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.

Low serum creatinine, a surrogate marker of muscle mass, correlates with insulin sensitivity in nonhuman primates.

OBJECTIVES: Decreased serum creatinine levels are associated with increased risk of type 2 diabetes (T2DM) in humans, however, its association with muscle mass and insulin sensitivity have not been studied in NHPs. METHODS: Retrospective data of 229 adult NHPs were studied for association of serum creatinine levels with muscle mass and onset of T2DM. RESULTS: Serum creatinine levels were positively correlated with lean muscle mass in nondiabetic (non-DM), male and female NHPs. Aged NHPs had significantly reduced lean muscle mass and corresponding creatinine levels compared to young age groups (p < .001). Creatinine was positively correlated with insulin sensitivity in nonDM male NHPs and significant decrease in creatinine was observed in T2DM (p < .001) compared to same age group nonDM NHPs. CONCLUSIONS: The pathophysiology of T2DM in NHPs is similar to humans, low creatinine further provides utility of surrogate biomarkers of lower muscle mass and risk factor for T2DM NHPs.

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.

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.

Iron deficiency, but not anemia, is identified in naturally occurring obesity and insulin resistance in male nonhuman primates.

OBJECTIVES: To understand the development of iron deficiency in obesity and its long-term impact on the profile of anemia in spontaneously obese nonhuman primates. METHODS: The study included 69 adult male nonhuman primates, (NHPs, Macaca mulatta, rhesus monkeys), ranging from normal to obese, and type 2 diabetes (T2D) as defined for humans. RESULTS: Iron deficiency was present in 31.9% and mild anemia in 13% of the rhesus monkey in the colony. Serum iron levels were significantly lower in obese (p < .01) and T2D (p < .01)) compared with normal NHP. Obese NHPs also had significantly higher hemoglobin (p < .05), and red blood cell count (p < .05) than normal weight NHPs, thus not related to anemia. CONCLUSIONS: Iron deficiency with increased hemoglobin and red blood cells was significantly associated with increased adiposity, insulin resistance, and diabetes. Iron deficiency does not cause and is not related to anemia in obese and T2D NHPs.

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.

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.

Mouse model for endometriosis is characterized by proliferation and inflammation but not epithelial-to-mesenchymal transition and fibrosis.

Endometriosis is a common disorder of unknown etiology, and non-surgical therapies are still a challenge. To understand the pathogenesis and preclinical testing of drugs for endometriosis, animal models are highly desirous. Herein, we carried out longitudinal characterization of a mouse model for endometriosis where uterine tissue was transplanted onto the intestinal mesentery. During the course of lesion development from day 15 to 60 post-induction, the ectopic endometrium became pale, fluid-filled and the animals developed peritoneal adhesions. Most lesions resembled a well-differentiated type of endometriosis and ~13% of animals had mixed type of lesions. There was extensive stromal compaction in the ectopic tissue. During the progression of endometriosis, there was increased proliferation of epithelial and stromal cells as evident by PCNA staining. Cyp19a1 (aromatase) mRNA was detected in the ectopic lesions on day 15 and 30 post-induction of endometriosis, by day 60 the expression was reduced. As compared to the control endometrium, the mRNA levels of Esr1 progressively reduced while the levels of inflammation associated genes (Esr2, Ifng, Tnf and Il1b) increased in the ectopic lesions. Infiltration of macrophages and polymorphonuclear leucocytes was also observed in the ectopic lesions indicative of inflammation. As compared to control, there was no change in levels of Cytokeratin and E-cadherin in the epithelial cells of ectopic endometrium. We did not observe excessive collagen deposition or α -SMA positive myofibroblasts in the stroma of the ectopic endometrium. Thus, epithelial-to-mesenchymal transition and fibrosis are not detected in the mouse model of endometriosis. Our results show that the mouse model of endometriosis mimics some but not all the features of human endometriosis.

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.

Altered expression of p63 isoforms and expansion of p63- and club cell secretory protein-positive epithelial cells in the lung as novel features of aging.

Aging is a key contributor for subclinical progression of late-onset lung diseases. Basal, club, and type II alveolar epithelial cells (AECs) are lung epithelial progenitors whose capacities of differentiation are extensively studied. The timely transition of these cells in response to environmental changes helps maintain the intricate organization of lung structure. However, it remains unclear how aging affects their behavior. This paper demonstrates that the protein expression profiles of a type II AEC marker, prosurfactant protein C (pro-SPC), and a basal cell marker, p63, are altered in the lungs of 14-mo-old versus 7- to 9-wk-old mice. Expression of NH2-terminal-truncated forms of p63 (ΔNp63), a basal cell marker, and claudin-10, a club cell marker, in cytoplasmic extracts of lungs of 14-mo-old mice was upregulated. In contrast, nuclear expression of full-length forms of p63 (TAp63) decreases with age. These alterations in protein expression profiles coincide with dramatic changes in lung functions including compliance. Whole tissue lysates of middle-aged versus aged rhesus monkey lungs display similar age-associated alterations in pro-SPC expression. An age-associated decrease of TAp63 in nuclear lysates was observed in aged monkey group. Moreover, the lungs of 14-mo-old versus 7- to 9-wk-old mice display a wider spreading of ΔNp63-positive CCSP-positive bronchiolar epithelial cells. This expansion did not involve upregulation of Ki67, a representative proliferation marker. Collectively, it is postulated that 1) this expansion is secondary to a transition of progenitor cells committed to club cells from ΔNp63-negative to ΔNp63-positive status, and 2) high levels of cytoplasmic ΔNp63 expression trigger club cell migration.

Use of ultrasound imaging for the diagnosis of abnormal uterine bleeding in the bonnet macaque ( Macaca radiata).

Ultrasound is a powerful, low-cost, non-invasive medical tool used by laboratory animal veterinarians for diagnostic imaging. Sonohysterography and transvaginal ultrasound are frequently used to assess uterine anomalies in women presenting with abnormal uterine bleeding (AUB). In the present study, we have evaluated the abdominal ultrasound of bonnet monkeys ( n = 8) showing spontaneous ovulatory ( n = 5) and anovulatory ( n = 3) AUB. The ovulatory ( n = 5) macaques showed cyclic AUB for 7-8 days. The anovulatory ( n = 3) macaques had irregular AUB with menstrual cycles of 40-45 days. The B-mode abdominal, colour Doppler and 3D ultrasound scans were performed during the proliferative phase of the menstrual cycle. Ultrasound examination revealed endometrial polyps in five macaques and endometrial hyperplasia in three animals. The width and length of endometrial polyps was around 0.5-1 cm (average 0.51 ± 0.23 cm × 0.96 ± 0.16 cm) with significant increase in endometrial thickness ( P < 0.0002). 3D ultrasound also showed a homogeneous mass in the uterine cavity and colour Doppler ultrasound showed increased vascularity in the endometrial polyps. Endometrial hyperplasia characteristically appeared as a thickened echogenic endometrium ( P < 0.0002). This study demonstrates the use of non-invasive ultrasound techniques in the diagnosis of AUB in macaques.

A triad of telomerase, androgen receptor and early growth response 1 in prostate cancer cells.

Telomerase activation is one of the key mechanisms that allow cells to bypass replicative senescence. Telomerase activity is primarily regulated at the level of transcription of its catalytic unit- hTERT. Prostate cancer (PCa), akin to other cancers, is characterized by high telomerase activity. Existing data suggest that hTERT expression and telomerase activity are positively regulated by androgenic stimuli in androgen-dependent prostate cancer (ADPC) cells. A part of the present study reaffirmed this by demonstrating a decline in the hTERT expression and telomerase activity on "loss of AR" in ADPC cells. The study further addressed 2 unresolved queries, i) whether AR-mediated signaling is of any relevance to hTERT expression in castration-resistant prostate cancer (CRPC) and ii) whether this signaling involves EGR1. Our data suggest that AR-mediated signaling negatively regulates hTERT expression in CRPC cells. Incidental support for the possibility of EGR1 being a regulator of hTERT expression in PCa was provided by i) immunolocalization of hTERT and EGR1 proteins in the same cell type (secretory epithelium) of PCa and BPH tissues; ii) significantly (p< 0.001) higher levels of both these proteins in CRPC (PC3 and DU145), compared with ADPC (LNCaP) cells. A direct evidence for the role of EGR1 in hTERT expression was evident by a significant (p<0.0001) decrease in the hTERT transcript levels in the EGR1-silenced CRPC cells. Further, "gain of AR" led to a significant reduction in the levels of hTERT and EGR1 in CRPC cells. However, restoration of EGR1 levels prevented the decline in the hTERT transcript levels in these cells. Taken together, our data indicate that AR regulates the expression of EGR1, which in turn acts as a positive regulator of hTERT expression in CRPC cells. Thus, AR exerts an inhibitory effect on hTERT expression and telomerase activity by modulating EGR1 levels in CRPC cells.

Secrets of Endometrial Receptivity: Some Are Hidden in Uterine Secretome.

PROBLEM: Endometrium, the innermost mucosal layer of the uterus, serves as a lodge for the embryo in eutherian mammals. The endometrium is constituted of various cell types, and each cell type executes specific functions to facilitate embryo implantation and development. It is well established that the endometrium, despite being non-permissive to the embryo for the major period of a menstrual cycle, is irreplaceable in the scheme of events essential for procreation. However, the embryo, before initiating physical contact with the endometrium, encounters the uterine cavity that remains bathed in uterine fluid. Uterine fluid is an admixture of endometrial secretions, plasma transudates, and oviductal fluid. Uterine fluid components are believed to play important roles in immunosuppression and embryo development during peri-implantation period. Uterine fluid is also involved in defense against pathogens, sperm migration, and lubrication of endometrium. The advent of high-throughput functional genomics tools has created enormous opportunities to investigate the uterine fluid for its protein repertoire and modulation during the receptive phase of an endometrial cycle in animals and humans. Towards this, few investigations have been conducted in recent years. The data obtained using non-targetted functional genomics approaches need to be assimilated with the existing information on specific components of uterine fluid. METHOD: This review compiles existing information on the composition of uterine fluid and its significance in endometrial functions and dysfunctions. RESULT: Collectively, investigations based on targetted and non-targetted approaches have revealed the presence of several cytokines, growth factors, ions, carbohydrates, and steroids, in human uterine fluid. CONCLUSION: Detailed investigations of human uterine fluid, especially directed towards the elucidation of functional relevance of different proteins in uterine fluid, will help identify novel markers of endometrial receptivity and also gain significant insights into the mechanisms underlying unexplained infertility, recurrent pregnancy losses, and other endometrial pathologies.

Proliferation and decidualization of endometrial stromal cells during embryo-attachment stage in bonnet monkeys (Macaca radiata).

We report embryo-induced alterations occurring in endometrial stromal cells (ESCs) during the embryo-attachment stage in bonnet monkeys (Macaca radiata). Laser micro-dissected ESCs obtained from pregnant and non-pregnant animals were compared for levels of selected proliferation and decidualization-associated factors by analysis with quantitative real-time polymerase chain reaction or immunohistochemistry. Stromal cells exhibited extensive cellular proliferation, as indicated by cellular compaction and significantly higher (P < 0.05) levels of proliferating cell nuclear antigen and of estrogen receptor 1, c-Myc, and Cyclin D1 transcripts in pregnant animals as compared with non-pregnant animals. A significant decrease (P < 0.05) was observed in the transcript levels of stromal interleukin-6 (IL-6) in pregnant animals. Cell proliferation was accompanied by a significant increase (P < 0.001) in the levels of decidualization-associated molecules such as IL-1ß in the luminal and glandular epithelium and of stromal insulin-like growth-factor-binding protein-1 (IGFBP-1) and prostaglandin-endoperoxide synthase-2 (PTGS-2) proteins. In pregnant animals, proliferation was evident throughout the gestational stroma, whereas decidualization was more pronounced in the embryo-attachment zone than in the non-attachment zone. To our knowledge, this is the first report of alterations in the endometrial stroma during the embryo-attachment stage in a non-human primate model.

Echography of the cervix and uterus during the proliferative and secretory phases of the menstrual cycle in bonnet monkeys (Macaca radiata).

We undertook the present study to investigate the echographic characteristics of the uterus and cervix of female bonnet monkeys ( Macaca radiata ) during the proliferative and secretory phases of the menstrual cycle. The cervix was tortuous in shape and measured 2.74 ± 0.30 cm (mean ± SD) in width by 3.10 ± 0.32 cm in length. The cervical lumen contained 2 or 3 colliculi, which projected from the cervical canal. The echogenicity of cervix varied during proliferative and secretory phases. The uterus was pyriform in shape (2.46 ± 0.28 cm × 1.45 ± 0.19 cm) and consisted of serosa, myometrium, and endometrium. The endometrium generated a triple-line pattern; the outer and central lines were hyperechogenic, whereas the inner line was hypoechogenic. The endometrium was significantly thicker during the secretory phase (0.69 ± 0.12 cm) than during the proliferative phase (0.43 ± 0.15 cm). Knowledge of the echogenic changes in the female reproductive organs of bonnet monkeys during a regular menstrual cycle may facilitate understanding of other physiologic and pathophysiologic changes.

Cell surfactomes of two endometrial epithelial cell lines that differ in their adhesiveness to embryonic cells.

Adhesiveness of the endometrial epithelium to an embryo plays a critical role in the initiation of pregnancy. Loss or gain of adhesiveness also dictates the potential of endometrial epithelial cells to metastasize, an event that can result from certain genetic insults. A proteomics-based exploration of the "adhesiveness" these epithelial cells was employed that could identify targets that could disrupt embryo-endometrium interactions and/or metastasis of endometrial cancer cells. The present study defined the surfactomes of two human endometrial epithelial cell lines known for their differential adhesiveness to embryonic cells. Comparative, two-dimensional electrophoretic analysis of the surfactomes of RL95-2 (exhibiting higher adhesiveness to the embryonic cell line JAr) and HEC-1A (exhibiting reduced adhesiveness to JAr cells) revealed 55 differentially enriched proteins. Of these, 10 proteins were identified by MALDI-TOF/TOF or LC-MS/MS. TUBB2C, ADAMTS3, and elongation factor beta were more abundant on the HEC-1A cell surface whereas HSP27, HSPA9, GP96, CRT, Tapasin-ERP57, PDI, and ß-actin were more abundant on the RL95-2 cell surface. Nano LC-MS/MS was also employed to generate a more comprehensive surfactomes of RL95-2 and HEC-1A. The study also demonstrated a pro-adhesive role of CRT and HSPA9 and an anti-adhesive role of TUBB2C populations found on the cell surface. In brief, this study identifies the cell-surface protein complements of two human endometrial epithelial cell lines, and reveals the role of three proteins in heterotypic cell adhesion.

Endometrial epithelial cell modifications in response to embryonic signals in bonnet monkeys (Macaca radiata).

The present investigation reports embryo-induced modifications in the epithelial cells of the endometrium in a primate species. In vivo, epithelial cell response to the embryonic signals was assessed at the embryo attachment stage in the gestational uterus of bonnet monkeys (Macaca radiata) and in vitro response was investigated by treating human endometrial epithelial cell line (Ishikawa) with human embryo conditioned media (CM). Endometrial epithelial (EE) cells at the embryo attachment stage in bonnet monkeys revealed higher proliferation accompanied by significant up regulation (p < 0.05) in the expression of estrogen receptor (ER)α and down regulation (p < 0.05) in ERß expression. Further gestational EE cells showed higher (p < 0.001) expression of mucin-1, except in the embryo attachment site. Also, observed were significantly higher expression (p < 0.05) and altered cytoplasmic distribution of α(v) and ß(3) integrins, when compared to non-pregnant animals. In pregnant animals, the embryo attachment zone showed differential expression of immunoreactive integrins as compared to the non-attachment zone. This suggested the role of embryo secreted factors in modulation of the epithelial cell profile. In vitro studies partially supported this assumption. Significantly higher proliferation (p < 0.05), as well as increased expression of ERα, integrin ß(3) and mucin-1 (p < 0.05) were observed in Ishikawa cells, on stimulation with CM. Taken together, these results indicated the proliferation and modulation in the expression of estrogen receptors and cell adhesion molecules in the EE cells; at the embryo attachment stage in bonnet monkeys. Further it is likely that embryo secreted factors contribute to some of these modifications in EE cells. This report is the first account of discrete cellular events, which occur in the uterine epithelium, at the embryo attachment stage in a primate species.

A simple method for isolation of genomic DNA from fresh and dry leaves of Terminalia arjuna (Roxb.) Wight and Arnot

Current protocols for isolation of genomic DNA from Terminalia arjuna have their own limitations due to the presence of high content of gummy polysaccharides and polyphenols. DNA isolated by these protocols is contaminated with a yellowish, sticky and viscous matrix. In our modified DNA isolation method polysaccharides and polyphenols are removed prior to the precipitation of the DNA. Then the genomic DNA was precipitated using isopropanol. This protocol yielded a high molecular weight DNA isolated from fresh as well as dry leaves of T. arjuna, which was free from contamination and colour. Isolated DNA can be used for restriction digestion and PCR amplification. The main objective of the present protocol is to provide a simple method of isolation of DNA, using in house prepared reagents.