incorporation of nanomaterials into the matrices are reported. We have also documented reports where these materials and matrices have been compared for their better biocompatibility and efficacy in supporting the damaged tissue. In addition to the recent developments, future directions and possible challenges in translating the findings from bench to bedside are outlined.

Identification of key contributory factors responsible for vascular dysfunction in idiopathic recurrent spontaneous miscarriage.

Poor endometrial perfusion during implantation window is reported to be one of the possible causes of idiopathic recurrent spontaneous miscarriage (IRSM). We have tested the hypothesis that certain angiogenic and vasoactive factors are associated with vascular dysfunction during implantation window in IRSM and, therefore, could play a contributory role in making the endometrium unreceptive in these women. This is a prospective case-controlled study carried out on 66 women with IRSM and age and BMI matched 50 fertile women serving as controls. Endometrial expression of pro-inflammatory (IL-1ß, TNF-α, IFN-γ, TGF-ß1), anti-inflammatory (IL-4, -10), angiogenesis-associated cytokines (IL-2, -6, -8), angiogenic and vasoactive factors including prostaglandin E2 (PGE2), vascular endothelial growth factor (VEGF), endothelial nitric oxide synthase (eNOS), nitric oxide (NO) and adrenomedullin (ADM) were measured during implantation window by ELISA. Subendometrial blood flow (SEBF) was assessed by color Doppler ultrasonography. Multivariate analysis was used to identify the significant factor(s) responsible for vascular dysfunction in IRSM women during window of implantation and further correlated with vascular dysfunction. Endometrial expression of pro-inflammatory cytokines and PGE2 were up-regulated and anti-inflammatory and angiogenesis-associated cytokines down-regulated in IRSM women as compared with controls. Further, the angiogenic and vasoactive factors including VEGF, eNOS, NO and ADM were found to be down-regulated and SEBF grossly affected in these women. Multivariate analysis identified IL-10, followed by VEGF and eNOS as the major factors contributing towards vascular dysfunction in IRSM women. Moreover, these factors strongly correlated with blood flow impairment. This study provides an understanding that IL-10, VEGF and eNOS are the principal key components having a contributory role in endometrial vascular dysfunction in women with IRSM. Down-regulation of these factors is also associated with impaired endometrial perfusion which possibly makes the endometrium unreceptive that may eventually cause early pregnancy loss.

Complex codon usage pattern and compositional features of retroviruses.

Retroviruses infect a wide range of organisms including humans. Among them, HIV-1, which causes AIDS, has now become a major threat for world health. Some of these viruses are also potential gene transfer vectors. In this study, the patterns of synonymous codon usage in retroviruses have been studied through multivariate statistical methods on ORFs sequences from the available 56 retroviruses. The principal determinant for evolution of the codon usage pattern in retroviruses seemed to be the compositional constraints, while selection for translation of the viral genes plays a secondary role. This was further supported by multivariate analysis on relative synonymous codon usage. Thus, it seems that mutational bias might have dominated role over translational selection in shaping the codon usage of retroviruses. Codon adaptation index was used to identify translationally optimal codons among genes from retroviruses. The comparative analysis of the preferred and optimal codons among different retroviral groups revealed that four codons GAA, AAA, AGA, and GGA were significantly more frequent in most of the retroviral genes inspite of some differences. Cluster analysis also revealed that phylogenetically related groups of retroviruses have probably evolved their codon usage in a concerted manner under the influence of their nucleotide composition.

Genus specific evolution of codon usage and nucleotide compositional traits of poxviruses.

Poxviruses are complex in their nucleotide compositional features of the coding regions. The codon usages in Poxviruses are in accordance with their compositional bias. In the Poxviridae family, codon usage patterns and nucleotide compositional traits are widely divergent across species but some conservation was observed within a genus. Viruses from six Chordopox genera, i.e., Avipoxvirus, Capripoxvirus, Cervidpoxvirus, Orthopoxvirus, Suipoxvirus, Yatapoxvirus, and one Entomopox genus- Betaentomopoxvirus, and some unclassified Entomopoxvirus are significantly rich in AT composition. Four other Chordopox genera- Molluscipoxvirus, Orthopoxvirus, Parapoxvirus, and some unclassified Chordopoxvirus are dominated by the GC rich viruses. Poxviruses from these AT rich and GC rich genera preferred AT or GC ending codons owing to their respective nucleotide compositional bias. For example, viruses from AT rich Orthopoxvirus, or GC rich Parapoxvirus have evolved with mutually exclusive type codon preferences following their genus-specific nucleotide compositions. Additional factors like gene length and expression level also influenced their codon usage patterns to some extent in some Poxvirus genera. Evidences from correspondence analysis and cluster analysis on the extent of divergence in codon usage also support this genus specific evolution of Poxvirus codon usage. Analyzes suggest that most of the Poxviruses from different genera, have evolved in almost two different evolutionary trajectory in context of their nucleotide composition and codon usage.

A detailed comparative analysis on the overall codon usage pattern in herpesviruses.

Herpesviruses infect a wide range of organisms including humans. Some of these viruses are potential gene transfer vectors for gene therapy. However no study has been reported on total codon usage of herpesvirus. In this study, the patterns of synonymous codon usage in herpesviruses have been studied through multivariate statistical methods on 4875 ORFs from the available 49 completely sequenced herpesvirus genomes. A general trend of weakly biased codon usage was observed among herpesviruses, but few among them showed some degree of strong bias. The principal determinants behind such notable variations within the patterns seemed to be the overall GC content and GC content at the 3rd base position of the viral genes. These determinants strongly correlated with the first major axis of correspondence analysis on relative synonymous codon usage (RSCU). This is an indication of mutational bias that dominates over translational selection in herpesvirus. Among the other determining factors, gene length also has some degree of influence on the codon usage pattern. The comparative analysis of the preferred and optimal codons among the clades revealed that different codons were preferred in different clades, though six codons CUG, CAC, CAG, AAC, GUG, and GAC were found to be more frequent in most of the herpesviral genes. Codon adaptation index (CAI) was used to predict highly expressed genes among herpesviruses, and to identify translationally optimal codons. Cluster analysis reveals that the majority of the members of a clade have similar codon usage and nucleotide composition, but with some notable exception. Additionally phylogenetic analysis indicates that codon usage of the viruses cannot be explicitly tied to their species evolution.