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1.  A prostaglandin E2 receptor antagonist prevents pregnancies during a preclinical contraceptive trial with female macaques 
Human Reproduction (Oxford, England)  2014;29(7):1400-1412.
Can administration of a prostaglandin (PG) E2 receptor 2 (PTGER2) antagonist prevent pregnancy in adult female monkeys by blocking periovulatory events in the follicle without altering menstrual cyclicity or general health?
This is the first study to demonstrate that a PTGER2 antagonist can serve as an effective non-hormonal contraceptive in primates.
The requirement for PGE2 in ovulation and the release of an oocyte surrounded by expanded cumulus cells (cumulus–oocyte expansion; C-OE) was established through the generation of PTGS2 and PTGER2 null-mutant mice. A critical role for PGE2 in primate ovulation is supported by evidence that intrafollicular injection of indomethacin in rhesus monkeys suppressed follicle rupture, whereas co-injection of PGE2 with indomethacin resulted in ovulation.
First, controlled ovulation protocols were performed in adult, female rhesus monkeys to analyze the mRNA levels for genes encoding PGE2 synthesis and signaling components in the naturally selected pre-ovulatory follicle at different times after the ovulatory hCG stimulus (0, 12, 24, 36 h pre-ovulation; 36 h post-ovulation, n = 3–4/time point). Second, controlled ovarian stimulation cycles were utilized to obtain multiple cumulus–oocyte complexes (COCs) from rhesus monkeys to evaluate the role of PGE2 in C-OE in vitro (n = 3–4 animals/treatment; ≥3 COCs/animal/treatment). Third, adult cycling female cynomolgus macaques were randomly assigned (n = 10/group) to vehicle (control) or PTGER2 antagonist (BAY06) groups to perform a contraceptive trial. After the first treatment cycle, a male of proven fertility was introduced into each group and they remained housed together for the duration of the 5-month contraceptive trial that was followed by a post-treatment reversibility trial.
Quantitative real-time PCR, COC culture and expansion, immunofluorescence/confocal microscopy, enzyme immunoassay, contraceptive trial, ultrasonography, complete blood counts, serum biochemistry tests and blood lipid profiles.
Several mRNAs encoding proteins involved in PGE2 synthesis, metabolism and signaling increase (P < 0.05) in the periovulatory follicle after administration of an ovulatory hCG bolus. PGE2 signaling through PTGER2 induces cumulus cell expansion and production of hyaluronic acid, which are critical events for fertilization. Moreover, chronic administration of a selective PTGER2 antagonist resulted in a significant (P < 0.05 versus vehicle-treated controls) contraceptive effect without altering steroid hormone patterns or menstrual cyclicity during a 5-months contraceptive trial. Fertility recovered as early as 1 month after ending treatment.
This is a proof-of-concept study in a non-human primate model. Further investigations are warranted to elucidate the mechanism(s) of PTGER2 antagonist action in the primate ovary. Although PTGER2 antagonist treatment did not produce any obvious undesirable effects, improvements in the mode of administration, as well as the efficacy of these compounds, are necessary to consider such a contraceptive for women.
Monitoring as well as improving the efficacy and safety of female contraceptives is an important public health activity. Even though hormonal contraceptives are effective for women, concerns remain regarding their side-effects and long-term use because of the widespread actions of such steroidal products in many tissues. Moreover, some women cannot take hormones for medical reasons. Thus, development of non-hormonal contraceptives for women is warranted.
Supported by Bayer HealthCare Pharmaceuticals, The Eunice Kennedy Shriver NICHD Contraceptive Development and Research Center (U54 HD055744), NIH Office of the Director (Oregon National Primate Research Center P51 OD011092), and a Lalor Foundation Postdoctoral Basic Research Fellowship (MCP). The use of the Leica confocal was supported by grant number S10RR024585. Some of the authors (N.B., A.R., K.-H.F., U.F., B.B. and B.L.) are employees of Bayer Healthcare Pharma.
PMCID: PMC4059334  PMID: 24781425
prostaglandin E2 receptor; cumulus oocyte expansion; contraceptive trial; primates
2.  The prostaglandin E2 EP1 receptor mediates pain perception and regulates blood pressure 
Journal of Clinical Investigation  2001;107(3):325-331.
The lipid mediator prostaglandin E2 (PGE2) has diverse biological activity in a variety of tissues. Four different receptor subtypes (EP1–4) mediate these wide-ranging effects. The EP-receptor subtypes differ in tissue distribution, ligand-binding affinity, and coupling to intracellular signaling pathways. To identify the physiological roles for one of these receptors, the EP1 receptor, we generated EP1-deficient (EP1–/–) mice using homologous recombination in embryonic stem cells derived from the DBA/1lacJ strain of mice. The EP1–/– mice are healthy and fertile, without any overt physical defects. However, their pain-sensitivity responses, tested in two acute prostaglandin-dependent models, were reduced by approximately 50%. This reduction in the perception of pain was virtually identical to that achieved through pharmacological inhibition of prostaglandin synthesis in wild-type mice using a cyclooxygenase inhibitor. In addition, systolic blood pressure is significantly reduced in EP1 receptor–deficient mice and accompanied by increased renin-angiotensin activity, especially in males, suggesting a role for this receptor in cardiovascular homeostasis. Thus, the EP1 receptor for PGE2 plays a direct role in mediating algesia and in regulation of blood pressure.
PMCID: PMC199184  PMID: 11160156
3.  Enhanced pressor response to acute angiotensin II infusion in mice lacking membrane-associated prostaglandin E2 synthase-1 
Acta pharmacologica Sinica  2010;31(10):1284-1292.
COX-2-mPGES-1-derived prostaglandin E2 (PGE2) plays important roles in regulating vascular tone and renal sodium excretion; however, little is known about the role of mPGES-1 during acute blood pressure regulation. The present study was designed to examine the contribution of vascular mPGES-1 to acute blood pressure homeostasis.
Angiotensin II (AngII, 75 pmol/kg/min) was continuously infused via the jugular vein into wild-type and mPGES-1−/− mice for 30 min, and blood pressure was measured by carotid arterial catheterization. RT-PCR and immunohistochemistry were performed to detect the expression and localization of mPGES-1 in the mouse arterial vessels. Mesenteric arteries were dissected from mice of both genotypes to study vessel tension and measure vascular PGE2 levels.
Wild-type and mPGES-1−/− mice showed similar blood pressure levels at baseline, and the acute intravenous infusion of AngII caused a greater increase in mean arterial pressure in the mPGES-1−/− group, with a similar diuretic and natriuretic response in both groups. mPGES-1 was constitutively expressed in the aortic and mesenteric arteries and vascular smooth muscle cells of wild-type mice. Strong staining was detected in the smooth muscle layer of arterial vessels. Ex vivo treatment of mesenteric arteries with AngII produced more vasodilatory PGE2 in wild-type than in mPGES-1−/− mice. In vitro tension assays further revealed that the mesenteric arteries of mPGES-1−/− mice exhibited a greater vasopressor response to AngII than those arteries of wild-type mice.
Vascular mPGES-1 acts as an important tonic vasodilator, contributing to acute blood pressure regulation.
PMCID: PMC3895497  PMID: 20871624
PGE2; mPGES-1; Angiotensin II; Blood pressure; Resistant vessel
4.  Microsomal prostaglandin E synthase-1 in both cancer cells and hosts contributes to tumour growth, invasion and metastasis 
Biochemical Journal  2009;425(Pt 2):361-371.
mPGES-1 (microsomal prostaglandin E synthase-1) is a stimulus-inducible enzyme that functions downstream of COX (cyclo-oxygenase)-2 in the PGE2 (prostaglandin E2)-biosynthesis pathway. Although COX-2-derived PGE2 is known to play a role in the development of various tumours, the involvement of mPGES-1 in carcinogenesis has not yet been fully understood. In the present study, we used LLC (Lewis lung carcinoma) cells with mPGES-1 knockdown or overexpression, as well as mPGES-1-deficient mice to examine the roles of cancer cell- and host-associated mPGES-1 in the processes of tumorigenesis in vitro and in vivo. We found that siRNA (small interfering RNA) silencing of mPGES-1 in LLC cells decreased PGE2 synthesis markedly, accompanied by reduced cell proliferation, attenuated Matrigel™ invasiveness and increased extracellular matrix adhesion. Conversely, mPGES-1-overexpressing LLC cells showed increased proliferating and invasive capacities. When implanted subcutaneously into wild-type mice, mPGES-1-silenced cells formed smaller xenograft tumours than did control cells. Furthermore, LLC tumours grafted subcutaneously into mPGES-1-knockout mice grew more slowly than did those grafted into littermate wild-type mice, with concomitant decreases in the density of microvascular networks, the expression of pro-angiogenic vascular endothelial growth factor, and the activity of matrix metalloproteinase-2. Lung metastasis of intravenously injected LLC cells was also significantly less obvious in mPGES-1-null mice than in wild-type mice. Thus our present approaches provide unequivocal evidence for critical roles of the mPGES-1-dependent PGE2 biosynthetic pathway in both cancer cells and host microenvironments in tumour growth and metastasis.
PMCID: PMC2825730  PMID: 19845504
knockout mouse; metastasis; microsomal prostaglandin E synthase-1; prostaglandin E2; tumorigenesis; COX, cyclo-oxygenase; cPGES, cytosolic prostaglandin E synthase; DMEM, Dulbecco's modified Eagle's medium; dmPGE2, 16,16-dimethyl prostaglandin E2; ECM, extracellular matrix; EP, prostaglandin E receptor; FCS, fetal calf serum; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; HEK, human embryonic kidney; KD, knockdown; KO, knockout; LLC, Lewis lung carcinoma; MMP, matrix metalloproteinase; mPGES, microsomal prostaglandin E synthase; NSAID, non-steroidal anti-inflammatory drug; PG, prostaglandin; PGES, PGE synthase; RT, reverse transcriptase; siRNA, small interfering RNA; TBS, Tris-buffered saline; TBS-Tween, TBS containing 0.05% Tween 20; VEGF, vascular endothelial growth factor; WT, wild-type
5.  Mammalian oocytes are targets for prostaglandin E2 (PGE2) action 
The ovulatory gonadotropin surge increases synthesis of prostaglandin E2 (PGE2) by the periovulatory follicle. PGE2 actions on granulosa cells are essential for successful ovulation. The aim of the present study is to determine if PGE2 also acts directly at the oocyte to regulate periovulatory events.
Oocytes were obtained from monkeys and mice after ovarian follicular stimulation and assessed for PGE2 receptor mRNA and proteins. Oocytes were cultured with vehicle or PGE2 and assessed for cAMP generation, resumption of meiosis, and in vitro fertilization.
Germinal vesicle intact (GV) oocytes from both monkeys and mice expressed mRNA for the PGE2 receptors EP2, EP3, and EP4. EP2 and EP4 proteins were detected by confocal microscopy in oocytes of both species. Monkey and mouse oocytes responded to PGE2 as well as agonists selective for EP2 and EP4 receptors with elevated cAMP, consistent with previous identification of EP2 and EP4 as Gαs/adenylyl cyclase coupled receptors. Incubation of mouse GV stage oocytes with PGE2 delayed oocyte nuclear maturation in vitro, but PGE2 treatment did not alter the percentage of mouse oocytes that fertilized successfully. PGE2 treatment also decreased the percentage of monkey oocytes that resumed meiosis in vitro. In contrast with mouse oocytes, the percentage of monkey oocytes which fertilized in vitro was lower after treatment with PGE2. Monkey oocytes with intact cumulus showed delayed nuclear maturation, but fertilization rate was not affected by PGE2 treatment.
Monkey and mouse oocytes express functional PGE2 receptors. PGE2 acts directly at mammalian oocytes to delay nuclear maturation. Surrounding cumulus cells modulate the effect of PGE2 to alter subsequent fertilization.
PMCID: PMC2988801  PMID: 21040553
6.  Enhanced pressor response to acute Ang II infusion in mice lacking membrane-associated prostaglandin E2 synthase-1 
Acta Pharmacologica Sinica  2010;31(10):1284-1292.
To examine the contribution of vascular membrane-associated prostaglandin E2 synthase-1 (mPGES-1) to acute blood pressure homeostasis.
Angiotensin II (AngII, 75 pmol·kg−1·min−1) was continuously infused via the jugular vein into wild-type and mPGES-1−/− mice for 30 min, and blood pressure was measured by carotid arterial catheterization. RT-PCR and immunohistochemistry were performed to detect the expression and localization of mPGES-1 in the mouse arterial vessels. Mesenteric arteries were dissected from mice of both genotypes to study vessel tension and measure vascular PGE2 levels.
Wild-type and mPGES-1−/− mice showed similar blood pressure levels at baseline, and the acute intravenous infusion of AngII caused a greater increase in mean arterial pressure in the mPGES-1−/− group, with a similar diuretic and natriuretic response in both groups. mPGES-1 was constitutively expressed in the aortic and mesenteric arteries and vascular smooth muscle cells of wild-type mice. Strong staining was detected in the smooth muscle layer of arterial vessels. Ex vivo treatment of mesenteric arteries with AngII produced more vasodilatory PGE2 in wild-type than in mPGES-1−/− mice. In vitro tension assays further revealed that the mesenteric arteries of mPGES-1−/− mice exhibited a greater vasopressor response to AngII than those arteries of wild-type mice.
Vascular mPGES-1 acts as an important tonic vasodilator, contributing to acute blood pressure regulation.
PMCID: PMC3895497  PMID: 20871624
prostaglandin E2; membrane-associated prostaglandin E2 synthase-1; angiotensin II; blood pressure; resistant vessel
7.  The periovulatory period in cattle: progesterone, prostaglandins, oxytocin and ADAMTS proteases 
Ovulation has long been recognized as one of the most dramatic reproductive processes. Decades of research on how the LH/FSH surge leads to ovulation have made it clear that the surge induces a very complex cascade of changes. Studies of genetically modified mice have pointed to progesterone (P4) and its receptor (PGR) and the prostaglandins (PGs) as critical components of the ovulatory cascade. In cattle, the gonadotropin surge also induces oxytocin (OT), which does not appear to increase in rodent periovulatory follicles. This review is an attempt to summarize studies by our laboratory on the temporal patterns, roles, regulation, and interrelationships among P4/PGR, PGs, and OT in bovine periovulatory follicles. Most of these results are based on an experimental model in which the dominant follicle of the first follicular wave of the estrous cycle is induced to develop into a preovulatory follicle by injection of PGF2α on Day 6 of the cycle, followed 36 h later by an injection of GnRH to induce the LH/FSH surge. The results suggest that the effects of the gonadotropin surge on PG production by bovine granulosa cells are mediated by the gonadotropin-induced increase in intrafollicular P4 and that P4 acts by binding to its nuclear receptor and increasing the abundance of mRNA for the enzyme PTGS2 (COX-2). Our data thus far also support the hypothesis that PGs, especially PGE2, can stimulate progesterone secretion by both follicular cell types and suggest a positive feedback relationship between P4/PGR and the PGs. Additional results suggest a positive feedback loop between P4/PGR and OT. The finding that levels of mRNA for several ADAMTS proteases are regulated by the LH/FSH surge in vivo and by P4/PGR and/or PGs in vitro suggests a role for this family of proteases in remodeling the bovine ovulatory follicle in preparation for ovulation and the formation of the corpus luteum. It is important to remember that a process essential for reproduction, such as ovulation, may involve redundant mechanisms and that these mechanisms may have evolved differently from rodents in larger mammalian species, such as ruminants and humans.
PMCID: PMC2853051  PMID: 20390049
ovary; ovarian follicle; ovulation; cattle; progesterone; prostaglandins; ADAMTS proteases; oxytocin
Hypertension  2010;55(2):531-538.
Prostaglandin (PG) E2 has multiple actions that may affect blood pressure. It is synthesized from arachidonic acid by the sequential actions of phospholipases, cyclooxygenases, and PGE synthases. While microsomal PGE synthase 1 (mPGES1) is the only genetically-verified PGE synthase, results of previous studies examining the consequences of mPGES1-deficiency on blood pressure (BP) are conflicting. To determine whether genetic background modifies the impact of mPGES1 on BP, we generated mPGES1−/− mice on two distinct inbred backgrounds, DBA/1lacJ and 129/SvEv. On the DBA/1 background, baseline BP was similar between wild-type (WT) and mPGES1−/− mice. By contrast, on the 129 background, baseline BPs were significantly higher in mPGES1−/− animals than WT controls. During angiotensin II infusion, the DBA/1 mPGES1−/− and WT mice developed mild hypertension of similar magnitude, while 129-mPGES1−/− mice developed more severe hypertension than WT controls. DBA/1 animals developed only minimal albuminuria in response to angiotensin II infusion. By contrast, WT 129 mice had significantly higher levels of albumin excretion than WT DBA/1 and the extent of albuminuria was further augmented in 129 mPGES1−/− animals. In WT mice of both strains, the increase in urinary excretion of PGE2 with angiotensin II was attenuated in mPGES1−/− animals. Urinary excretion of thromboxane was unaffected by angiotensin II in the DBA/1 lines but increased more than 4-fold in 129 mPGES1−/− mice. These data indicate that genetic background significantly modifies the BP response to mPGES1 deficiency. Exaggerated production of thromboxane may contribute to the robust hypertension and albuminuria in 129 mPGES1-deficient mice.
PMCID: PMC2836731  PMID: 20065147
prostanoids; PGE synthase; blood pressure; strain; hypertension
9.  Cyclooxygenases, microsomal prostaglandin E synthase-1, and cardiovascular function 
Journal of Clinical Investigation  2006;116(5):1391-1399.
We investigated the mechanisms by which inhibitors of prostaglandin G/H synthase-2 (PGHS-2; known colloquially as COX-2) increase the incidence of myocardial infarction and stroke. These inhibitors are believed to exert both their beneficial and their adverse effects by suppression of PGHS-2–derived prostacyclin (PGI2) and PGE2. Therefore, the challenge remains to identify a mechanism whereby PGI2 and PGE2 expression can be suppressed while avoiding adverse cardiovascular events. Here, selective inhibition, knockout, or mutation of PGHS-2, or deletion of the receptor for PGHS-2–derived PGI2, was shown to accelerate thrombogenesis and elevate blood pressure in mice. These responses were attenuated by COX-1 knock down, which mimics the beneficial effects of low-dose aspirin. PGE2 biosynthesis is catalyzed by the coordinate actions of COX enzymes and microsomal PGE synthase-1 (mPGES-1). We show that deletion of mPGES-1 depressed PGE2 expression, augmented PGI2 expression, and had no effect on thromboxane biosynthesis in vivo. Most importantly, mPGES-1 deletion affected neither thrombogenesis nor blood pressure. These results suggest that inhibitors of mPGES-1 may retain their antiinflammatory efficacy by depressing PGE2, while avoiding the adverse cardiovascular consequences associated with PGHS-2–mediated PGI2 suppression.
PMCID: PMC1435722  PMID: 16614756
10.  Vascular wall–produced prostaglandin E2 exacerbates arterial thrombosis and atherothrombosis through platelet EP3 receptors 
Prostanoids, bioactive lipids derived from arachidonic acid (AA), are important for vascular homeostasis. Among them, prostaglandin E2 (PGE2) enhances aggregation of platelets submaximally stimulated in vitro. This results from activation of EP3, one of the four PGE2 receptors, which decreases the threshold at which agonists activate platelets to aggregate. Although PGE2 altered venous thrombosis induced by administration of AA, its role in pathophysiopathological conditions has remained speculative. We report that arterial walls subjected to inflammatory stimuli produce PGE2. In several models, we show that PGE2 produced by the arterial wall facilitates arterial thrombosis. Next, we detected PGE2 in mouse atherosclerotic plaques. We demonstrate that this plaque-produced PGE2 is not altered and is still able to activate EP3. In addition, we present evidence that PGE2 can leave the plaque and activate EP3 on blood platelets. Consistent with these findings, we observed that atherothrombosis induced in vivo by mechanical rupture of the plaque was drastically decreased when platelets lacked EP3. In conclusion, PGE2 facilitates the initiation of arterial thrombosis and, hence, contributes to atherothrombosis. Inhibition of the platelet EP3 receptor should improve prevention of atherothrombosis.
PMCID: PMC2118736  PMID: 17242161
11.  The effects of 1α,25-dihydroxyvitamin D3 on matrix metalloproteinase and prostaglandin E2 production by cells of the rheumatoid lesion 
Arthritis Research  1999;1(1):63-70.
The biologically active metabolite of vitamin D3, 1α,25-dihydroxyvitamin D3 [1α,25(OH)2D3], acts through vitamin D receptors, which were found in rheumatoid tissues in the present study. IL-1β-activated rheumatoid synovial fibroblasts and human articular chondrocytes were shown to respond differently to exposure to 1α,25(OH)2D3, which has different effects on the regulatory pathways of specific matrix metalloproteinases and prostaglandin E2.
1α,25-dihydroxyvitamin D3 [1α,25(OH)2D3], the biologically active metabolite of vitamin D3, acts through an intracellular vitamin D receptor (VDR) and has several immunostimulatory effects. Animal studies have shown that production of some matrix metalloproteinases (MMPs) may be upregulated in rat chondrocytes by administration of 1α,25(OH)2D3; and cell cultures have suggested that 1α,25(OH)2D3 may affect chondrocytic function. Discoordinate regulation by vitamin D of MMP-1 and MMP-9 in human mononuclear phagocytes has also been reported. These data suggest that vitamin D may regulate MMP expression in tissues where VDRs are expressed. Production of 1α,25(OH)2D3 within synovial fluids of arthritic joints has been shown and VDRs have been found in rheumatoid synovial tissues and at sites of cartilage erosion. The physiological function of 1α,25(OH)2D3 at these sites remains obscure. MMPs play a major role in cartilage breakdown in the rheumatoid joint and are produced locally by several cell types under strict control by regulatory factors. As 1α,25(OH)2D3 modulates the production of specific MMPs and is produced within the rheumatoid joint, the present study investigates its effects on MMP and prostaglandin E2 (PGE2) production in two cell types known to express chondrolytic enzymes.
To investigate VDR expression in rheumatoid tissues and to examine the effects of 1α,25-dihydroxyvitamin D3 on cultured rheumatoid synovial fibroblasts (RSFs) and human articular chondrocytes (HACs) with respect to MMP and PGE2 production.
Rheumatoid synovial tissues were obtained from arthroplasty procedures on patients with late-stage rheumatoid arthritis; normal articular cartilage was obtained from lower limb amputations. Samples were embedded in paraffin, and examined for presence of VDRs by immunolocalisation using a biotinylated antibody and alkaline-phosphatase-conjugated avidin-biotin complex system. Cultured synovial fibroblasts and chondrocytes were treated with either 1α,25(OH)2D3, or interleukin (IL)-1β or both. Conditioned medium was assayed for MMP and PGE2 by enzyme-linked immunosorbent assay (ELISA), and the results were normalised relative to control values.
The rheumatoid synovial tissue specimens (n = 18) immunostained for VDRs showed positive staining but at variable distributions and in no observable pattern. VDR-positive cells were also observed in association with some cartilage-pannus junctions (the rheumatoid lesion). MMP production by RSFs in monolayer culture was not affected by treatment with 1α,25(OH)2D3 alone, but when added simultaneously with IL-1β the stimulation by IL-1β was reduced from expected levels by up to 50%. In contrast, 1α,25(OH)2D3 had a slight stimulatory effect on basal production of MMPs 1 and 3 by monolayer cultures of HACs, but stimulation of MMP-1 by IL-1β was not affected by the simultaneous addition of 1α,25(OH)2D3 whilst MMP-3 production was enhanced (Table 1). The production of PGE2 by RSFs was unaffected by 1α,25(OH)2D3 addition, but when added concomitantly with IL-1β the expected IL-1 β-stimulated increase was reduced to almost basal levels. In contrast, IL-1β stimulation of PGE2 in HACs was not affected by the simultaneous addition of 1α,25(OH)2D3 (Table 2). Pretreatment of RSFs with 1α,25(OH)2D3 for 1 h made no significant difference to IL-1β-induced stimulation of PGE2, but incubation for 16 h suppressed the expected increase in PGE2 to control values. This effect was also noted when 1α,25(OH)2D3 was removed after the 16h and the IL-1 added alone. Thus it appears that 1α,25(OH)2D3 does not interfere with the IL-1β receptor, but reduces the capacity of RSFs to elaborate PGE2 after IL-1β induction.
Cells within the rheumatoid lesion which expressed VDR were fibroblasts, macrophages, lymphocytes and endothelial cells. These cells are thought to be involved in the degradative processes associated with rheumatoid arthritis (RA), thus providing evidence of a functional role of 1α,25(OH)2D3 in RA. MMPs may play important roles in the chondrolytic processes of the rheumatoid lesion and are known to be produced by both fibroblasts and chondrocytes. The 1α,25(OH)2D3 had little effect on basal MMP production by RSFs, although more pronounced differences were noted when IL-1β-stimulated cells were treated with 1α,25(OH)2D3, with the RSF and HAC showing quite disparate responses. These opposite effects may be relevant to the processes of joint destruction, especially cartilage loss, as the ability of 1α,25(OH)2D3 to potentiate MMP-1 and MMP-3 expression by 'activated' chondrocytes might facilitate intrinsic cartilage chondrolysis in vivo. By contrast, the MMP-suppressive effects observed for 1α,25(OH)2D3 treatment of 'activated' synovial fibroblasts might reduce extrinsic chondrolysis and also matrix degradation within the synovial tissue. Prostaglandins have a role in the immune response and inflammatory processes associated with RA. The 1α,25(OH)2D3 had little effect on basal PGE2 production by RSF, but the enhanced PGE2 production observed following IL-1β stimulation of these cells was markedly suppressed by the concomitant addition of 1α,25(OH)2D3. As with MMP production, there are disparate effects of 1α,25(OH)2D3 on IL-1β stimulated PGE2 production by the two cell types; 1α,25(OH)2D3 added concomitantly with IL-1β had no effect on PGE2 production by HACs. In summary, the presence of VDRs in the rheumatoid lesion demonstrates that 1α,25(OH)2D3 may have a functional role in the joint disease process. 1α,25(OH)2D3 does not appear to directly affect MMP or PGE2 production but does modulate cytokine-induced production.
Comparative effects of 1 α,25-dihydroxyvitamin D3 (1 α,25D3) on interleukin (IL)-1-stimulated matrix metalloproteinase (MMP)-1 and MMP-3 production by rheumatoid synovial fibroblasts and human articular chondrocytes in vivo
Data given are normalized relative to control values and are expressed ± SEM for three cultures of each cell type.
Comparative effects of 1α,25-dihydroxyvitamin D3 (1α,25D3) on Interleukin (IL)-1-stimulated prostaglandin E2 production by rheumatoid synovial fibroblasts and human articular chondrocyte in vivo
Data given are normalized relative to control values and are expressed ± SEM for three cultures of each cell type.
PMCID: PMC17774  PMID: 11056661
1α,25-dihydroxyvitamin D3; matrix metalloproteinase; prostaglandin E2; rheumatoid arthritis
12.  In Vitro Fertilization and Multiple Pregnancies 
Executive Summary
The objective of this health technology policy assessment was to determine the clinical effectiveness and cost-effectiveness of IVF for infertility treatment, as well as the role of IVF in reducing the rate of multiple pregnancies.
Clinical Need: Target Population and Condition
Typically defined as a failure to conceive after a year of regular unprotected intercourse, infertility affects 8% to 16% of reproductive age couples. The condition can be caused by disruptions at various steps of the reproductive process. Major causes of infertility include abnormalities of sperm, tubal obstruction, endometriosis, ovulatory disorder, and idiopathic infertility. Depending on the cause and patient characteristics, management options range from pharmacologic treatment to more advanced techniques referred to as assisted reproductive technologies (ART). ART include IVF and IVF-related procedures such as intra-cytoplasmic sperm injection (ICSI) and, according to some definitions, intra-uterine insemination (IUI), also known as artificial insemination. Almost invariably, an initial step in ART is controlled ovarian stimulation (COS), which leads to a significantly higher rate of multiple pregnancies after ART compared with that following natural conception. Multiple pregnancies are associated with a broad range of negative consequences for both mother and fetuses. Maternal complications include increased risk of pregnancy-induced hypertension, pre-eclampsia, polyhydramnios, gestational diabetes, fetal malpresentation requiring Caesarean section, postpartum haemorrhage, and postpartum depression. Babies from multiple pregnancies are at a significantly higher risk of early death, prematurity, and low birth weight, as well as mental and physical disabilities related to prematurity. Increased maternal and fetal morbidity leads to higher perinatal and neonatal costs of multiple pregnancies, as well as subsequent lifelong costs due to disabilities and an increased need for medical and social support.
The Technology Being Reviewed
IVF was first developed as a method to overcome bilateral Fallopian tube obstruction. The procedure includes several steps: (1) the woman’s egg is retrieved from the ovaries; (2) exposed to sperm outside the body and fertilized; (3) the embryo(s) is cultured for 3 to 5 days; and (4) is transferred back to the uterus. IFV is considered to be one of the most effective treatments for infertility today. According to data from the Canadian Assisted Reproductive Technology Registry, the average live birth rate after IVF in Canada is around 30%, but there is considerable variation in the age of the mother and primary cause of infertility.
An important advantage of IVF is that it allows for the control of the number of embryos transferred. An elective single embryo transfer in IVF cycles adopted in many European countries was shown to significantly reduce the risk of multiple pregnancies while maintaining acceptable birth rates. However, when number of embryos transferred is not limited, the rate of IVF-associated multiple pregnancies is similar to that of other treatments involving ovarian stimulation. The practice of multiple embryo transfer in IVF is often the result of pressures to increase success rates due to the high costs of the procedure. The average rate of multiple pregnancies resulting from IVF in Canada is currently around 30%.
An alternative to IVF is IUI. In spite of reported lower success rates of IUI (pregnancy rates per cycle range from 8.7% to 17.1%) it is generally attempted before IVF due to its lower invasiveness and cost.
Two major drawbacks of IUI are that it cannot be used in cases of bilateral tubal obstruction and it does not allow much control over the risk of multiple pregnancies compared with IVF. The rate of multiple pregnancies after IUI with COS is estimated to be about 21% to 29%.
Ontario Health Insurance Plan Coverage
Currently, the Ontario Health Insurance Plan covers the cost of IVF for women with bilaterally blocked Fallopian tubes only, in which case it is funded for 3 cycles, excluding the cost of drugs. The cost of IUI is covered except for preparation of the sperm and drugs used for COS.
Diffusion of Technology
According to Canadian Assisted Reproductive Technology Registry data, in 2004 there were 25 infertility clinics across Canada offering IVF and 7,619 IVF cycles performed. In Ontario, there are 13 infertility clinics with about 4,300 IVF cycles performed annually.
Literature Review
Royal Commission Report on Reproductive Technologies
The 1993 release of the Royal Commission report on reproductive technologies, Proceed With Care, resulted in the withdrawal of most IVF funding in Ontario, where prior to 1994 IVF was fully funded. Recommendations of the Commission to withdraw IVF funding were largely based on findings of the systematic review of randomized controlled trials (RCTs) published before 1990. The review showed IVF effectiveness only in cases of bilateral tubal obstruction. As for nontubal causes of infertility, there was not enough evidence to establish whether IVF was effective or not.
Since the field of reproductive technology is constantly evolving, there have been several changes since the publication of the Royal Commission report. These changes include: increased success rates of IVF; introduction of ICSI in the early 1990’s as a treatment for male factor infertility; and improved embryo implantation rates allowing for the transfer of a single embryo to avoid multiple pregnancies after IVF.
Studies After the Royal Commission Report: Review Strategy
Three separate literature reviews were conducted in the following areas: clinical effectiveness of IVF, cost-effectiveness of IVF, and outcomes of single embryo transfer (SET) in IVF cycles.
Clinical effectiveness of IVF: RCTs or meta-analyses of RCTs that compared live birth rates after IVF versus alternative treatments, where the cause of infertility was clearly stated or it was possible to stratify the outcome by the cause of infertility.
Cost effectiveness of IVF: All relevant economic studies comparing IVF to alternative methods of treatment were reviewed
Outcomes of IVF with SET: RCTs or meta-analyses of RCTs that compared live birth rates and multiple birth rates associated with transfer of single versus double embryos.
OVID MEDLINE, MEDLINE In-Process & Other Non-Indexed Citations, EMBASE, Cochrane Library, the International Agency for Health Technology Assessment database, and websites of other health technology assessment agencies were searched using specific subject headings and keywords to identify relevant studies.
Summary of Findings
Comparative Clinical Effectiveness of IVF
Overall, there is a lack of well composed RCTs in this area and considerable diversity in both definition and measurement of outcomes exists between trials. Many studies used fertility or pregnancy rates instead of live birth rates. Moreover, the denominator for rate calculation varied from study to study (e.g. rates were calculated per cycle started, per cycle completed, per couple, etc...).
Nevertheless, few studies of sufficient quality were identified and categorized by the cause of infertility and existing alternatives to IVF. The following are the key findings:
A 2005 meta-analysis demonstrated that, in patients with idiopathic infertility, IVF was clearly superior to expectant management, but there were no statistically significant differences in live birth rates between IVF and IUI, nor between IVF and gamete-intra-Fallopian transfer.
A subset of data from a 2000 study showed no significant differences in pregnancy rates between IVF and IUI for moderate male factor infertility.
In patients with moderate male factor infertility, standard IVF was also compared with ICSI in a 2002 meta-analysis. All studies included in the meta-analysis showed superior fertilization rates with ICSI, and the pooled risk ratio for oocyte fertilization was 1.9 (95% Confidence Interval 1.4-2.5) in favour of ICSI. Two other RCTs in this area published after the 2002 meta-analysis had similar results and further confirmed these findings. There were no RCTs comparing IVF with ICSI in patients with severe male factor infertility, mainly because based on the expert opinion, ICSI might only be an effective treatment for severe male factor infertility.
Cost-Effectiveness of IVF
Five economic evaluations of IVF were found, including one comprehensive systematic review of 57 health economic studies. The studies compared cost-effectiveness of IVF with a number of alternatives such as observation, ovarian stimulation, IUI, tubal surgery, varicocelectomy, etc... The cost-effectiveness of IVF was analyzed separately for different types of infertility. Most of the reviewed studies concluded that due to the high cost, IVF has a less favourable cost-effectiveness profile compared with alternative treatment options. Therefore, IVF was not recommended as the first line of treatment in the majority of cases. The only two exceptions were bilateral tubal obstruction and severe male factor infertility, where an immediate offer of IVF/ICSI might the most cost-effective option.
Clinical Outcomes After Single Versus Double Embryo Transfer Strategies of IVF
Since the SET strategy has been more widely adopted in Europe, all RCT outcomes of SET were conducted in European countries. The major study in this area was a large 2005 meta-analysis, followed by two other published RCTs.
All of these studies reached similar conclusions:
Although a single SET cycle results in lower birth rates than a single double embryo transfer (DET) cycle, the cumulative birth rate after 2 cycles of SET (fresh + frozen-thawed embryos) was comparable to the birth rate after a single DET cycle (~40%).
SET was associated with a significant reduction in multiple births compared with DET (0.8% vs. 33.1% respectively in the largest RCT).
Most trials on SET included women younger than 36 years old with a sufficient number of embryos available for transfer that allowed for selection of the top quality embryo(s). A 2006 RCT, however, compared SET and DET strategies in an unselected group of patients without restrictions on the woman’s age or embryo quality. This study demonstrated that SET could be applied to older women.
Estimate of the Target Population
Based on results of the literature review and consultations with experts, four categories of infertile patients who may benefit from increased access to IVF/ICSI were identified:
Patients with severe male factor infertility, where IVF should be offered in conjunction with ICSI;
Infertile women with serious medical contraindications to multiple pregnancy, who should be offered IVF-SET;
Infertile patients who want to avoid the risk of multiple pregnancy and thus opt for IVF-SET; and
Patients who failed treatment with IUI and wish to try IVF.
Since, however, the latter indication does not reflect any new advances in IVF technology that would alter existing policy, it was not considered in this analysis.
Economic Analysis
Economic Review: Cost–Effectiveness of SET Versus DET
Conclusions of published studies on cost-effectiveness of SET versus DET were not consistent. While some studies found that SET strategy is more cost-effective due to avoidance of multiple pregnancies, other studies either did not find any significant differences in cost per birth between SET and DET, or favoured DET as a more cost-effective option.
Ontario-Based Economic Analysis
An Ontario-based economic analysis compared cost per birth using three treatment strategies: IUI, IVF-SET, and IVF-DET. A decision-tree model assumed three cycles for each treatment option. Two separate models were considered; the first included only fresh cycles of IVF, while the second had a combination of fresh and frozen cycles. Even after accounting for cost-savings due to avoidance of multiple pregnancies (only short-term complications), IVF-SET was still associated with a highest cost per birth. The approximate budget impact to cover the first three indications for IVF listed above (severe male factor infertility, women with medical contraindications to multiple pregnancy, and couples who wish to avoid the risk of multiple pregnancy) is estimated at $9.8 to $12.8 million (Cdn). Coverage of only first two indications, namely, ICSI in patients with severe male factor infertility and infertile women with serious medical contraindications to multiple pregnancy, is estimated at $3.8 to $5.5 million Cdn.
Other Considerations
International data shows that both IVF utilization and the average number of embryos transferred in IVF cycles are influenced by IVF funding policy. The success of the SET strategy in European countries is largely due to the fact that IVF treatment is subsidized by governments.
Surveys of patients with infertility demonstrated that a significant proportion (~40%) of patients not only do not mind having multiple babies, but consider twins being an ideal outcome of infertility treatment.
A women’s age may impose some restrictions on the implementation of a SET strategy.
Conclusions and Recommendations
A review of published studies has demonstrated that IVF-SET is an effective treatment for infertility that avoids multiple pregnancies.
However, results of an Ontario-based economic analysis shows that cost savings associated with a reduction in multiple pregnancies after IVF-SET does not justify the cost of universal IVF-SET coverage by the province. Moreover, the province currently funds IUI, which has been shown to be as effective as IVF for certain types of infertility and is significantly less expensive.
In patients with severe male factor infertility, IVF in conjunction with ICSI may be the only effective treatment.
Thus, 2 indications where additional IVF access should be considered include:
IVF/ICSI for patients with severe male factor infertility
IVF-SET in infertile women with serious medical contraindications to multiple pregnancy
PMCID: PMC3379537  PMID: 23074488
13.  GnRH signaling, the gonadotrope and endocrine control of fertility 
Frontiers in neuroendocrinology  2010;31(3):322-340.
Mammalian reproductive cycles are controlled by an intricate interplay between the hypothalamus, pituitary and gonads. Central to the function of this axis is the ability of the pituitary gonadotrope to appropriately respond to stimulation by gonadotropin-releasing hormone (GnRH). This review focuses on the role of cell signaling and in particular, mitogen-activated protein kinase (MAPK) activities regulated by GnRH that are necessary for normal fertility. Recently, new mouse models making use of conditional gene deletion have shed new light on the relationships between GnRH signaling and fertility in both male and female mice. Within the reproductive axis, GnRH signaling is initiated through discrete membrane compartments in which the receptor resides leading to the activation of the extracellular signal-regulated kinases (ERKs 1/2). As defined by gonadotrope-derived cellular models, the ERKs appear to play a central role in the regulation of a cohort of immediate early genes that regulate the expression of late genes that, in part, define the differentiated character of the gonadotrope. Recent data would suggest that in vivo, conditional, pituitary-specific disruption of ERK signaling by GnRH leads to a gender-specific perturbation of fertility. Double ERK knockout in the anterior pituitary leads to female infertility due to LH biosynthesis deficiency and a failure in ovulation. In contrast, male mice are modestly LH deficient; however, this does not have an appreciable impact on fertility.
PMCID: PMC2923852  PMID: 20451543
Hypothalamic-Pituitary-Gonadal axis; Reproduction; Gonadotropin-releasing hormone; Gonadotrope; Cell signaling; Mitogen-activated protein kinase; ERKs; Gonadotropins; Fertility; Mouse models
14.  Stimulation of protease-activated receptor-2 inhibits airway eosinophilia, hyperresponsiveness and bronchoconstriction in a murine model of allergic inflammation 
British Journal of Pharmacology  2005;144(8):1100-1108.
An emerging body of evidence indicates that PGE2 has a privileged anti-inflammatory role within the airways. Stimulants of protease-activated receptor-2 (PAR2) inhibit airway smooth muscle tone in vitro and in vivo predominantly via cyclooxygenase (COX)-dependent generation of prostaglandin E2 (PGE2). Thus, the current study tested the hypothesis that PAR2-induced generation of PGE2 inhibits the development of allergic airways inflammation and hyperresponsiveness.Bronchoalveolar lavage (BAL) fluid recovered from ovalbumin (OVA)-sensitised and -challenged (allergic) mice contained elevated numbers of eosinophils, which peaked at 48 h postchallenge. Intranasal (i.n.) administration of a PAR2-activating peptide (PAR2-AP) SLIGRL (25 mg kg−1, at the time of OVA challenge) caused a 70% reduction in the numbers of BAL eosinophils (compared to the scrambled peptide LSIGRL, 25 mg kg−1).Pretreatment of allergic mice with either indomethacin (1 mg kg−1, dual COX inhibitor) or nimesulide (3 mg kg−1, COX-2-selective inhibitor) blocked SLIGRL-induced reductions in BAL eosinophils.I.n. SLIGRL, but not LSIGRL, inhibited the development of antigen-induced airways hyperresponsiveness. The inhibitory effect of SLIGRL was blocked by indomethacin.Exposure of isolated tracheal preparations from allergic mice to 100 μM SLIGRL was associated with a 5.0-fold increase in PGE2 levels (P<0.05, compared to 100 μM LSIGRL). SLIGRL induced similar increases in PGE2 levels in control mice (OVA-sensitised, saline-challenged).I.n. administration of PGE2 (0.15 mg kg−1) to allergic mice significantly inhibited eosinophilia and airways hyperresponsiveness to methacholine.In anaesthetised, ventilated allergic mice, SLIGRL (5 mg kg−1, i.v.) inhibited methacholine-induced increases in airways resistance. Consistent with this bronchodilator effect, SLIGRL induced pronounced relaxation responses in isolated tracheal preparations obtained from allergic mice. LSIGRL did not inhibit bronchomotor tone in either of these in vivo or in vitro experiments.In summary, a PAR2-AP SLIGRL inhibited the development of airway eosinophilia and hyperresponsiveness in allergic mice through a COX-dependent pathway involving COX-2-mediated generation of the anti-inflammatory mediator PGE2. SLIGRL also displayed bronchodilator activity in allergic mice. These studies support the concept that PAR2 exerts predominantly bronchoprotective actions within allergic murine airways.
PMCID: PMC1576095  PMID: 15700024
Protease-activated receptor; allergic inflammation; airway hyperresponsiveness; eosinophils; SLIGRL; cyclooxygenase, prostaglandin E2; indomethacin; bronchoalveolar lavage
15.  Differential effects of 2- and 3-series E-prostaglandins on in vitro expansion of Lgr5+ colonic stem cells 
Carcinogenesis  2013;35(3):606-612.
This study demonstrates that relative to AA-derived PGE2, a known promoter of colon tumorigenesis, EPA-derived PGE3 has diminished ability to support colonic stem cell expansion in mouse colonic organoids
Arachidonic acid (20:4∆5,8,11,14, AA)-derived prostaglandin E2 (PGE2) promotes colon cancer development. In contrast, chemoprotective n-3 polyunsaturated fatty acids supplant AA, thereby decreasing PGE2 biosynthesis in colonocytes, with eicosapentaenoic acid (20:5∆5,8,11,14,17, EPA) in particular being metabolized to a novel 3-series E-prostaglandin (PGE3), a putative anti-tumorigenic-cyclooxygenase metabolite. Because transformation of adult stem cells is an extremely important route toward initiating intestinal cancer, we utilized the leucine-rich-repeat-containing G-protein-coupled receptor 5 (Lgr5)-enhanced green fluorescent protein-internal ribosome entry site (IRES)-creERT2 knock-in mouse model to isolate and culture colonic organoids, in order to document ex vivo responses to exogenous PGE2 and PGE3. Colonic crypts were isolated from transgenic mice and cultured in a Matrigel-based three-dimensional platform. Organoids were treated with exogenous PGE2, PGE3 or dimethyl sulfoxide (vehicle control) for 5 days and the number of viable organoids was recorded daily. Subsequently, samples were processed for immunohistochemistry, flow cytometry and real-time PCR analyses. PGE2 promoted optimal organoid growth and induced significantly higher levels of cell proliferation (P < 0.05) compared with PGE3 and control. In contrast, the Lgr5-green fluorescent protein-positive stem cell number was uniquely elevated by >2-fold in PGE2-treated cultures compared with PGE3 and control. This coincided with the upregulation of stem-cell-related Sox9, Axin2 and Cd44 messenger RNAs. Our results demonstrate that relative to AA-derived PGE2, a known promoter of colon tumorigenesis, EPA-derived PGE3 has diminished ability to support colonic stem cell expansion in mouse colonic organoids.
PMCID: PMC3941743  PMID: 24336194
16.  The influence of prostaglandins on noradrenaline-induced vasoconstriction in isolated perfused mesenteric blood vessels of the rat 
1 The report of the depression by indomethacin of vasoconstrictor responses to noradrenaline and their partial restoration by prostaglandin E2 (PGE2) and PGE1 in rat isolated perfused mesenteric blood vessels was investigated. The further suggestion that prostaglandins may be necessary for the combination of noradrenaline with the α-adrenoceptor in this tissue was also studied.
2 The reported depression by indomethacin was confirmed and was further shown to be in the form of a concentration-dependent flattening of the noradrenaline concentration-effect curve.
3 A concentration-dependent restorative effect was observed for all prostaglandins studied. The decreasing order of potency for the restoration towards normal of the indomethacin-depressed responses to noradrenaline was: PGE2, PGE1, PGA1, PGF2α, PGA2.
4 The prostaglandins studied were not uniform in their restorative actions and could be separated into two groups. PGE2 and PGE1 restored responses towards the control level whereas PGA1, PGA2 and PGF2α increased responses to an above control level and did so over a smaller concentration range. The possibility of several prostaglandin receptors is discussed.
5 At concentrations equi-effective in restoring depressed responses to control levels PGA1 but not PGE2, caused a parallel shift of the noradrenaline concentration-effect curve to the left and a small, gradual rise in the basal perfusion pressure.
6 The reason for the differing effects remains obscure but does not seem to involve a change in the α-adrenoceptor as indicated by the pA2 of phentolamine. Furthermore, the restorative and potentiating effect of PGA1 is not mediated by blockade of neuronal uptake of noradrenaline.
7 It appears that prostaglandins are required for the vasoconstrictor action of noradrenaline in rat mesenteric blood vessels and that this effect is distal to the drug-receptor interaction. The possible involvement of prostaglandins with intracellular calcium ions is discussed.
PMCID: PMC1667790  PMID: 202365
17.  Sensitization of TRPV1 by EP1 and IP reveals peripheral nociceptive mechanism of prostaglandins 
Molecular Pain  2005;1:3.
Prostaglandin E2 (PGE2) and prostaglandin I2 (PGI2) are major inflammatory mediators that play important roles in pain sensation and hyperalgesia. The role of their receptors (EP and IP, respectively) in inflammation has been well documented, although the EP receptor subtypes involved in this process and the underlying cellular mechanisms remain to be elucidated. The capsaicin receptor TRPV1 is a nonselective cation channel expressed in sensory neurons and activated by various noxious stimuli. TRPV1 has been reported to be critical for inflammatory pain mediated through PKA- and PKC-dependent pathways. PGE2 or PGI2increased or sensitized TRPV1 responses through EP1 or IP receptors, respectively predominantly in a PKC-dependent manner in both HEK293 cells expressing TRPV1 and mouse DRG neurons. In the presence of PGE2 or PGI2, the temperature threshold for TRPV1 activation was reduced below 35°C, so that temperatures near body temperature are sufficient to activate TRPV1. A PKA-dependent pathway was also involved in the potentiation of TRPV1 through EP4 and IP receptors upon exposure to PGE2 and PGI2, respectively. Both PGE2-induced thermal hyperalgesia and inflammatory nociceptive responses were diminished in TRPV1-deficient mice and EP1-deficient mice. IP receptor involvement was also demonstrated using TRPV1-deficient mice and IP-deficient mice. Thus, the potentiation or sensitization of TRPV1 activity through EP1 or IP activation might be one important mechanism underlying the peripheral nociceptive actions of PGE2 or PGI2.
PMCID: PMC1074353  PMID: 15813989
18.  Transforming Growth Factor β Receptor Type 1 Is Essential for Female Reproductive Tract Integrity and Function 
PLoS Genetics  2011;7(10):e1002320.
The transforming growth factor β (TGFβ) superfamily proteins are principle regulators of numerous biological functions. Although recent studies have gained tremendous insights into this growth factor family in female reproduction, the functions of the receptors in vivo remain poorly defined. TGFβ type 1 receptor (TGFBR1), also known as activin receptor-like kinase 5, is the major type 1 receptor for TGFβ ligands. Tgfbr1 null mice die embryonically, precluding functional characterization of TGFBR1 postnatally. To study TGFBR1–mediated signaling in female reproduction, we generated a mouse model with conditional knockout (cKO) of Tgfbr1 in the female reproductive tract using anti-Müllerian hormone receptor type 2 promoter-driven Cre recombinase. We found that Tgfbr1 cKO females are sterile. However, unlike its role in growth differentiation factor 9 (GDF9) signaling in vitro, TGFBR1 seems to be dispensable for GDF9 signaling in vivo. Strikingly, we discovered that the Tgfbr1 cKO females develop oviductal diverticula, which impair embryo development and transit of embryos to the uterus. Molecular analysis further demonstrated the dysregulation of several cell differentiation and migration genes (e.g., Krt12, Ace2, and MyoR) that are potentially associated with female reproductive tract development. Moreover, defective smooth muscle development was also revealed in the uteri of the Tgfbr1 cKO mice. Thus, TGFBR1 is required for female reproductive tract integrity and function, and disruption of TGFBR1–mediated signaling leads to catastrophic structural and functional consequences in the oviduct and uterus.
Author Summary
Approximately 20% of infertile couples in the United States have unexplained causes. Many vital aspects of female fertility are regulated by a family of growth factors called the transforming growth factor β (TGFβ) superfamily. These factors exert their functions via specific receptors and downstream signal mediators. Perturbation of components in this pathway can lead to reproductive dysfunction. We identified a novel role for a TGFβ receptor (called TGFBR1) in female fertility. We demonstrated that female mice with disruption of Tgfbr1 in the reproductive tract are unable to successfully conceive, although they can ovulate and produce fertilizable oocytes. Most importantly, these mice have a striking deformity in the oviduct, marked by the formation of oviductal outpouchings (diverticula) that prevent embryos from reaching the uterus. Concomitant aberrations in the uterine smooth muscle layers are additional features of mice lacking TGFBR1. Therefore, TGFBR1 is critical for the structural integrity and function of the female reproductive tract. Our model can be further exploited to study the development of smooth muscle cells of the female reproductive tract. Genetic mutations in TGFBR1 or other TGFβ signaling machinery may lead to fertility defects in women.
PMCID: PMC3197682  PMID: 22028666
19.  Expression of COX-1 and COX-2 in the endometrium of cyclic, pregnant and in a model of pseudopregnant rats and their regulation by sex steroids 
Cyclooxygenases (COXs) are the rate limiting enzymes in the process of prostaglandins (PGs) synthesis, which are critical regulators of a number of reproductive processes, including ovulation, implantation, decidualization and parturition. The aim of the present study was to investigate the expression and regulation of COX-1 and COX-2 and levels of prostaglandins during rat pregnancy, in a model of pseudopregnancy and estrous cycle.
Uteri were collected from the cyclic rats on each day of estrous cycle, after every two days for pregnant (days 2 to 22) and pseudopregnant rats (days 1 to 9). In vitro primary endometrial stromal cells were cultured in the presence of steroid hormones and their respective inhibitors for the possible modulation of COX-1 and COX-2. Endometrial protein extracts were used for western blot analysis and tissue sections were prepared for protein localization using immunofluorescence. Measurements of PGF2alpha and PGE2 metabolites in serum were performed by enzyme immunoassay (EIA).
COX-1 expression was found to be elevated during implantation and parturition, however, the levels of COX-1 decreased during decidualization periods. COX-2 was detected during early pregnancy from day 2 to 5, increased during decidual regression, and was also expressed at the time of parturition. COX-2 protein expression was found to be increased at estrus phase in cyclic rats. Both enzymes were found to be modulated in the endometrium of pseudopregnant rats, suggesting that they are regulated by 17beta-estradiol and progesterone. A significant increase in PGE2 metabolite levels was observed on day 10, 12 and 14 of pregnancy. However, an increase in PGF2alpha metabolite levels was observed only on day 14. The concentration of both these metabolites changed during pseudopregnancy and maximum levels were observed at day 7. Significant increase in PGE2 metabolite was observed at proestrus phase, on the other hand, PGF2alpha metabolite was significantly increased at proestrus and metestrus phase. COX-2 protein was regulated by 17beta-estradiol in cultured endometrial stromal cells which was blocked in the presence of ICI-182,780.
Taken together, these results suggest that COX-1 and COX-2 could be differentially regulated by steroid hormones and might be the key factors involved in embryo implantation, decidualization, decidua basalis regression and parturition in rats.
PMCID: PMC2936314  PMID: 20735829
20.  Renal β-intercalated cells maintain body fluid and electrolyte balance 
The Journal of Clinical Investigation  2013;123(10):4219-4231.
Inactivation of the B1 proton pump subunit (ATP6V1B1) in intercalated cells (ICs) leads to type I distal renal tubular acidosis (dRTA), a disease associated with salt- and potassium-losing nephropathy. Here we show that mice deficient in ATP6V1B1 (Atp6v1b1–/– mice) displayed renal loss of NaCl, K+, and water, causing hypovolemia, hypokalemia, and polyuria. We demonstrated that NaCl loss originated from the cortical collecting duct, where activity of both the epithelial sodium channel (ENaC) and the pendrin/Na+-driven chloride/bicarbonate exchanger (pendrin/NDCBE) transport system was impaired. ENaC was appropriately increased in the medullary collecting duct, suggesting a localized inhibition in the cortex. We detected high urinary prostaglandin E2 (PGE2) and ATP levels in Atp6v1b1–/– mice. Inhibition of PGE2 synthesis in vivo restored ENaC protein levels specifically in the cortex. It also normalized protein levels of the large conductance calcium-activated potassium channel and the water channel aquaporin 2, and improved polyuria and hypokalemia in mutant mice. Furthermore, pharmacological inactivation of the proton pump in β-ICs induced release of PGE2 through activation of calcium-coupled purinergic receptors. In the present study, we identified ATP-triggered PGE2 paracrine signaling originating from β-ICs as a mechanism in the development of the hydroelectrolytic imbalance associated with dRTA. Our data indicate that in addition to principal cells, ICs are also critical in maintaining sodium balance and, hence, normal vascular volume and blood pressure.
PMCID: PMC3784520  PMID: 24051376
21.  Increased renal vasodilator prostanoids prevent hypertension in mice lacking the angiotensin subtype-2 receptor 
Journal of Clinical Investigation  1999;104(2):181-188.
The angiotensin subtype-1 (AT1) receptor mediates renal prostaglandin E2 (PGE2) production, and pharmacological blockade of the angiotensin subtype-2 (AT2) receptor potentiates the action of angiotensin II (Ang II) to increase PGE2 levels. We investigated the role of the AT2 receptor in prostaglandin metabolism in mice with targeted deletion of the AT2 receptor gene. Mice lacking the AT2 receptor (AT2-null) had normal blood pressure that was slightly elevated compared with that of wild-type (WT) control mice. AT2-null mice had higher renal interstitial fluid (RIF) 6-keto-PGF1α (a stable hydrolysis product of prostacyclin [PGI2]) and PGE2 levels than did WT mice, and had similar increases in PGE2 and 6-keto-PGF1α in response to dietary sodium restriction and Ang II infusion. In contrast, AT2-null mice had lower PGF2α levels compared with WT mice during basal conditions and in response to dietary sodium restriction or infusion of Ang II. RIF cAMP was markedly higher in AT2-null mice than in WT mice, both during basal conditions and during sodium restriction or Ang II infusion. AT1 receptor blockade with losartan decreased PGE2, PGI2, and cAMP to levels observed in WT mice. To determine whether increased vasodilator prostanoids prevented hypertension in AT2-null mice, we treated AT2-null and WT mice with indomethacin for 14 days. PGI2, PGE2, and cAMP were markedly decreased in both WT and AT2-null mice. Blood pressure increased to hypertensive levels in AT2-null mice but was unchanged in WT. These results demonstrate that in the absence of the AT2 receptor, increased vasodilator prostanoids protect against the development of hypertension.
PMCID: PMC408474  PMID: 10411547
22.  A Maladaptive Role for EP4 Receptors in Mouse Mesangial Cells 
PLoS ONE  2014;9(8):e104091.
Roles of the prostaglandin E2 E-prostanoid 4 receptor (EP4) on extracellular matrix (ECM) accumulation induced by TGF-β1 in mouse glomerular mesangial cells (GMCs) remain unknown. Previously, we have identified that TGF-β1 stimulates the expression of FN and Col I in mouse GMCs. Here we asked whether stimulation of EP4 receptors would exacerbate renal fibrosis associated with enhanced glomerular ECM accumulation. We generated EP4Flox/Flox and EP4+/− mice, cultured primary WT, EP4Flox/Flox and EP4+/− GMCs, AD-EP4 transfected WT GMCs (EP4 overexpression) and AD-Cre transfected EP4Flox/Flox GMCs (EP4 deleted). We found that TGF-β1-induced cAMP and PGE2 synthesis decreased in EP4 deleted GMCs and increased in EP4 overexpressed GMCs. Elevated EP4 expression in GMCs augmented the coupling of TGF-β1 to FN, Col I expression and COX2/PGE2 signaling, while TGF-β1 induced FN, Col I expression and COX2/PGE2 signaling were down-regulated in EP4 deficiency GMCs. 8 weeks after 5/6 nephrectomy (Nx), WT and EP4+/− mice exhibited markedly increased accumulation of ECM compared with sham-operated controls. Albuminuria, blood urea nitrogen and creatinine (BUN and Cr) concentrations were significantly increased in WT mice as compared to those of EP4+/− mice. Urine osmotic pressure was dramatically decreased after 5/6 Nx surgery in WT mice as compared to EP4+/− mice. The pathological changes in kidney of EP4+/− mice was markedly alleviated compared with WT mice. Immunohistochemical analysis showed significant reductions of Col I and FN in the kidney of EP4+/− mice compared with WT mice. Collectively, this investigation established EP4 as a potent mediator of the pro-TGF-β1 activities elicited by COX2/PGE2 in mice GMCs. Our findings suggested that prostaglandin E2, acting via EP4 receptors contributed to accumulation of ECM in GMCs and promoted renal fibrosis.
PMCID: PMC4133176  PMID: 25122504
23.  Proteomic analysis of human follicular fluid from fertile women 
Clinical Proteomics  2015;12(1):5.
Follicular fluid is a unique biological fluid in which the critical events of oocyte and follicular maturation and somatic cell-germ cell communication occur. Because of the intimate proximity of follicular fluid to the maturing oocyte, this fluid provides a unique window into the processes occurring during follicular maturation. A thorough identification of the specific components within follicular fluid may provide a better understanding of intrafollicular signaling, as well as reveal potential biomarkers of oocyte health for women undergoing assisted reproductive treatment. In this study, we used high and low pH HPLC peptide separations followed by mass spectrometry to perform a comprehensive proteomic analysis of human follicular fluid from healthy ovum donors. Next, using samples from a second set of patients, an isobaric mass tagging strategy for quantitative analysis was used to identify proteins with altered abundances after hCG treatment.
A total of 742 follicular fluid proteins were identified in healthy ovum donors, including 413 that have not been previously reported. The proteins belong to diverse functional groups including insulin growth factor and insulin growth factor binding protein families, growth factor and related proteins, receptor signaling, defense/immunity, anti-apoptotic proteins, matrix metalloprotease related proteins, and complement activity. In a quantitative analysis, follicular fluid samples from age-matched women undergoing in vitro fertilization oocyte retrieval were compared and 17 follicular fluid proteins were found at significantly altered levels (p < 0.05) between pre-hCG and post-hCG samples. These proteins belong to a variety of functional processes, including protease inhibition, inflammation, and cell adhesion.
This database of FF proteins significantly extends the known protein components present during the peri-ovulatory period and provides a useful basis for future studies comparing follicular fluid proteomes in various fertility, disease, and environmental exposure conditions. We identified 17 differentially expressed proteins after hCG treatment and together these data showed the feasibility for defining biomarkers that illuminate how the ovarian follicle microenvironment is altered in various infertility-related conditions.
Electronic supplementary material
The online version of this article (doi:10.1186/s12014-015-9077-6) contains supplementary material, which is available to authorized users.
PMCID: PMC4357057  PMID: 25838815
Follicle development; Follicular fluid; Human; Proteomics
24.  PGE2 produced by the lung augments the effector phase of allergic inflammation 
Elevated PGE2 is a hallmark of most inflammatory lesions. This lipid mediator can induce the cardinal signs of inflammation, and the beneficial actions of non-steroidal anti-inflammatory drugs are attributed to inhibition of cyclooxygenase COX-1 and COX-2, enzymes essential in the biosynthesis of PGE2 from arachidonic acid. However, both clinical studies and rodent models suggest that, in the asthmatic lung, PGE2 acts to restrain the immune response and limit physiological change secondary to inflammation. To directly address the role of PGE2 in the lung, we examined the development of disease in mice lacking microsomal prostaglandin E synthase 1 (mPGES1), which converts COX-1/COX-2 derived PGH2 to PGE2. We show that mPGES1 determines PGE2 levels in the naïve lung and is required for increases in PGE2 after ovalbumin (OVA) induced allergy. While loss of either COX-1 or COX-2 increases the disease severity, surprisingly mPGES1 −/− mice show reduced inflammation. However, an increase in serum IgE is still observed in the mPGES1 −/− mice, suggesting that loss of PGE2 does not impair induction of a TH2 response. Furthermore, mPGES1 −/− mice expressing a transgenic OVA-specific T cell receptor are also protected, indicating that PGE2 acts primarily after challenge with inhaled antigen. PGE2 produced by the lung plays the critical role in this response, as loss of lung mPGES1 is sufficient to protect against disease. Together this supports a model in which mPGES1-dependent PGE2 produced by populations of cells native to the lung contributes to the effector phase of some allergic responses.
PMCID: PMC3324636  PMID: 22412193
25.  Effects of a single administration of prostaglandin F2alpha, or a combination of prostaglandin F2alpha and prostaglandin E2, or placebo on fertility variables in dairy cows 3–5 weeks post partum, a randomized, double-blind clinical trial 
Delayed uterine involution has negative effects on the fertility of cows; use of prostaglandin F2alpha alone as a single treatment has not been shown to consistently improve fertility. Combined administration of PGF2alpha and PGE2 increased uterine pressure in healthy cows. We hypothesized, that the combination of both prostaglandins would accelerate uterine involution and have, therefore, a positive effect on fertility variables. In commercial dairy farming, the benefit of a single post partum combined prostaglandin treatment should be demonstrated.
383 cows from commercial dairy farms were included in this study. Uterine size and secretion were evaluated at treatment 21–35 days post partum and 14 days later. Cows were randomly allocated to one of three treatment groups: PGF2alpha and PGE2, PGF2alpha or placebo. For every animal participating in the study, the following reproduction variables were recorded: Interval from calving to first insemination, days open, number of artificial inseminations (AI) to conception; subsequent treatment of uterus, subsequent treatment of ovaries. Plasma progesterone level at time of treatment was used as a covariable. For continuous measurements, analysis of variance was performed. Fisher's exact test for categorical non-ordered data and exact Kruskal-Wallis test for ordered data were used; pairwise group comparisons with Bonferroni adjustment of significance level were performed.
There was no significant difference among treatment groups in uterine size. Furthermore, there was no significant difference among treatments concerning days open, number of AI, and subsequent treatment of uterus and ovaries. Days from calving to first insemination tended to be shorter for cows with low progesterone level given PGF2alpha and PGE2 in combination than for the placebo-group (P = 0.024).
The results of this study indicate that the administration of PGF2alpha or a combination of PGF2alpha and PGE2 21 to 35 days post partum had no beneficial effect upon measured fertility variables. The exception was a tendency for a shorter interval from calving to first insemination after administration of the combination of PGF2alpha and PGE2, as compared to the placebo group. Further research should be done in herds with reduced fertility and/or an increased incidence of postpartum vaginal discharge.
PMCID: PMC1764748  PMID: 17184541

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