Prescription-based cost analysis of medicines for cardiovascular risk factors at Indian Council of Medical Research-Rational Use of Medicine Centre Hospitals of India.

OBJECTIVES: India has taken several initiatives to provide health care to its population while keeping the related expenditure minimum. Since cardiovascular diseases are the most prevalent chronic conditions, in the present study, we aimed to analyze the difference in prices of medicines prescribed for three cardiovascular risk factors, based on (a) listed and not listed in the National List of Essential Medicines (NLEM) and (b) generic and branded drugs. MATERIALS AND METHODS: Outpatient prescriptions for diabetes mellitus, hypertension, and dyslipidemia were retrospectively analyzed from 12 tertiary centers. The prices of medicines prescribed were compared based on presence or absence in NLEM India-2015 and prescribing by generic versus brand name. The price was standardized and presented as average price per medicine per year for a given medicine. The results are presented in Indian rupee (INR) and as median (range). RESULTS: Of the 4,736 prescriptions collected, 843 contained oral antidiabetic, antihypertensive, and/or hypolipidemic medicines. The price per medicine per year for NLEM oral antidiabetics was INR 2849 (2593-3104) and for non-NLEM was INR 5343 (2964-14364). It was INR 806 (243-2132) for generic and INR 3809 (1968-14364) for branded antidiabetics. Antihypertensives and hypolipidemics followed the trend. The price of branded non-NLEM medicines was 5-22 times higher compared to generic NLEM which, for a population of 1.37 billion, would translate to a potential saving of 346.8 billion INR for statins. The variability was significant for sulfonylureas, angiotensin receptor blockers, beta-blockers, diuretics, and statins (P < 0.0001). CONCLUSION: The study highlights an urgent need for intervention to actualize the maximum benefit of government policies and minimize the out-of-pocket expenditure on medicines.

Robotic Resection in Succinate Dehydrogenase Subunit B (SDHB)-Mutated Hereditary Paraganglioma: A Case Report of Two Patients and A Literature Review.

Autosomal dominant hereditary paraganglioma-pheochromocytoma syndrome (HPPS) is a rare genetic disorder characterized by neuroendocrine tumor development associated with pathogenic variants in succinate dehydrogenase (SDH) enzyme complex genes. Particularly, HPPS linked to SDHB mutation poses a significant clinical challenge due to its association with aggressive tumor features and a high risk of malignancy. Our report underscores the diversity in the presentation of patients with SDHB-mutated paraganglioma and the feasibility of managing it with a minimally invasive surgical approach. In the first case, a 17-year-old female was diagnosed with a metabolically active, poorly differentiated extra-adrenal retroperitoneal paraganglioma that required challenging robotic resection. Cascade genetic testing revealed an SDHB mutation not only in her but also in three family members, pointing to the inherited nature of the syndrome. Conversely, the second case involves a 37-year-old male with an asymptomatic well-differentiated left paraaortic paraganglioma incidentally found during an unrelated medical examination. Robotic converted-to-open resection allowed the successful removal of the mass. Subsequent germline testing confirmed a deleterious SDHB mutation, initiating a process of familial cascade testing. Both patients remained symptom- and recurrence-free at 12 and six months, respectively. Through these cases, and supported by a literature review, we highlight the variable clinical presentations of HPPS, arising from the same genetic alteration. The successful application of minimally invasive surgical techniques, combined with genetic evaluation, emphasizes the necessity for a comprehensive, tailored approach to treatment and surveillance. This strategy not only addresses the immediate clinical needs but also fosters proactive management of at-risk family members, ensuring a multidisciplinary approach to this complex hereditary condition.

Financial Toxicity in Radiation Oncology.

Financial toxicity details the financial burden patients face due to a variety of medical costs. Cancer patients, especially those receiving radiation therapy, are at a much higher risk of experiencing economic hardships than healthy people or people with other conditions. There are a variety of risk factors associated with financial toxicity as well as numerous tools to assess the toxicity experienced by patients. In this review article, we present a concise overview of contributors, risk factors, case studies, tools, impacts, and potential interventions of financial toxicity.

Evaluation of prescriptions from tertiary care hospitals across India for deviations from treatment guidelines & their potential consequences.

BACKGROUND OBJECTIVES: Irrational prescribing practices have major consequences on patient safety and also increase the economic burden. Real-life examples of impact of irrational prescription have potential to improve prescribing practices. In this context, the present study aimed to capture and evaluate the prevalence of deviations from treatment guidelines in the prescriptions, potential consequence/s of the deviations and corrective actions recommended by clinicians. METHODS: It was a cross-sectional observational study conducted in the outpatient departments of tertiary care hospitals in India wherein the 13 Indian Council of Medical Research Rational Use of Medicines Centres are located. Prescriptions not compliant with the standard treatment guidelines and incomplete prescriptions with respect to formulation, dose, duration and frequency were labelled as 'prescriptions having deviations'. A deviation that could result in a drug interaction, lack of response, increased cost, preventable adverse drug reaction (ADR) and/or antimicrobial resistance was labelled as an 'unacceptable deviation'. RESULTS: Against all the prescriptions assessed, about one tenth of them (475/4838; 9.8%) had unacceptable deviations. However, in 2667/4838 (55.1%) prescriptions, the clinicians had adhered to the treatment guidelines. Two thousand one hundred and seventy-one prescriptions had deviations, of which 475 (21.9%) had unacceptable deviations with pantoprazole (n=54), rabeprazole+domperidone (n=35) and oral enzyme preparations (n=24) as the most frequently prescribed drugs and upper respiratory tract infection (URTI) and hypertension as most common diseases with unacceptable deviations. The potential consequences of deviations were increase in cost (n=301), ADRs (n=254), drug interactions (n=81), lack of therapeutic response (n=77) and antimicrobial resistance (n=72). Major corrective actions proposed for consideration were issuance of an administrative order (n=196) and conducting online training programme (n=108). INTERPRETATION CONCLUSIONS: The overall prevalence of deviations found was 45 per cent of which unacceptable deviations was estimated to be 9.8 per cent. To minimize the deviations, clinicians recommended online training on rational prescribing and administrative directives as potential interventions.

Intravascular large B-cell lymphoma masquerading as stroke successfully treated with R-Hyper-CVAD.

Intravascular large B cell lymphoma (IVLBCL) is exceedingly rare and difficult to diagnose. We describe a case of IVLBCL in a 56-year-old male which was identified after recurrent strokes. Right partial nephrectomy was then performed which demonstrated renal oncocytoma and IVLBCL. Chemotherapy was initiated with standard R-Hyper-CVAD which included intrathecal methotrexate and cytarabine. R-CHOP is largely considered the treatment of choice in IVLBCL, however low doses of chemotherapy in this regimen do not cross the blood brain barrier like in R-Hyper-CVAD. The patient achieved complete remission after completion of treatment and has remained in remission for 5 years after diagnosis.

HDAC8-mediated inhibition of EP300 drives a transcriptional state that increases melanoma brain metastasis.

Melanomas can adopt multiple transcriptional states. Little is known about the epigenetic drivers of these cell states, limiting our ability to regulate melanoma heterogeneity. Here, we identify stress-induced HDAC8 activity as driving melanoma brain metastasis development. Exposure of melanocytes and melanoma cells to multiple stresses increases HDAC8 activation leading to a neural crest-stem cell transcriptional state and an amoeboid, invasive phenotype that increases seeding to the brain. Using ATAC-Seq and ChIP-Seq we show that increased HDAC8 activity alters chromatin structure by increasing H3K27ac and enhancing accessibility at c-Jun binding sites. Functionally, HDAC8 deacetylates the histone acetyltransferase EP300, causing its enzymatic inactivation. This, in turn, increases binding of EP300 to Jun-transcriptional sites and decreases binding to MITF-transcriptional sites. Inhibition of EP300 increases melanoma cell invasion, resistance to stress and increases melanoma brain metastasis development. HDAC8 is identified as a mediator of transcriptional co-factor inactivation and chromatin accessibility that drives brain metastasis.

Orbital Apex Syndrome Due to Aspergillus flavus Infection in Immunocompetent Patients: A Report of Two Cases.

Aspergillus species are fungi that are commonly found in soil and decaying vegetation and have the potential to cause an orbital apex syndrome that is marked by ophthalmoplegia or vision loss. We report the clinical and investigational findings and outcomes of two patients with orbital apex syndrome. The first patient was a 26-year-old female, premorbidly healthy, who presented with a gradually increasing proptosis of the left eye with a reduction in vision. An MRI revealed findings consistent with proptosis, pansinusitis with a soft tissue opacity involving the left orbital apex with optic nerve compression, extending to the cavernous sinus with an associated temporal meningeal enhancement. Following functional endoscopic sinus surgery (FESS), Aspergillus flavus was grown in culture, and oral voriconazole was initiated. The second patient was a 53-year-old male who presented with bilateral reduction of vision and ptosis, proptosis with total ophthalmoplegia (third, fourth, and sixth nerve palsies) of the right eye. An MRI study revealed extensive involvement of the apex of the right orbit, the right cavernous sinus, the medial aspect of the left cavernous sinus, and the pituitary gland. A FESS was done, and the histopathology specimen was suggestive of aspergillosis, and the tissue fungal polymerase chain reaction (PCR) test was positive for Aspergillus flavus. He was treated with amphotericin B and oral voriconazole with significant improvement. Physicians need to have a high index of suspicion for invasive fungal sino-orbital infections, even in immunocompetent patients. The presence of nasal congestion, recurrent sinusitis, facial pain, headache, orbital cellulitis, proptosis, or ophthalmoplegia should prompt early investigations.

Mutations within the selectivity filter reveal that Kv1 channels have distinct propensities to slow inactivate.

Voltage-activated potassium (Kv) channels open in response to membrane depolarization and subsequently inactivate through distinct mechanisms. For the model Shaker Kv channel from Drosophila, fast N-type inactivation is thought to occur by a mechanism involving blockade of the internal pore by the N-terminus, whereas slow C-type inactivation results from conformational changes in the ion selectivity filter in the external pore. Kv channel inactivation plays critical roles in shaping the action potential and regulating firing frequency, and has been implicated in a range of diseases including episodic ataxia and arrhythmias. Although structures of the closely related Shaker and Kv1.2 channels containing mutations that promote slow inactivation both support a mechanism involving dilation of the outer selectivity filter, mutations in the outer pores of these two Kv channels have been reported to have markedly distinct effects on slow inactivation, raising questions about the extent to which slow inactivation is related in both channels. In this study, we characterized the influence of a series of mutations within the external pore of Shaker and Kv1.2 channels and observed many distinct mutant phenotypes. We find that mutations at four positions near the selectivity filter promote inactivation less dramatically in Kv1.2 when compared to Shaker, and they identify one key variable position (T449 in Shaker and V381 in Kv1.2) underlying the different phenotypes in the two channels. Collectively, our results suggest that Kv1.2 is less prone to inactivate compared to Shaker, yet support a common mechanism of inactivation in the two channels.

Peroxiredoxin 6 Plays Essential Role in Mediating Fertilization and Early Embryonic Development in Rabbit Oviduct.

The oviduct is a site for early reproductive events including gamete maturation, fertilization, and early embryo development. Secretory cells lining the oviduct lumen synthesize and secrete proteins that interact with gametes and developing embryos. Although previous studies have identified some of the secretory proteins in the oviduct, however, knowledge and their precise specific functions in the oviduct are poorly understood. In this study, by using proteomic approach, we identified a secretory protein, Peroxiredoxin 6 (PRDX6), and evaluated its role in mediating early pregnancy events, fertilization, and embryo development in rabbit oviduct. The expression of PRDX6 was significantly higher in ampulla and isthmus sections of the oviduct in mated animal groups compared to non-mated controls. Furthermore, significant reduction in number of embryos recovered from PRDX6 siRNA-transfected oviductal horn was observed compared to the control contralateral horn. Moreover, in animals receiving PRDX6 siRNA in their oviductal horn, the number of implanted blastocysts was significantly less in the uterus as observed on day 9 post-coital (p.c.). Further, during embryo-rabbit oviduct epithelial cell (ROEC) co-culture, siRNA-mediated PRDX6 silencing attenuated the early embryonic development. Mechanistically, increased levels of ROS and expression of oxidative stress- and inflammation-related proteins were found in PRDX6 siRNA-treated ROEC cells as compared to control cells, implicating that ablation of PRDX6 in the oviduct creates a stress-induced micro-environment detrimental to early embryonic development in oviduct. Taken together, our data suggest that PRDX6 maintains an optimal micro-environment conducive to successful embryo development and can be considered as a candidate to evaluate its therapeutic potential in IVF strategies.

Use of drugs not listed in the National List of Essential Medicines: Findings from a prescription analysis by the Indian Council of Medical Research-Rational Use of Medicines Centres Network in tertiary care hospitals across India.

BACKGROUND: The concept of listing essential medicines can lead to improved supply and access, more rational prescribing, and lower costs of drugs. However, these benefits hinge on the prescription of drugs from an Essential Medicines List (EML). Several studies have highlighted the problem of underutilization of EMLs by prescribers. Therefore, as part of prescription research by the Indian Council of Medical Research-Rational Use of Medicines Centres Network, we evaluated the extent of prescription of drugs not listed in the National List of Essential Medicines (NLEM). MATERIALS AND METHODS: Prescriptions of outpatients from participating centers were included after obtaining verbal/written informed consent as approved by the Ethics Committee, and evaluated for prescription of drugs from the NLEM 2015. RESULTS: Analysis of 4838 prescriptions from 13 tertiary health-care institutes revealed that 2677 (55.33%) prescriptions had at least one non-NLEM drug prescribed. In all, 5215 (31.12%) of the total 16,758 drugs prescribed were not in NLEM. Of these, 2722 (16.24%) were single drugs and 2493 (14.88%) were fixed-dose combinations (FDCs). These comprised 700 different drug products - 346 single drugs and 354 FDCs. The average number of non-NLEM drugs prescribed per prescription was 1.08, while the average number of all drugs prescribed was 3.35 per prescription. It was also found that some of the non-NLEM drugs prescribed had the potential to result in increased cost (for example, levocetirizine), increased adverse effects (dextromethorphan), and less effectiveness (losartan) when compared to their NLEM counterparts. Nonavailability of an essential drug (oral hydroxocobalamin) was another important finding of our study. CONCLUSION: This study highlights the extent and pattern of drugs prescribed from outside the NLEM at the tertiary health-care level and the need for training and enhanced awareness among prescribers for greater utilization of the NLEM.

Rho signaling inhibition mitigates lung injury via targeting neutrophil recruitment and selectin-AKT signaling.

Neutrophils, the early responders of the immune system, eliminate intruders, but their over-activation can also instigate tissue damage leading to various autoimmune and inflammatory disease conditions. As approaches causing neutropenia are associated with immunodeficiency, targeting aberrant neutrophil infiltration offers an attractive strategy in neutrophil-centered diseases including acute lung injury. Rho GTPase family proteins Rho, Rac and Cdc42 play important role as regulators of chemotaxis in diverse systems. Rho inhibitors protected against lung injuries, while genetic Rho-deficiency exhibited neutrophil hyperactivity and exacerbated lung injury. These differential outcomes might be due to distinct effects on different cell types or activation/ inhibition of specific signaling pathways responsible for neutrophil polarity, migration and functions. In this study, we explored neutrophil centric effects of Rho signaling mitigation. Consistent with previous reports, Rho signaling inhibitor Y-27632 provided protection against acute lung injury, but without regulating LPS mediated systemic increase of neutrophils in the circulation. Interestingly, the adoptive transfer approach identified a specific defect in neutrophil migration capacity after Rho signaling mitigation. These defects were associated with loss of polarity and altered actin dynamics identified using time-lapse in vitro studies. Further analysis revealed a rescue of stimulation-dependent L-selectin shedding on neutrophils with Rho signaling inhibitor. Surprisingly, functional blocking of L-selectin (CD62L) led to defective recruitment of neutrophils into inflamed lungs. Further, single-cell level analyses identified MAPK signaling as downstream mechanism of Rho signaling and L-selectin mediated effects. p-AKT levels were diminished in detergent resistance membrane-associated signalosome upon Rho signaling inhibition and blockade of selectin. Moreover, inhibition of AKT signaling as well as selectin blocking led to defects in neutrophil polarity. Together, this study identified Rho-dependent distinct L-selectin and AKT signaling mediated regulation of neutrophil recruitment to inflamed lung tissue.

Simultaneous Use of Two Different Tools to Assess Compliance with Antiepileptic Drugs: Experience in A Community-Based Study.

Objective Antiepileptic drug (AED) therapy remains the primary form of treatment for epilepsy, noncompliance to which can result in breakthrough seizure, emergency department visits, fractures, head injuries, and increased mortality. Various tools like self-report measures, pill-counts, medication refills, and frequency of seizures can assess compliance with varying extent. Thus, assessment of compliance with AEDs is crucial to be studied. Materials and Methods Compliance was assessed using pill-count and Morisky medication adherence scale (MMAS) during home visits. A pill-count (pills dispensed-pills remaining)/(pills to be consumed between two visits) value of 0.85 to ≤1.15 was recorded as appropriate compliance. Underdose (<0.85) and overdose (>1.15) was labeled as noncompliance. Score of 1 was given to each positive answer in MMAS. Score of ≥1 was labeled as noncompliance. Statistical analysis: Relationship of demographic factors between compliant and noncompliant patients was analyzed using Chi-square test (SPSS version 21.0, IBM). Rest of the data was analyzed with the help of descriptive statistics using Microsoft Excel. p < 0.05 was considered statistically significant. Results Out of 105 patients, 54 patients were noncompliant with both pill-count and MMAS. 10 patients were noncompliant with pill-count only, while 10 were noncompliant with MMAS. Conclusion Both tools complement each other when used in combination, as use of a single tool was not able to completely detect compliance.

Exploring structural dynamics of a membrane protein by combining bioorthogonal chemistry and cysteine mutagenesis.

The functional mechanisms of membrane proteins are extensively investigated with cysteine mutagenesis. To complement cysteine-based approaches, we engineered a membrane protein with thiol-independent crosslinkable groups using azidohomoalanine (AHA), a non-canonical methionine analogue containing an azide group that can selectively react with cycloalkynes through a strain-promoted azide-alkyne cycloaddition (SPAAC) reaction. We demonstrate that AHA can be readily incorporated into the Shaker Kv channel in place of methionine residues and modified with azide-reactive alkyne probes in Xenopus oocytes. Using voltage-clamp fluorometry, we show that AHA incorporation permits site-specific fluorescent labeling to track voltage-dependent conformational changes similar to cysteine-based methods. By combining AHA incorporation and cysteine mutagenesis in an orthogonal manner, we were able to site-specifically label the Shaker Kv channel with two different fluorophores simultaneously. Our results identify a facile and straightforward approach for chemical modification of membrane proteins with bioorthogonal chemistry to explore their structure-function relationships in live cells.

Naringenin ameliorates progression of endometriosis by modulating Nrf2/Keap1/HO1 axis and inducing apoptosis in rats.

Endometriosis is mainly characterized by the presence of endometrial tissue exterior to the uterus, however, the exact pathophysiology of this disease still remains uncertain. Moreover, the incidence significantly contributes to infertility among women and hence, a novel treatment for endometriosis is widely investigated. Naringenin is a plant-derived flavonoid having anti-proliferative, anti-inflammatory, and anti-angiogenic properties in chronic and metabolic diseases. The current study was planned with an objective to demonstrate the anti-endometriotic therapeutic potential of naringenin in rats and to examine its impact on various cellular aspects with a view to define the mechanism involved. The endometrial lesion volumes, weight, serum TNF-α level and the histopathologic scores were significantly reduced in the naringenin- treated group as compared to the endometriotic control group. Naringenin ameliorated the expression of prognostic markers (TAK1, PAK1, VEGF and PCNA) involved in development and progression of endometriotic cells. Naringenin caused dose-dependent loss of mitochondrial membrane potential, induced apoptosis and inhibited proliferation in these cells. Further, a significant increase in level of Nrf2 and its downstream molecules (NQO1, HO-1) was found in endometriotic lesion, with a subsequent decrease in its repressor molecule Keap-1. Naringenin significantly modulated the expression of Nrf2 and its effector molecules downstream. It also inhibited the invasion of endometrial cells by reducing the expression of MMP-2 and MMP-9 in in-vitro primary culture. We conclude that naringenin may have a therapeutic potential in the treatment of endometriosis via induction of ROS-mediated apoptosis and its anti-invasive effects.

Sortase-click strategy for defined protein conjugation on a heptavalent cyclodextrin scaffold.

Multivalent proteins or protein dendrimers are useful for clinical and biotechnological applications. However, assembly of chemically defined protein dendrimers is a challenging endeavor. In the past, majority of protein dendrimers have been developed on branched lysine scaffolds and are usually limited to a valency of two to four. The naturally occurring cyclodextrin (CD) scaffold composed of 6-8 glucose units offers the possibility of expanding the valency. Here we have adapted a chemoenzymatic-click strategy for displaying heptavalent peptides and large proteins on the ß-cyclodextrin (ß-CD) scaffold. We demonstrate that recombinant proteins (engineered with a LPXTG pentapeptide motif at the carboxy terminus), labeled with an alkyne moiety by sortase-mediated ligation, can be easily clicked on to the azide-derivatized ß-cyclodextrin through the Huisgen cycloaddition reaction yielding a well-defined heptavalent display of proteins.

MicroRNA-145 targets Smad1 in endometrial stromal cells and regulates decidualization in rat.

Decidualization of endometrial stromal cells is the pre-requisite for the embryo implantation and establishment of pregnancy. Although known to be regulated by several factors, the process of regulation of decidualization by miRNAs is largely unknown. Previous reports suggest that the upregulated expression of miR-145 is associated with repeated implantation failure. The current study was aimed to identify and validate the role of miR-145 in regulating stromal cell decidualization and the mechanism involved therein. Expression of miR-145 was found to be downregulated during the decidualization period of early pregnancy and also in artificially induced decidualization in rat uterus. During in vitro decidualization in rat endometrial stromal cells (ESCs), the overexpression of mimic miR-145 attenuated the progression of decidualization. Biochemical marker alkaline phosphatase and protein markers (insulin-like growth factor binding protein, cyclin D3) were also suppressed in miR-145 mimic-transfected cells as compared to normal decidualized cells. Bioinformatic analysis and luciferase reporter assay confirmed that Smad1 is the direct target of miR-145. Differentiation of ESCs was inhibited in miR-145 mimic-transfected cells which occurred via downregulating the target Smad1 along with its downstream p-Smad1/5/8 and Wnt-4. Pre-treatment of ESCs with Smad1 siRNA resulted in downregulated expression of p-Smad1/5/8, Wnt-4, Cox-2, and VEGF. In addition, miR-145 overexpression resulted in the loss of angiogenic factors Cox-2, MMP-9, and VEGF, indicating suppression of the process of angiogenesis. Migration of human umbilical vein endothelial cells was also attenuated in the presence of conditioned media obtained from miR-145-transfected decidualizing cells. In conclusion, the study demonstrated the role of miR-145 in regulation of progression of decidualization which is mediated through inhibition of Smad1. KEY MESSAGES: MiR-145 expression is downregulated during decidualization in the rat uterus. Overexpression of miR-145 inhibited the decidualization progression. MiR-145 suppressed the migration and invasion of HUVECs. MiR-145 downregulated Smad1 which suppresses Smad1/5/8, Wnt-4, MMP-9, Cox-2, and VEGF.

Uterine TPPP3 plays important role in embryo implantation via modulation of ß-catenin.

Tubulin polymerization promoting protein 3 (TPPP3) is known to be expressed in the endometrium in a cyclic manner, and its functional role in the physiology of implantation remains unknown. Here we demonstrate a novel function of TPPP3 during the window of implantation and in the establishment of pregnancy using a mouse model. The increased protein expression of TPPP3 and ß-catenin during peri-implantation period, i.e. D5 (receptive phase, 0800 h), was observed as compared to that on D1 (nonreceptive phase, 0800 h). SiRNATPPP3-mediated knockdown of uterine TPPP3 resulted in implantation failure and inhibited the expression of receptivity markers: LIF, Integrin-ß3, IHH, and Wnt4. TPPP3 silencing in mouse endometrial epithelial cells also prevented blastocyst attachment and the adhesion reaction. In delayed implantation experiment, expression of TPPP3 was increased in active implantation group (E2 + P4) compared to delayed implantation group (P4). The increased expression of TPPP3 in E2 + P4-treated Ishikawa cells compared to vehicle or P4 or E2 alone-treated Ishikawa cells also revealed its upregulation by E2. The suppression of ß-catenin in uterus under the condition of transient knockdown of TPPP3 and the co-immunoprecipitation experiment revealed that regulation of ß-catenin was mediated via TPPP3 during implantation. Additionally, in order to gain insight into TPPP3 collaborators, we identified TPPP3 interacting proteins by nanoLC-MS analysis in mouse uterus which might be involved during implantation. In conclusion, our study suggests that TPPP3 is important for embryo implantation and for the establishment of early pregnancy through modulation of ß-catenin.

Selective estrogen receptor modulator ormeloxifene suppresses embryo implantation via inducing miR-140 and targeting insulin-like growth factor 1 receptor in rat uterus.

Ormeloxifene, the non-steroidal SERM contraceptive, inhibits endometrial receptivity and embryo implantation via countering nidatory estrogen. However, the molecular mechanism of ormeloxifene action responsible for its contraceptive efficacy still remains unclear. Herein, we aimed to identify the miRNAs modulated under the influence of ormeloxifene and to explore their role in endometrial receptivity and embryo implantation. By doing microRNA sequencing analysis, a total of 168 miRNAs were found to be differentially expressed in uterine tissue of ormeloxifene-treated rats, on day 5 (10:00 h) of pregnancy i.e. peri-implantation period. Out of differentially expressed miRNAs, miR-140 expression was found to be elevated in ormeloxifene administered groups and was selected for detailed investigation. In-vivo gain-of-function of miR-140 resulted in a significant reduction of implantation sites indicating its role in embryo implantation. The experiment on delayed implantation showed that estradiol caused down-regulation of miR-140. It also suppressed the attachment and outgrowth of BeWo spheroids to RL95-2 endometrial cells. In transwell migration assay, miR-140 was found to be involved in suppression of migration and invasion of endometrial epithelial cells. The ormeloxifene treatment caused up-regulation of miR-140 along with down-regulated expression of its target IGF1R in endometrial epithelial and stromal cells which also led to the suppression of downstream effectors integrin ß3 and FAK. In mimic miR-140 receiving horn, the reduced expression of IGF1R was observed along with suppressed downstream integrin ß3 and FAK similar to that observed in uteri of ormeloxifene- treated rats. Taken together, these findings suggest that ormeloxifene-induced inhibition of embryo implantation occurs via inducing miR-140 and altering its target IGF1R in rat uterus.

Sorcin is involved during embryo implantation via activating VEGF/PI3K/Akt pathway in mice.

Our earlier studies have demonstrated the cyclic variation and also the altered expression of sorcin in endometrium during early-to-mid-secretory phase transition in women with unexplained infertility. The current study was undertaken to establish the functional role of sorcin in endometrial receptivity in mice. Results indicated that sorcin was highly expressed during the window of implantation in mice and functional blockage of sorcin caused significant reduction in number of implanted blastocyst. The receptivity markers (i.e.Integrin ß3, HBEGF, IGFBP1, WNT4 and Cyclin E)) were found to be downregulated in sorcin knocked down uterine horn on day 5 as compared to untreated horn. The reduced attachment and expansion of BeWo spheroids on RL95-2 endometrial cells with sorcin knock down, in in vitro model of endometrium-trophoblast interaction further supported these findings. Uterine sorcin expression pattern during estrous cycle and in delayed implantation mice model suggested the upregulation of sorcin by estrogen. The functional blockade of sorcin induced the intracellular Ca+2 levels in endometrial epithelial cells (EECs), which indicated that altered Ca+2 homeostasis might be responsible for implantation failure. Sorcin silencing led to significant reduction in the expression of angiogenic factor VEGF and its downstream effector molecules i.e. PI3K, Akt and NOS. The migratory and invasive properties of HUVECs were abrogated by anti-VEGF or by adding culture media from sorcin blocked EECs, which indicated that sorcin might mediate angiogenesis during implantation. Taken together, sorcin is involved in the regulation of Ca+2-mediated angiogenesis via VEGF/PI3K/Akt pathway in endometrial cells and plays a crucial role in preparing the endometrium for implantation.