Metformin ameliorates endometrial thickness in a rat model of thin endometrium.

Metformin, a well-established anti-diabetic drug, is also used in managing various other metabolic disorders including polycystic ovarian syndrome (PCOS). There are evidences to show that metformin improves endometrial functions in PCOS women. However, fewer studies have explored the direct effects of metformin on endometrium. Previous in vitro studies have shown that therapeutic serum concentrations of metformin enhance endometrial epithelial cell proliferation. The present study was undertaken to investigate in vivo effects of metformin on endometrial proliferation in a rat model of thin endometrium. Toward this, a rat model of thin endometrium was developed. Metformin (0.1% or 1% w/v) was administrated orally for 15 days in rats with thin endometrium. Oral metformin administration for three consecutive estrous cycles (15 days) in the thin endometrium rat model led to an increase in endometrial thickness compared to sham endometrium. Histological analysis showed a significant increase in the number of endometrial glands (P < 0.05), stromal cells (P < 0.01) and blood vessels (P < 0.01) in metformin-treated (n = 10 in each group) uterine horns compared to sham (saline-treated) uterine horns in rats. The expression of proliferating cell nuclear antigen and vascular epithelial growth factor was found to be upregulated on treatment with 1% metformin-treated group (n = 7). However, pregnancy outcomes in the rats treated with metformin remained unaltered despite the restoration of endometrial thickness. In conclusion, the study demonstrated that metformin ameliorates endometrial thickness in a rat model of thin endometrium by increasing endometrial proliferation and angiogenesis, without restoration of embryo implantation.

Secretomes from Conventional Insemination and Intra-Cytoplasmic Sperm Injection Derived Embryos Differentially Modulate Endometrial Cells In Vitro.

Conventional Insemination (CI) and Intra-Cytoplasmic Sperm Injection (ICSI) are routinely used insemination methods in clinical Assisted Reproductive Technologies (ART) settings. However, the existing data on the developmental competence and implantation potential of CI and ICSI derived embryos are not unequivocal. This prospective study on 23 patients undergoing ART treatment explored whether the secretomes of CI- and ICSI-derived embryo differentially alter the expression of integrins (αv and ß3 integrin) and MUCIN-1 (MUC-1) in a human endometrial epithelial cell line (Ishikawa). Immunocytochemical data demonstrated that the secretome of CI-derived top quality (GI) embryos induced higher (p < 0.05) expression of ɑv ß3 compared to sibling ICSI derived G1 embryos in Ishikawa cells. Though, relative levels of the transcript for MUC-1, anti-adhesion molecule did not show a significant difference between the study groups, immunocytochemical analysis demonstrated significantly (p < 0.0001) higher expression of MUC-1 in cells treated with ICSI-derived embryo secretome, compared to that treated with CI -derived embryo secretome. These results suggest that secretomes from CI and ICSI embryos differentially modulate the endometrial cells in vitro. This hints at differences in the ability of CI- and ICSI- derived embryos to alter endometrial profile.

The dynamics of SARS-CoV-2 infection in unvaccinated and vaccinated populations in Mumbai, India, between 28 December 2020 and 30 August 2021.

The emergence and evolution of severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) variants that could compromise vaccine efficacy (VE) with re-infections in immunized individuals have necessitated continuous surveillance of VE. Here, the occurrence and dynamics of SARS-CoV-2 infections in the context of vaccination during the second wave of infection in Mumbai were evaluated. RT-PCR cycle threshold (Ct) values of the open reading frame (ORF)/envelope (E)/nucleocapsid (N) genes obtained from a total of 42415 samples, comprising unvaccinated (96.88%) and vaccinated cases (3.12%) were analyzed between December 28, 2020, and August 30, 2021. A lower incidence of SARS-CoV-2 infection in fully vaccinated cases (5.07%) compared to partially vaccinated cases (6.5%) and unvaccinated cases (13.453%) was recorded. VE was significant after the first dose of vaccination (ORF gene p-value = 0.003429, and E/N gene p-value = 0.000866). Furthermore, VE was observed to be significant when the post-immunization (first dose) period was stratified to within 30 days (ORF gene p-value = 0.0094 and E/N gene p-value = 0.0023) and to 60 days following the second dose of vaccination (ORF gene p-value = 0.0238). Also, significantly higher efficacy was observed within individuals receiving two doses compared to a single dose (ORF gene p-value = 0.0132 and E/N gene p-value = 0.0387). The emergence of breakthrough infections was also evident (odds ratio= 0.34; 95% confidence interval= 0.27-0.43). Interestingly, viral loads trended towards being higher in some groups of partially vaccinated individuals compared to completely vaccinated and unvaccinated populations. Finally, our results delineated a significantly higher incidence of SARS-CoV-2 acquisition in males, asymptomatic individuals, individuals with comorbidities, and those who were unvaccinated.

Pregnancy outcomes among Indian women: increased prevalence of miscarriage and stillbirth during 2015-2021.

BACKGROUND: Pregnancy outcome is an important health indicator of the quality of maternal health. Adverse pregnancy outcomes is a major public health problem, which can lead to poor maternal and neonatal outcomes. This study investigates the trends in pregnancy outcomes prevalent during 2015-2021 in Indian women. METHODS: The study analysed the data presented in the fourth (2015-16) and fifth (2019-21) rounds of National Family Health Survey (NFHS). The absolute and relative changes in the birth outcomes of last pregnancy during the five years preceding the surveys were estimated using data collected from 195,470 women in NFHS-4 and from 255,549 women in NFHS-5. RESULTS: Livebirth decreased by 1.3 points (90.2% vs. 88.9%), and nearly half of the Indian states/UTs (n = 17/36) had lower than the national average of livebirth (88.9%) reported during 2019-21. A higher proportion of pregnancy loss was noted, particularly miscarriages increased in both urban (6.4% vs. 8.5%) and rural areas (5.3% vs. 6.9%), and stillbirth increased by 28.6% (0.7% vs. 0.9%). The number of abortions decreased (3.4% vs. 2.9%) among Indian women. Nearly half of the abortions were due to unplanned pregnancies (47.6%) and more than one-fourth (26.9%) of abortions were performed by self. Abortions among adolescent women in Telangana was eleven times higher during 2019-21 as compared to 2015-16 (8.0% vs. 0.7%). CONCLUSION: Our study presents evidence of a decrease in the livebirth and an increase in the frequency of miscarriage and stillbirth among Indian women during 2015-2021. This study emphasises that there is a need of regional-specific, comprehensive and quality maternal healthcare programs for improving livebirth among Indian women.

Cytomegalovirus reactivation in a SARS-CoV-2 infected woman experiencing fetal demise in the first trimester with fetal trisomy 21: A case report.

Cytomegalovirus (CMV) is the most common cause of congenital viral infections. Women seropositive for CMV prior to pregnancy can develop a non-primary CMV infection. Here, we present a case of first trimester pregnancy loss during active SARS-CoV-2 infection. There was no evidence of SARS-CoV-2 RNA in placenta and fetal tissue, but there was presence of congenital cytomegalovirus infection by nested PCR. To the best of our knowledge, this is the first report demonstrating association of early congenital CMV infection due to reactivation and fetal demise in a SARS-CoV-2 positive woman with fetal trisomy 21.

Therapeutic metformin concentrations positively regulate proliferation in endometrial epithelial cells via mTOR activation and augmented mitochondrial strength.

Metformin, an antidiabetic drug, has recently been repositioned in the treatment of several nondiabetic disorders, including reproductive disorders such as polycystic ovarian syndrome, where it improves endometrial functions. In vitro studies employing supratherapeutic concentrations (5-20 mmol/L) of metformin have reported antiproliferative effects on endometrial epithelial and stromal cells. However, animal and human studies have revealed that therapeutic serum concentrations of metformin range between 20 and 70 µmol/L. In the present study, the effect of therapeutic concentrations of metformin was studied on endometrial epithelial cells (EECs). Therapeutic concentrations of metformin induced proliferation in Ishikawa and HEC-1A cells. The proliferation of EECs was found to be mammalian target of rapamycin (mTOR) dependent. Interestingly, therapeutic metformin concentrations were not able to activate the classical AMP-activated protein kinase (AMPK) signaling. On the contrary, supratherapeutic metformin concentration (10 mmol/L) inhibited mTOR and activated AMPK signaling. Microarray analysis of metformin-treated HEC-1A cells revealed dose-dependent differential effects on biological pathways associated with translation, ribosomal RNA processing, mitochondrial translation, and cell proliferation. Therapeutic concentrations of metformin upregulated mitochondrial number as demonstrated by increased MitoTracker™ Red staining and enhanced succinate dehydrogenase expression; however, higher concentration (10 mmol/L) abrogated the same. Our results suggest that therapeutic concentrations of metformin augment mitochondrial strength and induce mTOR-dependent endometrial cell proliferation.

Clinical presentation, pregnancy complications, and outcomes of pregnant women with COVID-19 during the Omicron-dominant third wave in Mumbai, India.

OBJECTIVE: To study clinical presentation, disease severity, pregnancy complications, and maternal outcomes in women affected with coronavirus disease 2019 (COVID-19) during the third wave compared with the first and second waves of COVID-19. METHODS: A retrospective, observational cohort study was conducted among 2058 pregnant and postpartum women with COVID-19 admitted during three wave periods at a tertiary care COVID-19-dedicated hospital. RESULTS: The number of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) -infected pregnant and postpartum women with symptoms of COVID-19 was four times higher during the third wave compared with the first (odds ratio [OR] 4.6, 95% confidence interval [CI] 3.5-6.0, P < 0.001). There was a significantly lower proportion of pregnant and postpartum women with moderate to severe COVID-19 during the third wave (0.6%, 2/318) compared with those during the first wave (2.4%, 27/1143, P < 0.001) and second wave (14.4%, 86/597, P < 0.001). The intensive care/high dependency unit admissions during the third wave were significantly lower (2.5%, 8/318) than during the second wave (14.7%, 88/597; OR 0.2, 95% CI 0.1-0.3, P < 0.001) but similar to the first wave (2.4%, 27/1143). CONCLUSIONS: Decreased severity of COVID-19, reduced maternal mortality, and morbidity were reported in the third wave compared with the first wave and second wave of COVID-19 in the Mumbai Metropolitan Region, India. TRIAL REGISTRATION: The study is registered with the Clinical Trial Registry of India (Registration no: CTRI/2020/05/025423).

Estrogen receptor expression is modulated in human and mouse prostate epithelial cells during cancer progression.

Substantial data posit estrogen receptors (ERs) as promising targets for prostate cancer (PCa) therapeutics. However, the trials on assessing the chemo-preventive or therapeutic potential of ER targeting drugs or selective estrogen receptor modulators (SERMs) have not yet established their clinical benefits. This could be ascribed to a possible modulation in the ER expression during PCa progression. Further it is warranted to test various ER targeting drugs in appropriate preclinical models that simulate human ER expression pattern during PCa progression. The study was undertaken to revisit the existing data on the epithelial ER expression pattern in human cancerous prostates and experimentally determine whether these patterns are replicated in TRAMP (Transgenic Adenocarcinoma of Mouse Prostate) mice, a model for human PCa. Estradiol (E2) binding to the plasma membrane of the epithelial cells and its modulation during the PCa progression in TRAMP were also investigated. A reassessment of the existing data revealed a trend towards downregulation in the epithelial expression of wild-type ESR1 transcripts in high-grade PCa, compared to non-cancerous prostate in humans. Next, epithelial cell-enriched populations from TRAMP prostates (TP) displaying low-grade prostatic intraepithelial neoplasia (LGPIN), high-grade PIN (HGPIN), HGPIN with well-differentiated carcinoma (PIN + WDC), WDC (equivalent to grade 2/3 human PCa), and poorly-differentiated carcinoma (PDC-equivalent to grade 4/5 human PCa) revealed significantly higher Esr1 and Esr2 levels in HGPIN and significantly reduced levels in WDC, compared to respective age-matched control prostates. These patterns for the nuclear ERs were similar to the trend shown by E2 binding to the plasma membrane of the epithelial cells during PCa progression in TRAMP. E2 binding to epithelial cells (EpCAM+), though significantly higher in TPs displaying LGPIN, decreased significantly as the disease progressed to WDC. The study highlights a reduction in the epithelial ESR level with the PCa progression and this pattern was evident in both humans and TRAMP. These observations may have major implications in refining PCa therapeutics targeting ER.

Clinical characteristics and surgical management of endometriosis-associated infertility: A multicenter prospective cohort study.

OBJECTIVE: To study clinical, surgical characteristics and the relationship between endometriosis lesion types and conception rate after surgery in infertile women with endometriosis. METHODS: A prospective, multicenter cohort of 204 women (age 20-35 years) with endometriosis was followed up post-surgery between November 2017 and February 2020 at three tertiary-care hospitals. RESULTS: Based on the severity of endometriosis lesion type, deep infiltrating endometriosis (DIE) (81/204, 39.7%) was the most common lesion; followed by ovarian endometriosis (OMA) (64/204, 31.4%), and superficial peritoneal endometriosis (SUP) (59/204, 28.9%). Endometriosis patients had a single lesion type (94/204, 46.1%), two lesion types (77/204, 37.7%), or three lesion types (33/204, 16.2%) with significant differences between regions (P < 0.001). Around 40% (37/95) of obese women had SUP (P = 0.003) whereas 78% (14/18) of underweight women had DIE (P < 0.001). Significant differences in mean Endometriosis Fertility Index scores between endometriosis lesion types and patients with one, two, and three types of lesions were observed (P < 0.001). The majority (22/32, 68.8%) of the women conceived naturally after the surgery. Half (16/32; 50%) of the women with a single lesion type conceived after the surgery; of which most (13/16, 81.2%) had SUP, followed by OMA (2/16, 12.5%), and DIE (1/16, 6.3%). CONCLUSION: Women with SUP and only one type of endometriotic lesion were more likely to conceive post-surgery.

Dysregulation of X-ray repair cross-complementing 4 expression in the eutopic endometrium of women with endometriosis.

Recent data suggest that the DNA damage response (DDR) is altered in the eutopic endometrium (EE) of women with endometriosis and this probably ensues in response to higher DNA damage encountered by the EE in endometriosis. DDR operates in a tissue-specific manner and involves different pathways depending on the type of DNA lesions. Among these pathways, the non-homologous end joining (NHEJ) pathway plays a critical role in the repair of dsDNA breaks. The present study was undertaken to explore whether NHEJ is affected in the EE of women with endometriosis. Toward this, we focused on the X-ray repair cross-complementing 4 (XRCC4) protein, one of the core components of the NHEJ pathway. Endometrial XRCC4 protein levels in the mid-proliferative phase were found significantly (P < 0.05) downregulated in women with endometriosis, compared to control women. Investigation of a microarray-based largest dataset in the Gene Expression Omnibus database (GSE51981) revealed a similar trend at the transcript level in the EE of women with endometriosis, compared to control women. Further in vitro studies were undertaken to explore the effects of H2O2-induced oxidative stress on DNA damage, as assessed by γ-H2AX and 8-hydroxy-2'-deoxyguanosine (8-OHdG) immunolocalization, and XRCC4 protein levels in endometrial stromal (hTERT immortalized human endometrial stromal cell line (ThESCs)) and epithelial (Ishikawa) cells. A significant decrease in XRCC4 protein levels and significantly higher localization of γ-H2AX and 8-OHdG were evident in ThESCs and Ishikawa cells experiencing oxidative stress. Overall, the study demonstrates that the endometrial XRCC4 expression is dysregulated in women with endometriosis and this could be due to higher oxidative stress in endometriosis.

Soluble human leukocyte antigen-G is a potential embryo viability biomarker and a positive predictor of live-births in humans.

PROBLEM: Human infertility affects 15-20% of reproductive-age couples and it is mitigated by assisted reproductive technology (ART) approaches. Poor biological viability of embryos contributes to implantation failure and live birth rate (LBR). This study is aimed to examine whether or not embryo-secreted soluble human leukocyte antigen-G (sHLA-G) is (i) associated with developing embryos and (ii) able to predict successful pregnancy outcome. METHOD OF STUDY: A retrospective, multicentric study using 539 human embryo spent medium samples (E-SMs), analysed for sHLA-G levels by ELISA. Correlation analysis was performed on sHLA-G levels with developing embryonic stages, their quality scores and pregnancy outcome in terms of LBR. RESULTS: Of 539 E-SMs analysed, 445 had detectable sHLA-G (83%) with levels varying within and across clinics and, between stages of embryonic development. Levels of sHLA-G (ng/mL) were significantly (P < .05) different in E-SMs of cleavage-stage embryos versus blastocysts. There was an insignificant correlation between the sHLA-G levels and morphology scores of embryos. But, sHLA-G levels showed a positive correlation with grades of blastocysts and importantly, its levels were significantly (P < .05) higher in live-birth vis-a-vis no-birth cases. Also, levels were higher in live-births out of blastocysts-ETs versus cleavage-stage-embryo transfers. Altered levels were observed with embryos, which resulted in miscarriages. Overall, a significant (P < .0001) association of sHLA-G with live births was observed. CONCLUSION: Embryo-derived sHLA-G can be a valuable embryo viability, independent, biomarker, which can predict live-birth outcome and it could be useful as an adjunct to existing criteria for elective single embryo transfer.

An overview of preclinical animal models for SARS-CoV-2 pathogenicity.

Severe acute respiratory syndrome coronavirus (SARS-CoV)-2 has caused millions of fatalities globally since its origin in November 2019. The SARS-CoV-2 shares 79 and 50 per cent genome similarity with its predecessors, severe SARS-CoV and Middle East respiratory syndrome (MERS) coronavirus, all belonging to the same genus, Betacoronavirus. This relatively new virus has stymied the effective control of COVID-19 pandemic and caused huge social and economic impact worldwide. The FDA-approved drugs were re-purposed to reduce the number of fatalities caused by SARS-CoV-2. However, controversy surrounds about the efficacy of these re-purposed antiviral drugs against SARS-CoV-2.This necessitates the identification of new drug targets for SARS-CoV-2. Hence, the development of pre-clinical animal model is warranted. Such animal models may help us gain better understanding of the pathophysiology of SARS-CoV-2 infection and will be effective tools for the evaluation and licensure of therapeutic strategies against SARS-CoV-2. This review provides a summary of the attempts made till to develop a suitable animal model to understand pathophysiology and effectiveness of therapeutic agents against SARS-CoV-2.

Endometrial DNA damage response is modulated in endometriosis.

STUDY QUESTION: Is the DNA damage response (DDR) dysregulated in the eutopic endometrium of women with endometriosis? SUMMARY ANSWER: Endometrial expression of genes involved in DDR is modulated in women with endometriosis, compared to those without the disease. WHAT IS KNOWN ALREADY: Ectopic endometriotic lesions are reported to harbour somatic mutations, thereby hinting at dysregulation of DDR and DNA repair pathways. However, it remains inconclusive whether the eutopic endometrium also manifests dysregulated DDR in endometriosis. STUDY DESIGN, SIZE, DURATION: For this case-control study conducted between 2015 and 2019, eutopic endometrial (E) samples (EE- from women with endometriosis, CE- from women without endometriosis) were collected in either mid-proliferative (EE-MP, n = 23; CE-MP, n = 17) or mid-secretory (EE-MS, n = 17; CE-MS, n = 9) phases of the menstrual cycle. This study compares: (i) DNA damage marker localization, (ii) expression of DDR genes and (iii) expression of DNA repair genes in eutopic endometrial samples from women with and without endometriosis. PARTICIPANTS/MATERIALS, SETTING, METHODS: The study included (i) 40 women (aged 31.9 ± 0.81 years) with endometriosis and (ii) 26 control women (aged 31.4 ± 1.02 years) without endometriosis. Eutopic endometrial samples from the two groups were divided into different parts for histological analysis, immunohistochemistry, RNA extraction, protein extraction and comet assays. Eighty-four genes of relevance in the DNA damage signalling pathway were evaluated for their expression in eutopic endometrial samples, using RT2 Profiler PCR arrays. Validations of the expression of two GADD (Growth Arrest DNA Damage Inducible) proteins - GADD45A and GADD45G were carried out by immunoblotting. DNA damage was assessed by immunohistochemical localization of γ-H2AFX (a phosphorylated variant of histone H2AX) and 8-OHdG (8-hydroxy-2'-deoxyguanosine). RNA sequencing data from mid-proliferative (EE-MP, n = 4; CE-MP, n = 3) and mid-secretory phase (EE-MS and CE-MS, n = 4 each) endometrial samples were scanned to compare the expression status of all the genes implicated in human DNA repair. PCNA (Proliferating Cell Nuclear Antigen) expression was determined to assess endometrial proliferation. Residual DNA damage in primary endometrial cells was checked by comet assays. Public datasets were also scanned for the expression of DDR and DNA repair genes as our RNASeq data were limited by small sample size. All the comparisons were made between phase-matched endometrial samples from women with and without endometriosis. MAIN RESULTS AND THE ROLE OF CHANCE: Endometrial expression of DDR genes and intensity of immunolocalized γ-H2AFX were significantly (P < 0.05) higher in EE, compared to CE samples. DDR proteins, especially those belonging to the GADD family, were found to be differentially abundant in EE, as compared to CE. These patterns were evident in both mid-proliferative and mid-secretory phases. Intriguingly, higher DDR was associated with increased cell proliferation in EE-MP, compared to CE-MP. Furthermore, among the differentially expressed transcripts (DETs) encoded by DNA repair genes, the majority showed up-regulation in EE-MP, compared to CE-MP. Interestingly, CE-MP and EE-MP had a comparable percentage (P > 0.05) of cells with residual DNA damage. However, unlike the mid-proliferative phase data, many DETs encoded by DNA repair genes were down-regulated in EE-MS, compared to CE-MS. An analysis of the phase-matched control and endometriosis samples included in the GSE51981 dataset available in the Gene Expression Omnibus database also revealed significant (P < 0.05) alterations in the expression of DDR and DNA repair genes in EE, compared to CE. LARGE-SCALE DATA: N/A. LIMITATIONS, REASONS FOR CAUTION: The study was conducted on a limited number of endometrial samples. Also, the study does not reveal the causes underlying dysregulated DDR in the eutopic endometrium of women with endometriosis. WIDER IMPLICATIONS OF THE FINDINGS: Alterations in the expression of DDR and DNA repair genes indirectly suggest that eutopic endometrium, as compared to its healthy counterpart, encounters DNA damage-inducing stimuli, either of higher strength or for longer duration in endometriosis. It will be worthwhile to identify the nature of such stimuli and also explore the role of higher genomic insults and dysregulated DDR/DNA repair in the origin and/or progression of endometriosis. STUDY FUNDING/COMPETING INTEREST(S): The study was supported by the Department of Biotechnology and Indian Council of Medical Research, Government of India. No conflict of interest is declared.

Plasma membrane proteome of adhesion-competent endometrial epithelial cells and its modulation by Rab11a.

Human endometrial epithelium (EE) is composed of a multitude of proteins, amongst which those localized on the plasma membrane [plasma membrane proteins (PMPs)] are of critical relevance in the early stages of implantation. Evidence supports the key role of few PMPs in implantation. However, many remain unidentified, as efforts have not been made till date to generate the plasma membrane proteome of human EE cells, using a gel-free approach. This study presents a protein catalog of the PMP enriched fraction of Ishikawa cell line; often used as an in vitro model for embryo-adhesive EE. Liquid chromatography with tandem mass spectrometry identified 3,598 proteins. Of these, 1,963 proteins were annotated for their membrane localization. Of 1,963 proteins, 1,321 were found to have a transmembrane domain and 43 proteins had glycophosphatidylinositol (GPI) anchor. Extensive data mining revealed endometrial expression of 943 proteins reported in humans and/or rodents. Further, quantitative alterations were observed in the plasma membrane proteome on the perturbation of intracellular trafficking. Silencing of Rab11a (known for its role in plasma membrane organization) expression caused alteration in the abundance of 74 proteins. Caveolin-1 and EpCAM levels were reduced whereas Rab4a abundance increased in the PMP extracts of Rab11a deficient cells, compared with control cells. Briefly, the study reports the identity of several novel plasma membrane-localized proteins. A major spin-off of the study is the identification of novel proteins trafficked by Rab11a to the plasma membrane. Targeted analysis of novel PMPs may reveal their specific roles in endometrial receptivity and implantation.

Membrane-initiated estrogen signaling in prostate cancer: A route to epithelial-to-mesenchymal transition.

The plasma membrane (PM) is considered as a major druggable site. More than 50% of the existing drugs target PM proteins. In the wake of emerging data indicating a key role of estrogens in prostate cancer (PCa) pathogenesis, the study was undertaken to explore whether the estrogen binding sites exist on the PM and if such sites are functionally relevant in PCa. Estradiol (E2) binding to the PM was detected in androgen-dependent (LNCaP), androgen-independent (PC3, DU145) PCa cell lines, nontumorigenic (RWPE1) prostate epithelial cell line, and rat prostate cells. Conventional estrogen receptors (nuclear estrogen receptors), known for their nuclear localization, were detected in the PM enriched extracts. This was indirectly confirmed by reduced localization of ERs on the PM of cells, silenced for the expression of their cognate genes. Further, unlike cell-permeable E2, stimulation with cell-impermeable estradiol (E2-BSA) did not induce proliferation in LNCaP cells. However, stimulation with E2-BSA led to alterations in the phosphorylation status of several kinases including GSK3 and AKT, along with the hyperphosphorylation of cytoskeletal proteins such as ß-actin and cytokeratin 8 in LNCaP. This was accompanied by epithelial-to-mesenchymal (EMT) features such as increased migration and invasion; higher vimentin expression, and a concomitant decrease in the E-cadherin expression. These effects were not observed in RWPE1 cells. Interestingly, cell-permeable E2 failed to induce EMT in PCa cells. This in vitro study is the first to suggest that the PM-initiated estrogen signaling contributes to higher invasiveness in PCa cells. Plasma membrane ERs may act as novel targets for PCa therapeutics.

Rab11a drives adhesion molecules to the surface of endometrial epithelial cells.

STUDY QUESTION: Is Rab11a GTPase, a regulator of intracellular trafficking, of significance in endometrial functions? SUMMARY ANSWER: Rab11a is an important component of the cascades involved in equipping the endometrial epithelium (EE) with 'adhesiveness' and 'cohesiveness'. WHAT IS KNOWN ALREADY: Cell adhesion molecules (CAMs) have been investigated extensively for modulation in their endometrial expression during the peri-implantation phase. However, the mechanisms by which CAMs are transported to the EE surface have not received the same attention. Rab11a facilitates transport of specific proteins to the plasma membrane in endothelial cells, fibroblasts, embryonic ectodermal cells, etc. However, its role in the transport of CAMs in EE remains unexplored. STUDY DESIGN, SIZE, DURATION: In-vitro investigations were directed towards deciphering the role of Rab11a in trafficking of CAMs (integrins and E-cadherin) to the cell surface of Ishikawa, an EE cell line. Towards this, Rab11a stable knockdown (Rab-kd) and control clones of Ishikawa were generated. JAr (human trophoblastic cell line) cells were used to form multicellular spheroids. Pre-receptive (n = 6) and receptive (n = 6) phase endometrial tissues from women with proven fertility and receptive phase (n = 6) endometrial tissues from women with unexplained infertility were used. PARTICIPANTS/MATERIALS, SETTING, METHODS: Rab-kd and control clones were used for in-vitro assays. Live cells were used for biotinylation, JAr spheroid assays, flow cytometry, trans-epithelial electrical resistance assays and wound-healing assays. Lysosome and Golgi membranes were isolated by ultracentrifugation. Confocal microscopy, immunoblotting, qRT-PCR and immunohistochemistry were employed for assessing the expression of Rab11a, integrins and E-cadherin. MAIN RESULTS AND THE ROLE OF CHANCE: shRNA-mediated attenuation of Rab11a expression led to a significant (P < 0.01) decline in the surface localization of αVß3 integrin. Cell surface protein extracts of Rab-kd clones showed a significant (P < 0.05) reduction in the levels of αV integrin. Further, a significant (P < 0.01) decrease was observed in the percent JAr spheroids attached to Rab-kd clones, compared to control clones. Rab-kd clones also showed a significant (P < 0.001) decline in the total levels of E-cadherin. This was caused neither by reduced transcription nor by increased lysosomal degradation. The role of Rab11a in maintaining the epithelial nature of the cells was evident by a significant increase in the migratory potential, presence of stress-fibres and a decrease in the trans-epithelial resistance in Rab-kd monolayers. Further, the levels of endometrial Rab11a and E-cadherin in the receptive phase were found to be significantly (P < 0.05) lower in women with unexplained infertility compared to that in fertile women. Taken together, these observations hint at a key role of Rab11a in the trafficking of αVß3 integrin and maintenance of E-cadherin levels at the surface of EE cells. LARGE-SCALE DATA: N/A. LIMITATIONS, REASONS FOR CAUTION: The in-vitro setting of the study is a limitation. Further immunohistochemical localizations of Rab11a and CAMs were conducted on a limited number of human endometrial samples. WIDER IMPLICATIONS OF THE FINDINGS: Rab11a-mediated trafficking of endometrial CAMs in EE cells can be explored further for its potential as a target for fertility regulation or infertility management. STUDY FUNDING/COMPETING INTEREST(S): This study was funded by the Indian Council of Medical Research (ICMR), the Department of Science and Technology (DST), the Council of Scientific and Industrial Research (CSIR), Government of India. No competing interests are declared.

Association of high-density lipoprotein, triglycerides, and homocysteine with bone mineral density in young Indian tribal women.

This study investigated association between lipids and homocysteine (Hcy) with bone mineral density (BMD) in young women as opposed to previous studies on elderly women. HDL, triglyceride, and Hcy are significantly associated with BMD in young women and tobacco and alcohol consumption have no effect on this association. PURPOSE: The present study investigates whether the association of serum lipids and homocysteine (Hcy) with bone mineral density (BMD) reported mostly in elderly population can be generalized to young or premenopausal women, consequently suggesting screening of young women with low BMD for dyslipidemia or any cardiovascular events and vice versa. METHODS: Women (n = 293, aged 20-47 years) from Northeast India belonging to Tibeto-Burman origin were enrolled. Information about their physical and clinical attributes were collected by a structured questionnaire. Their BMDs at lumbar spine and femur were measured by dual-energy X-ray absorptiometry (DXA) and sera were profiled for lipid parameters and Hcy by auto-analyzer and ELISA, respectively. Women consuming tobacco and/or alcohol were grouped as consumers and others as non-consumers for the analysis. RESULTS: Positive correlation of BMD with HDL (spine and femur r = 0.38, p < 0.0001) and triglyceride (spine r = 0.534, p < 0.0001; femur r = 0.423, p < 0.0001) was observed, whereas Hcy correlated negatively with BMD (spine r = - 0.189, p = 0.0026; femur r = - 0.273, p < 0.0001). LDL showed a weak negative correlation with BMD (spine r = - 0.128, p = 0.0283; femur r = - 0.199, p = 0.0006). However, after adjusting for age, BMI, and consumption, HDL, triglyceride, and Hcy continued to show significant correlation with BMD at both the sites. Logistic regression analyses indicated that HDL, triglyceride, and Hcy were significant predictors of osteopenia and osteoporosis in our study cohort; however, consumption did not contribute to its prediction. CONCLUSION: Low levels of HDL and triglyceride and high levels of Hcy are significantly associated with osteopenia and osteoporosis in young Northeast Indian women.

Estrogen matters in metastasis.

Metastatic cancer cells meet several physical, biochemical and immunological barriers before colonizing a new territory. Cancerous cells turn invasive, mobile and eventually disengage from their native niche. This is followed by their intravasation, extravasation, survival, proliferation, and colonization into distant organs. Unlike well-confined tumors, which respond favorably to anti-cancer therapeutics, metastatic tumors are life-threatening and incurable. More than 90% of cancer-related mortality is caused by metastases, hence the emphasis is now on developing the strategies to block or reverse the process of metastasis. This has ensued intensive research with a focus on the mechanisms underlying metastasis. Substantial work carried out in this direction has led to the identification of specific enzymes, proteins, cytokines, chemokines, growth factors, exosomes, miRNA and lipids, etc. as the facilitators of metastasis. Metastatic cells are exposed to a diverse array of local and systemic signals. Among these, estrogens are of great relevance. Estrogens have been strongly linked to cancers, especially of breast and uterine origin. Recent data hint that estrogens, well recognized for their role in proliferation, may have a role in metastasis also. It is proposed that influence of estrogen on metastasis may be independent of its proliferation-inducing ability. Data are emerging to suggest that estrogens have potential to modulate various events of the metastatic cascade such as local invasion, intravasation, anoikis, immune evasion, extravasation, angiogenesis and metastatic colonization. This review summarizes some of the recent advances in our knowledge on the role of estrogens in the metastatic cascade of cancerous cells.

Investigation of HSA as a biocompatible coating material for arsenic trioxide nanoparticles.

The anticancer properties of arsenic trioxide (As2O3) are accompanied by highly cytotoxic effects on normal cells. This necessitates developing modalities towards the targeted delivery of As2O3. Albumins, on account of their large structure and presence of several interacting groups, are ideal for encapsulating or carrying various drugs. In the present study, human serum albumin (HSA) was chosen as a coating agent to increase the biocompatibility of As2O3. An in situ chemical precipitation method was adopted for the synthesis of HSA-coated As2O3 nanoparticles (HSA-As2O3NPs) that were further characterized by Fourier-transform infrared spectroscopy (FT-IR), X-ray diffraction (XRD), ultraviolet-visible (UV-vis) spectroscopy, inductively coupled plasma atomic emission spectrometry (ICP-AES), zeta potential and transmission electron microscopy (TEM). HSA-As2O3NPs were assessed for their biocompatibility using mouse fibroblast cells (NIH-3T3) and human dermal fibroblast (HDF) cells by a time- and dose-dependent cytocompatibility MTT assay. The safety of the HSA-As2O3 nanoparticles was assessed using haemolysis and blood cell aggregation studies. Molecular simulation studies provided evidence of interaction between HSA and As2O3. Herein, we report the development of a protein-based delivery system for As2O3 with improved biocompatibility.