Ahmad Chehaitly

Emilie Vessieres

Anne-Laure Guihot

Dr Daniel Henrion
email: daniel.henrion@inserm.fr

Ph.D Mitovasc

Flow-mediated outward arterial remodeling in aging

Keywords: blood flow, collateral growth, Arterial outward remodeling, aging, estrogens, gender, mitochondria Highlights

Introduction

Flow-mediated outward remodeling (FMR) promotes the growth of pre-existing arteries. It occurs in somatic growth, along with angiogenesis and arteriogenesis [START_REF] Silvestre | Postischemic revascularization: from cellular and molecular mechanisms to clinical applications[END_REF] . In adulthood, uterine arteries undergo regular outward remodeling during the menstrual cycle and a pronounced enlargement in diameter during pregnancy [START_REF] Mandala | Physiological remodelling of the maternal uterine circulation during pregnancy[END_REF] . In pathological situations, such as diabetes [START_REF] De Chantemele | Type 2 diabetes severely impairs structural and functional adaptation of rat resistance arteries to chronic changes in blood flow[END_REF][START_REF] Vessieres | Flow (shear stress)mediated remodeling of resistance arteries in diabetes[END_REF][START_REF] Ito | Differential impact of diabetes mellitus type II and arterial hypertension on collateral artery growth and concomitant macrophage accumulation[END_REF][START_REF] Shen | Reduced coronary collateralization in type 2 diabetic patients with chronic total occlusion[END_REF] and hypertension [START_REF] Zhou | NAD(P)H oxidase-derived peroxide mediates elevated basal and impaired flow-induced NO production in SHR mesenteric arteries in vivo[END_REF][START_REF] Dumont | Time-related alteration in flow-(shear stress-) mediated remodeling in resistance arteries from spontaneously hypertensive rats[END_REF] , FMR and thus the growth of collateral arteries is altered. Recovery after an ischemic event is, therefore, more difficult when associated with these health risk factors. Furthermore, as has been shown in rats [START_REF] Tuttle | Impaired collateral development in mature rats[END_REF] and mice [START_REF] Wang | Aging-induced collateral dysfunction: impaired responsiveness of collaterals and susceptibility to apoptosis via dysfunctional eNOS signaling[END_REF] , aging itself is also associated with reduced FMR or collateral growth. Similarly, aging has been reported to be a critical issue in elderly individuals for the management of revascularization [START_REF] Halandras | Vascular Surgery and Geriatric Patients[END_REF] .

Nevertheless, as has been shown in rats, the pathway involved in remodeling remains efficient and can be rescued by the administration of a vasodilator drug [START_REF] Dumont | Alteration in flow (shear stress)-induced remodelling in rat resistance arteries with aging: improvement by a treatment with hydralazine[END_REF] . This suggests that while the threshold for the response to flow is shifted, the pathway itself may not be affected [START_REF] Dumont | Alteration in flow (shear stress)-induced remodelling in rat resistance arteries with aging: improvement by a treatment with hydralazine[END_REF] .

Interestingly, the extent of the decrease in FMR in old female rats is much less compared to age-matched males [START_REF] Tarhouni | Role of estrogens and age in flow-mediated outward remodeling of rat mesenteric resistance arteries[END_REF][START_REF] Tarhouni | Determinants of flow-mediated outward remodeling in female rodents: respective roles of age, estrogens, and timing[END_REF] . Indeed, it is well known and documented that pre-menopausal women have a lower incidence of cardiovascular diseases compared to men. This low incidence of cardiovascular diseases has been largely attributed to the protective effect of estrogens, which exert many beneficial effects in arteries, including arterial dilation, acceleration of healing after arterial injury, and atheroprotection [START_REF] Arnal | Membrane and Nuclear Estrogen Receptor Alpha Actions: From Tissue Specificity to Medical Implications[END_REF] . Thus, estrogens are thought to reduce the effects of vascular aging. Indeed, estrogens are also potent regulators of mitochondrial functions and they exert a protective effect on mitochondria [START_REF] Vasconsuelo | Actions of 17beta-estradiol and testosterone in the mitochondria and their implications in aging[END_REF] . However, endothelial cells (ECs), which sense shear stress due to blood flow, contain a low number of mitochondria and they depend mainly on glycolysis for ATP production [START_REF] Groschner | Endothelial mitochondria--less respiration, more integration[END_REF] . This results in ECs having a low level of reactive oxygen species (ROS) as aerobic oxidation is limited in these cells. This is important as ROS play a major role in endothelial dysfunction [START_REF] Levy | Impaired tissue perfusion: a pathology common to hypertension, obesity, and diabetes mellitus[END_REF] . In addition, ECs are not exposed to a hypoxic environment during angiogenesis under ischemic conditions, and glycolysis produces lactate, which is a strong activator of angiogenesis [START_REF] Rohlenova | Endothelial Cell Metabolism in Health and Disease[END_REF] . Thus, a role for mitochondria in flow (shear stress) sensing, which is the trigger for FMR, is unlikely, although the key role of oxidative stress in FMR [START_REF] De Chantemele | Reactive oxygen species are necessary for high flow (shear stress)-induced diameter enlargement of rat resistance arteries[END_REF] could nonetheless be associated with mitochondrial functioning. On the other hand, excessive oxidative stress can also involve mitochondrial ROS production, which could impair FMR in pathological situations, such as diabetes [START_REF] De Chantemele | Type 2 diabetes severely impairs structural and functional adaptation of rat resistance arteries to chronic changes in blood flow[END_REF][START_REF] Vessieres | Flow (shear stress)mediated remodeling of resistance arteries in diabetes[END_REF][START_REF] Ito | Differential impact of diabetes mellitus type II and arterial hypertension on collateral artery growth and concomitant macrophage accumulation[END_REF][START_REF] Shen | Reduced coronary collateralization in type 2 diabetic patients with chronic total occlusion[END_REF] or hypertension [START_REF] Zhou | NAD(P)H oxidase-derived peroxide mediates elevated basal and impaired flow-induced NO production in SHR mesenteric arteries in vivo[END_REF][START_REF] Dumont | Time-related alteration in flow-(shear stress-) mediated remodeling in resistance arteries from spontaneously hypertensive rats[END_REF][START_REF] Fontaine | The Impact of Estrogen Receptor in Arterial and Lymphatic Vascular Diseases[END_REF] . Interestingly, mitochondria are thought to be the biological clock of cells. The American Heart Association has issued broad overview of the key role of mitochondria in cardiovascular physiology that provides insight into how mitochondrial defects contribute to cardiovascular diseases [START_REF] Murphy | Mitochondrial Function, Biology, and Role in Disease: A Scientific Statement From the American Heart Association[END_REF] .

Revascularization: angiogenesis, arteriogenesis, collateral growth, and the role of flow-mediated outward remodeling

Post-ischemic revascularization is the result of several processes that occur simultaneously or sequentially [START_REF] Silvestre | Postischemic revascularization: from cellular and molecular mechanisms to clinical applications[END_REF] . First, angiogenesis is triggered by the ischemia and starts with the sprouting of preexisting capillaries. Arteriogenesis then allows capillaries to form arterioles after stabilization of the sprouts by smooth muscle cells and pericytes.

Arteriogenesis hence increases the arteriolar density, which then results in more blood being available to feed the new capillaries. In parallel, collateral artery growth results from the outward remodeling of preexisting arteriolar anastomoses, which had little flow. Collateral growth increases the number of conducting arteries. Finally, upstream arteries also become subjected to increased blood flow and undergo outward remodeling, leading to an increase in their diameter. Ultimately, blood flow is restored, and the tissue recovers normal functioning.

An increase in blood flow is the main trigger for the outward remodeling that occurs in arteriogenesis, collateral growth, and for the increase in diameter of larger feeding arteries.

The effect of age and the associated risk factors are described in several review articles [START_REF] Silvestre | Postischemic revascularization: from cellular and molecular mechanisms to clinical applications[END_REF][START_REF] Lahteenvuo | Effects of aging on angiogenesis[END_REF][START_REF] Chen | The Roles of AMPK in Revascularization[END_REF]

-Flow-mediated remodeling in somatic growth and other physiological processes

During somatic growth, the diameter of blood vessels increases along with vessel wall hypertrophy so that mechanical strength and vasomotor tone allow proper control of blood flow to tissues with growing needs [START_REF] Majesky | Vascular Development[END_REF] . As in pregnancy, this remodeling has a number of features in common with the remodeling that occurs in post-ischemic revascularization. It is, however, mainly governed by growth factors and hormones at a level not observed later in life.

Remodeling of the uterine arteries allows for the pronounced increase in uterine blood flow that occurs during pregnancy (for a review, see Mandala & Osol 2 ). This is threedimensional remodeling with enlargement in the diameter along with a substantial axial elongation and changes in vessel biomechanical properties, although wall thickening remains limited. Both local and systemic humoral systems regulate uterine artery remodeling, with a major role for shear stress and steroid-induced upregulation of endothelial NO production [START_REF] Van Der Heijden | Uterine artery remodeling and reproductive performance are impaired in endothelial nitric oxide synthase-deficient mice[END_REF] .

The placenta also activates this remodeling by producing VEGF or PlGF, with venoarterial communication as a plausible pathway for the placenta to control the outward remodeling of afferent arteries 2 . Although estrogens control outward remodeling in peripheral arteries [START_REF] Tarhouni | Key role of estrogens and endothelial estrogen receptor alpha in blood flow-mediated remodeling of resistance arteries[END_REF] , their role in the remodeling of uterine arteries and veins remains uncertain.

Exercise induces a strong angiogenic stimulus in the active muscles where the capillaries are functionally recruited due to the increase in blood flow [START_REF] Prior | Exercise-induced vascular remodeling[END_REF] . Indeed, exercise increases blood flow (shear stress), blood pressure, and wall stress, thereby leading to outward hypertrophic remodeling. Numerous studies have demonstrated that the direct hemodynamic impact of exercise training on the health of the artery wall decreases cardiovascular incidents (for a review, see Green et al. 2017 [START_REF] Green | Vascular Adaptation to Exercise in Humans: Role of Hemodynamic Stimuli[END_REF] ). A degree of caution is warranted, however, as excessive exercise has been shown to have deleterious effects on the cardiovascular system [START_REF] Gerche | Exercise-induced right ventricular dysfunction and structural remodelling in endurance athletes[END_REF] , with a reversal in the balance between NO and superoxide and inflammatory cytokines production [START_REF] Durand | Exercise and vascular function: how much is too much?[END_REF] . Excessive exercising has an impact on estrogen production in women thus inducing vascular changes that are usually seen in post-menopausal women with reduced FMD [START_REF] Hoch | Association between the female athlete triad and endothelial dysfunction in dancers[END_REF] and pro-inflammatory profile [START_REF] Durand | Exercise and vascular function: how much is too much?[END_REF] .

-Mechanism of flow-mediated remodeling

Shear stress sensing

Many well-documented review articles are available on flow (shear stress)-mediated signal transduction [START_REF] Chatterjee | Endothelial Mechanotransduction, Redox Signaling and the Regulation of Vascular Inflammatory Pathways[END_REF][START_REF] Fang | Mechanosensing and Mechanoregulation of Endothelial Cell Functions[END_REF] , including reviews on disturbed flow, which alters large artery walls and induces atherosclerosis [START_REF] Chiu | Effects of disturbed flow on vascular endothelium: pathophysiological basis and clinical perspectives[END_REF][START_REF] Souilhol | Endothelial responses to shear stress in atherosclerosis: a novel role for developmental genes[END_REF] . Nevertheless, the dilation induced by an acute increase in flow involves a rapid response of the ECs, leading to immediate relaxation of the adjacent smooth muscle cells or flow-mediated dilation (FMD). FMD mainly involves the production of NO and a range of COX-derivatives and other hyperpolarizing factors (EDHF) depending on the vessel type, the species, and the disease. Interestingly, FMD can be assessed in patients using a non-invasive technique and it correlates well with endothelial dysfunction and risk factors, although a number of limitations have recently been highlighted [START_REF] Tremblay | Flow-mediated dilation stimulated by sustained increases in shear stress: a useful tool for assessing endothelial function in humans?[END_REF] . FMR starts with FMD, which then becomes chronic and changes the arterial wall structure. A first approach to understand the effect of age on FMR is hence to analyze the effect of age on FMD. The main effect of aging on FMD is a reduction in the production of NO in response to increased shear stress and overexpression of inflammatory factors associated with excessive oxidative stress [START_REF] Collins | Hemodynamic forces in endothelial dysfunction and vascular aging[END_REF] . These alterations also characterize the changes in endothelial function observed in cardiovascular and metabolic diseases, which are common, and present as premature vascular (endothelial) aging.

Interestingly, mitochondria are thought to be the biological clock of cells, and ECs are sensitive to oxidative stress of mitochondrial origin [START_REF] Mikhed | Mitochondrial Oxidative Stress, Mitochondrial DNA Damage and Their Role in Age-Related Vascular Dysfunction[END_REF][START_REF] Tang | Mitochondria, endothelial cell function, and vascular diseases[END_REF] . Furthermore, the mitochondria of ECs undergo hyper-fragmentation in cardiovascular diseases [START_REF] Marin-Garcia | Mitochondrial dynamics and cell death in heart failure[END_REF] . Although vascular ECs do not depend on mitochondria for their energy demands [START_REF] Culic | Energy turnover of vascular endothelial cells[END_REF] , it has been hypothesized that the mitochondria of ECs play a regulatory rather than an energy-producing role [START_REF] Kadlec | PGC-1alpha (Peroxisome Proliferator-Activated Receptor gamma Coactivator 1-alpha) Overexpression in Coronary Artery Disease Recruits NO and Hydrogen Peroxide During Flow-Mediated Dilation and Protects Against Increased Intraluminal Pressure[END_REF] . As demonstrated in HUVECs exposed to laminar shear stress [START_REF] Tang | Mitochondria, endothelial cell function, and vascular diseases[END_REF] , shear stress induces a more quiescent and less oxidative phenotype in ECs. Indeed, mitochondrial fusion and fission are sensitive to shear stress in cultured human and bovine ECs [START_REF] Breton-Romero | Laminar shear stress regulates mitochondrial dynamics, bioenergetics responses and PRX3 activation in endothelial cells[END_REF] , and shear stress-dependent Ca 2+ mobilization in human ECs depends on mitochondria-dependent activation of endoplasmic reticulum channels [START_REF] Scheitlin | Endothelial mitochondria regulate the intracellular Ca2+ response to fluid shear stress[END_REF] . Thus, shear stress most probably reduces EC metabolism, with a reduction in mitochondrial ROS production [START_REF] Doddaballapur | Laminar shear stress inhibits endothelial cell metabolism via KLF2-mediated repression of PFKFB3[END_REF][START_REF] Wu | Laminar shear stress promotes mitochondrial homeostasis in endothelial cells[END_REF] . It is, therefore, tempting to hypothesize that the reduction in flow sensing and FMD observed with age is associated with increased production of ROS of mitochondrial origin, as discussed below (5.1). Indeed, free radicals produced by the mitochondria in vascular cells have a major role in the peripheral vascular disorders that occur with age [START_REF] Gioscia-Ryan | Mitochondria-targeted antioxidant (MitoQ) ameliorates age-related arterial endothelial dysfunction in mice[END_REF][START_REF] Camara | Potential therapeutic benefits of strategies directed to mitochondria[END_REF] .

Vascular response to chronic changes in blood flow

For flow-mediated remodeling (FMR) to occur, there must first be a moderate yet also requisite inflammatory response [START_REF] Bakker | Blood flow-dependent arterial remodelling is facilitated by inflammation but directed by vascular tone[END_REF][START_REF] Caillon | The angiotensin II type 2 receptor activates flow-mediated outward remodelling through T cells-dependent interleukin-17 production[END_REF] as well as a degree of oxidative stress [START_REF] De Chantemele | Reactive oxygen species are necessary for high flow (shear stress)-induced diameter enlargement of rat resistance arteries[END_REF] . As has been shown in rat mesenteric arteries, the chronic increase in blood flow is followed by an accumulation of perivascular macrophages during the early stage (the first 2 days), as outlined in Figure 1. This has been demonstrated using macrophage inactivation with clodronate, which prevented remodeling of rat mesenteric arteries subjected in vivo to a doubling of blood flow after surgical ligation of the adjacent arteries [START_REF] Bakker | Blood flow-dependent arterial remodelling is facilitated by inflammation but directed by vascular tone[END_REF][START_REF] Freidja | Heme oxygenase 1 is differentially involved in blood flow-dependent arterial remodeling: role of inflammation, oxidative stress, and nitric oxide[END_REF] . Interestingly, in these studies, both arteries submitted to high flow and arteries submitted to low flow were investigated and both the outward remodeling induced by high flow and the inward remodeling induced by a low flow were prevented by clodronate [START_REF] Bakker | Blood flow-dependent arterial remodelling is facilitated by inflammation but directed by vascular tone[END_REF] . Both type of remodeling depend on the presence of macrophage and the vascular tone determines the direction of the remodeling. Indeed, the chronic change in flow is associated with MCP1 production [START_REF] Tarhouni | Key role of estrogens and endothelial estrogen receptor alpha in blood flow-mediated remodeling of resistance arteries[END_REF][START_REF] Frosen | Flow-induced, inflammation-mediated arterial wall remodeling in the formation and progression of intracranial aneurysms[END_REF] which certainly increase macrophages infiltration in the perivascular space and this inflammatory state induces oxidative stress with superoxide production [START_REF] Vessieres | Flow (shear stress)mediated remodeling of resistance arteries in diabetes[END_REF][START_REF] De Chantemele | Reactive oxygen species are necessary for high flow (shear stress)-induced diameter enlargement of rat resistance arteries[END_REF] by both NADPH-oxidase and macrophages (Figure 1). This oxidative stress induces peroxinitrite (ONOO-) production which initiates metalloproteinases (MMPs) activation and thus extracellular matrix (ECM) digestion [START_REF] Caillon | The angiotensin II type 2 receptor activates flow-mediated outward remodelling through T cells-dependent interleukin-17 production[END_REF] (Figure 1). Low flow-mediated inward remodeling results of an unbalanced contractility due to the reduction in flow-dependent production of vasodilator agents followed by extracellular matrix remodeling and transglutaminase-2 activation [START_REF] Bakker | Blood flow-dependent arterial remodelling is facilitated by inflammation but directed by vascular tone[END_REF][START_REF] Pourageaud | Structural properties of rat mesenteric small arteries after 4-wk exposure to elevated or reduced blood flow[END_REF][START_REF] Van Den Akker | The redox state of transglutaminase 2 controls arterial remodeling[END_REF][START_REF] Tuna | Relation between active and passive biomechanics of small mesenteric arteries during remodeling[END_REF] .

Bone marrow cells are also involved in collateral artery growth, as has been documented in the hind limb of mice [START_REF] Wang | Aging-induced collateral dysfunction: impaired responsiveness of collaterals and susceptibility to apoptosis via dysfunctional eNOS signaling[END_REF] . These findings are in keeping with two studies that have shown that revascularization of the femoral artery after ligation is severely reduced in mice lacking CD4+ cells [START_REF] Van Weel | Natural killer cells and CD4+ T-cells modulate collateral artery development[END_REF] , CD8+ cells [START_REF] Stabile | CD8+ T lymphocytes regulate the arteriogenic response to ischemia by infiltrating the site of collateral vessel development and recruiting CD4+ mononuclear cells through the expression of interleukin-16[END_REF] , or natural killer cells [START_REF] Van Weel | Natural killer cells and CD4+ T-cells modulate collateral artery development[END_REF] , thus highlighting a possible role for T lymphocyte activation in FMR and collateral artery growth. Indeed, in mice lacking T cells (nude mice), FMR of the mesenteric arteries is abrogated [START_REF] Caillon | The angiotensin II type 2 receptor activates flow-mediated outward remodelling through T cells-dependent interleukin-17 production[END_REF] . Our analysis of the immune cells surrounding the arteries subjected to increased blood flow revealed an accumulation of T cells two days after the increase in blood flow. These cells expressed markers of Th17 and a high level of angiotensin II type-2 receptor (AT2R) [START_REF] Caillon | The angiotensin II type 2 receptor activates flow-mediated outward remodelling through T cells-dependent interleukin-17 production[END_REF] . Interleukin-17 (IL-17) has a protective effect in post-ischemic revascularization in the mouse hindlimb [START_REF] Hata | Critical role of Th17 cells in inflammation and neovascularization after ischaemia[END_REF] , and AT2R activation induces the production of IL-17 by memory T cells [START_REF] Caillon | The angiotensin II type 2 receptor activates flow-mediated outward remodelling through T cells-dependent interleukin-17 production[END_REF] (Figure 1). This sequence of events has been confirmed by the observation that FMR does not occur in the absence of AT2R or IL-17 in mice and the finding that the injection of IL-17 into nude mice or mice lacking AT2R restores FMR in mesenteric arteries [START_REF] Caillon | The angiotensin II type 2 receptor activates flow-mediated outward remodelling through T cells-dependent interleukin-17 production[END_REF] . The involvement of AT2R in FMR provides a new therapeutic perspective in age-related vascular dysfunction (Figure 2). This could at least in part explain why the AT2R agonist C21 improves ischemic damage in stroke [START_REF] Oliveira | Influence of aerobic training on the reduced vasoconstriction to angiotensin II in rats exposed to intrauterine growth restriction: possible role of oxidative stress and AT2 receptor of angiotensin II[END_REF] and has a protective effect in neurodegenerative disorders [START_REF] Mogi | Effect of angiotensin II type 2 receptor on stroke, cognitive impairment and neurodegenerative diseases[END_REF] . Indeed, we have shown that C21 restored FMR in old mice mesenteric arteries subjected to a chronic increase in blood flow [START_REF] Caillon | The angiotensin II type 2 receptor activates flow-mediated outward remodelling through T cells-dependent interleukin-17 production[END_REF] .

Indeed, these studies could suggest that the outward remodeling depends on the production of Th1 cytokines leading to a lytic phenotype and that the inward remodeling depends on Th2 cytokines producing a fibrotic phenotype. Nevertheless, a more complex profile characterizes FMR of small arteries with a mixture of pro-and anti-inflammatory factors involved during the 2 or 3 days following the increase in blood flow with a rapid return to normal when shear stress is normalized [START_REF] Bakker | Blood flow-dependent arterial remodelling is facilitated by inflammation but directed by vascular tone[END_REF] . The involvement of the pro-inflammatory cytokine IL-17 and of the anti-inflammatory receptor AT2R is a good example of this complex pathway involved in FMR [START_REF] Caillon | The angiotensin II type 2 receptor activates flow-mediated outward remodelling through T cells-dependent interleukin-17 production[END_REF] .

The next step after this short inflammatory phase comprises activation of matrix metalloproteinases (MMPs), which results in partial digestion of the extracellular matrix in the vascular wall. This process facilitates expansion of the diameter and the ensuing compensatory hypertrophy (Figure 1). MMP9 is most likely involved in FMR, as has been shown in both large [START_REF] Tronc | Role of NO in flowinduced remodeling of the rabbit common carotid artery[END_REF][START_REF] Scott | The multifunctional Ca(2)(+)/calmodulin-dependent kinase IIdelta (CaMKIIdelta) regulates arteriogenesis in a mouse model of flow-mediated remodeling[END_REF] and small arteries [START_REF] Caillon | The angiotensin II type 2 receptor activates flow-mediated outward remodelling through T cells-dependent interleukin-17 production[END_REF][START_REF] Dumont | Key role of the NO-pathway and matrix metalloprotease-9 in high blood flow-induced remodeling of rat resistance arteries[END_REF] . An increase in diameter is hence possible and is governed by the artery tone, as demonstrated by Bakker et al. [START_REF] Bakker | Blood flow-dependent arterial remodelling is facilitated by inflammation but directed by vascular tone[END_REF] . The main stimulus for the expansion of the diameter is NO that is produced after eNOS activation by flow, as has been shown in both large [START_REF] Tronc | Role of NO in flowinduced remodeling of the rabbit common carotid artery[END_REF] and small arteries [START_REF] Dumont | Key role of the NO-pathway and matrix metalloprotease-9 in high blood flow-induced remodeling of rat resistance arteries[END_REF][START_REF] Dai | Endothelial nitric oxide synthase deficiency causes collateral vessel rarefaction and impairs activation of a cell cycle gene network during arteriogenesis[END_REF] . The expression level of eNOS is increased and, in parallel, caveolin-1 expression is reduced so that the NO pathway is more efficient. These changes are activated by IL-17 during the inflammatory phase of the remodeling [START_REF] Caillon | The angiotensin II type 2 receptor activates flow-mediated outward remodelling through T cells-dependent interleukin-17 production[END_REF] . Indeed, IL-17 has a protective effect at low concentrations and it is able to induce eNOS phosphorylation in cultured human ECs [START_REF] Liu | Induction of endothelial nitric oxide synthase expression by IL-17 in human vascular endothelial cells: implications for vascular remodeling in transplant vasculopathy[END_REF] . Thus, IL-17 could also directly activate NO production and thereby increase the arterial diameter (Figure 1). Adenosine monophosphate-activated protein kinase (AMPK) has also been proposed to play a role in arteriogenesis as described in a recent review article [START_REF] Chen | The Roles of AMPK in Revascularization[END_REF] the activation FSS and hypoxia (through reduction of the ratio of ATP to AMP) also activate 5′-Adenosine monophosphate-activated protein kinase (AMPK) phosphorylation which activates eNOS and arteriogenesis in collateral arterioles. Similarly, exercising also activates arteriogenesis through AMPK activation thus promoting FMR and arteriogenesis [START_REF] Chen | The Roles of AMPK in Revascularization[END_REF] .

More surprisingly, estrogens are also involved in this phase of the remodeling, as described below and as depicted in Figure 1. Aside from NO, other vasodilating agents are also recruited in this latter phase, such as CO produced by heme-oxygenase 1 [START_REF] Freidja | Heme oxygenase 1 is differentially involved in blood flow-dependent arterial remodeling: role of inflammation, oxidative stress, and nitric oxide[END_REF][START_REF] Freidja | Heme oxygenase-1 induction restores high-blood-flow-dependent remodeling and endothelial function in mesenteric arteries of old rats[END_REF] and prostanoids synthetized after cyclooxygenase 2 activation, at least in obese rats [START_REF] De Chantemele | Cyclooxygenase-2 preserves flow-mediated remodeling in old obese Zucker rat mesenteric arteries[END_REF] .

Finally, FMR requires an increase in wall thickness in order to normalize tensile stress, which is increased by the expansion of the diameter [START_REF] Pourageaud | Structural properties of rat mesenteric small arteries after 4-wk exposure to elevated or reduced blood flow[END_REF][START_REF] Tuttle | Shear level influences resistance artery remodeling: wall dimensions, cell density, and eNOS expression[END_REF] . The increase in diameter leads to excessive wall stress and stretching of smooth muscle cells that activates the local renin-angiotensin system and particularly the angiotensin II type 1 receptor (AT1R) [START_REF] Cousin | Role of angiotensin II in the remodeling induced by a chronic increase in flow in rat mesenteric resistance arteries[END_REF] .

Consequently, AT1R activation leads to ERK1/2 phosphorylation and smooth muscle hypertrophy [START_REF] Cousin | Role of angiotensin II in the remodeling induced by a chronic increase in flow in rat mesenteric resistance arteries[END_REF] (Figure 1).

Aside from the chronic increase in flow, which triggers FMR, reversal of the blood flow direction often occurs in collateral vascular segments after occlusion of a feed artery. A study performed on HUVECs cultured in a flow chamber has shown that flow reversal activates a wide panel of pro-arteriogenic signaling pathways [START_REF] Heuslein | Mechanisms of Amplified Arteriogenesis in Collateral Artery Segments Exposed to Reversed Flow Direction[END_REF] .

Role of estrogens and estrogen receptor alpha in flow-mediated remodeling

Female sex hormones play a major role in the uteroplacental changes that occur during pregnancy, with major uterine artery outward remodeling associated with trophoblast invasion, hyperplasia, hypertrophy, and modification of the extracellular matrix [START_REF] Mandala | Physiological remodelling of the maternal uterine circulation during pregnancy[END_REF] . Estrogens activate endometrial secretion of factors that promote vascular smooth muscle cell migration, which is an early step in outward arterial remodeling [START_REF] Babischkin | Estrogen stimulates the human endometrium to express a factor(s) that promotes vascular smooth muscle cell migration as an early step in microvessel remodeling[END_REF] . Endogenous 17-beta-estradiol (E2) is essential for increasing endothelium (NO)-mediated dilatation in rat mesenteric arteries [START_REF] Tarhouni | Estrogens are needed for the improvement in endothelium-mediated dilation induced by a chronic increase in blood flow in rat mesenteric arteries[END_REF] subjected to chronic high flow, and activation of estrogen receptor alpha (ERα) is essential for FMR in the mesenteric arteries of young female rats [START_REF] Tarhouni | Key role of estrogens and endothelial estrogen receptor alpha in blood flow-mediated remodeling of resistance arteries[END_REF] . Indeed, both ovariectomy of female rats and the absence of ERα in mice prevent FMR in mesenteric arteries [START_REF] Tarhouni | Key role of estrogens and endothelial estrogen receptor alpha in blood flow-mediated remodeling of resistance arteries[END_REF] . Furthermore, endothelial ERα is involved in this FMR, as shown in mice that selectively lack ERα in ECs (Tie2Cre(+)ERf/f mice) [START_REF] Tarhouni | Key role of estrogens and endothelial estrogen receptor alpha in blood flow-mediated remodeling of resistance arteries[END_REF] . Furthermore, FMR is controlled by the nuclear activating function 2 (AF2) of ERα. Indeed, ERα-AF2° mice, which selectively lack the AF2 function, do not develop FMR, whereas mice lacking only the membrane-associated form of ERα (C451A-ERα mice) respond to the chronic increase in flow with outward remodeling [START_REF] Guivarc'h E | Predominant Role of Nuclear Versus Membrane Estrogen Receptor alpha in Arterial Protection: Implications for Estrogen Receptor alpha Modulation in Cardiovascular Prevention/Safety[END_REF] . Interestingly, E2 blood level control AT2R expression in blood vessel and this could provide a link between Erα and AT2R, both involved in FMR [START_REF] Vessieres | Estrogens and the Angiotensin II Type 2 Receptor Control Flow-Mediated Outward Remodeling in the Female Mouse Mesenteric Artery[END_REF] .

The decline of the amplitude of FMR with age occurs later in female rats than in male rats and is correlated with a progressive decrease in E2 blood levels [START_REF] Tarhouni | Determinants of flow-mediated outward remodeling in female rodents: respective roles of age, estrogens, and timing[END_REF] . This could induce an equivalent decrease in ERα, the expression of which is governed by the level of E2 [START_REF] Arnal | Membrane and Nuclear Estrogen Receptor Alpha Actions: From Tissue Specificity to Medical Implications[END_REF] . While FMR did not occur in young ERα-/-mice, it was reduced by 50% in young ERα+/-mice, thus confirming the importance of gene dosage in FMR. Importantly, increasing E2 blood levels in old female rats could restore FMR to the level observed in young animals [START_REF] Tarhouni | Determinants of flow-mediated outward remodeling in female rodents: respective roles of age, estrogens, and timing[END_REF] . The role of E2 in post-ischemic revascularization has also been demonstrated using a skin flap model in mice [START_REF] Toutain | Prevention of skin flap necrosis by estradiol involves reperfusion of a protected vascular network[END_REF] . This effect of E2 on FMR could at least in part explain its protective action in central and peripheral ischemic disorders [START_REF] Toutain | Prevention of skin flap necrosis by estradiol involves reperfusion of a protected vascular network[END_REF][START_REF] Booth | Estrogen-mediated protection in myocardial ischemiareperfusion injury[END_REF][START_REF] Spyridopoulos | Estrogen-receptormediated inhibition of human endothelial cell apoptosis. Estradiol as a survival factor[END_REF] . Interestingly, we also observed that E2 lost its capacity to restore FMR in female mice after a 9-month long ovariectomy, thus demonstrating nonreversible loss of the effect of E2 on FMR after prolonged estrogen deprivation [START_REF] Tarhouni | Determinants of flow-mediated outward remodeling in female rodents: respective roles of age, estrogens, and timing[END_REF] . This observation supports the previous works that highlighted the importance of the timing in treating postmenopausal women with E2. Indeed, after a long interruption, the effect of E2 may be more deleterious than beneficial on the cardiovascular system [START_REF] Valera | From the Women's Health Initiative to the combination of estrogen and selective estrogen receptor modulators to avoid progestin addition[END_REF] .

Flow-mediated remodeling in aging

Vascular aging

While the probability of an ischemic event increases with age, the capacity of the vascular tree to undergo proper revascularization is reduced. Indeed, aging is usually associated with a progressive pro-inflammatory state which render the elderly more prone to develop metabolic, cardiovascular and neurovascular disorders. Endothelial cells senescence is the hallmark of vascular aging with increased production of pro-inflammatory cytokines and adhesion molecules [START_REF] Brandes | Endothelial aging[END_REF][START_REF] Jia | Endothelial cell senescence in aging-related vascular dysfunction[END_REF] . This slowly developing pro-inflammatory state facilitates the occurrence of metabolic and cardiovascular diseases which frequency increases with aging [START_REF] Brandes | Endothelial aging[END_REF][START_REF] Jia | Endothelial cell senescence in aging-related vascular dysfunction[END_REF] . Estrogens provide a protective effect in women before menopause against cardiovascular disorders in part through anti-inflammatory effects [START_REF] Trenti | Estrogen, Angiogenesis, Immunity and Cell Metabolism: Solving the Puzzle[END_REF] .

First, flow-mediated dilation (FMD), which initiates the remodeling, is early affected by aging. Ultrasound measurement of FMD in brachial arteries has revealed that both gender and age are independent risk factors for reduced endothelial function [START_REF] Deanfield | Endothelial function and dysfunction: testing and clinical relevance[END_REF] . Thus, the initial stimulus involved in FMR induces less of a dilatory response with age. Indeed, no FMR of first-order mesenteric arteries subjected to a chronic increase in blood flow was noted in healthy male rats that were 12 [START_REF] Tuttle | Impaired collateral development in mature rats[END_REF] or 24 months 12 old. Aging also induced a progressive decrease in the collateral number, the diameter, and remodeling after femoral ligation in mice aged 3 to 31 months [START_REF] Faber | Aging causes collateral rarefaction and increased severity of ischemic injury in multiple tissues[END_REF] . In this study, aging was accompanied by reduced eNOS signaling, with more nitrosylation and less phosphorylation of eNOS. However, this age-dependent reduction in collateral growth was not associated with a decrease in T cell or macrophage recruitment [START_REF] Faber | Aging causes collateral rarefaction and increased severity of ischemic injury in multiple tissues[END_REF] .

Another study has shown that aging in mice (3 versus 18 months) reduces bone marrow cell recruitment and homing [START_REF] Wang | Aging-induced collateral dysfunction: impaired responsiveness of collaterals and susceptibility to apoptosis via dysfunctional eNOS signaling[END_REF] . However, transplantation of young bone marrow into old mice did not result in recovery of blood flow in the leg subjected to ischemia after femoral ligation. A similar result was obtained by another group using an ischemic skin flap model [START_REF] Chang | Age decreases endothelial progenitor cell recruitment through decreases in hypoxia-inducible factor 1alpha stabilization during ischemia[END_REF] .

Importantly, aside from the absence of enlargement of the diameter of the arteries subjected to high flow in old male rats, the wall to lumen ratio, which is a key index of the hypertrophy associated with cardiovascular disorders [START_REF] Rizzoni | Small artery remodeling in hypertension and diabetes[END_REF][START_REF] Van Den Akker | Small artery remodeling: current concepts and questions[END_REF] , was greater than in young animals.

Thus, the consequences are two-fold, with a hypertrophied artery without normalization of shear stress and, above all, no increase in flow, which makes it impossible to compensate for ischemia.

Nevertheless, in relatively healthy aging, despite the absence of an increase in the diameter of arteries subjected to enhanced blood flow, endothelium-mediated dilation in these arteries is enhanced by the chronic increase in flow, as shown in 2-year-old male rats [START_REF] Yan | Chronic high blood flow potentiates shear stressinduced release of NO in arteries of aged rats[END_REF] . In addition, treatment with the vasodilator hydralazine at a dose that does not reduce blood pressure [START_REF] Gorny | Chronic hydralazine improves flow (shear stress)-induced endothelium-dependent dilation in mouse mesenteric resistance arteries in vitro[END_REF] restored FMR in the mesenteric arteries of 2-year-old male rats [START_REF] Dumont | Alteration in flow (shear stress)-induced remodelling in rat resistance arteries with aging: improvement by a treatment with hydralazine[END_REF] . These observations suggest that the pathways involved in FMR remain efficient, with possibly a shift of the threshold for flow (shear stress) sensitivity of the flow-sensors. This issue remains to be further investigated in order to better define which element could be targeted to improve tissue perfusion in the elderly. One possible explanation could be that the bioavailability of the NO necessary for the increase in diameter in FMR is impaired in aging and that a higher stimulus is needed, as this is can be obtained with hydralazine. In old mice subjected to femoral artery ligation, the level of eNOS and phosphorylated eNOS in collateral arteries is lower than in young mice [START_REF] Wang | Aging-induced collateral dysfunction: impaired responsiveness of collaterals and susceptibility to apoptosis via dysfunctional eNOS signaling[END_REF] . Furthermore, in old male rats, treatment with an antioxidant drug or with an AT1R blocker can restore FMR of the mesenteric artery to the level obtained in young rats [START_REF] Tarhouni | Role of estrogens and age in flow-mediated outward remodeling of rat mesenteric resistance arteries[END_REF] , which suggests that excessive oxidative stress counteracts the NO pathway involved in FMR. Indeed, oxidative stress increases with age and reduces FMR in aged rats [START_REF] Miller | Antioxidants reverse age-related collateral growth impairment[END_REF] .

Oxidative stress increases with age through several mechanisms such as increased NADPHoxidase levels and activity [START_REF] Miller | Antioxidants reverse age-related collateral growth impairment[END_REF] and through an increase in ROS production by mitochondria [START_REF] Doria | Relationship between human aging muscle and oxidative system pathway[END_REF] .

In old mice treated with the mitochondrial superoxide scavenger MitoTEMPO, the blood flow recovery after femoral occlusion was equivalent to that in young animals [START_REF] Miura | Mitochondrial-Targeted Antioxidant Maintains Blood Flow, Mitochondrial Function, and Redox Balance in Old Mice Following Prolonged Limb Ischemia[END_REF] . These studies performed in mice are in keeping with the finding that patients with peripheral artery disease exhibit mitochondrial dysfunction and increased oxidative stress-induced damage [START_REF] Brass | Multiple skeletal muscle mitochondrial DNA deletions in patients with unilateral peripheral arterial disease[END_REF][START_REF] Thompson | Protein concentration and mitochondrial content in the gastrocnemius predicts mortality rates in patients with peripheral arterial disease[END_REF] .

Interestingly, E2 has been shown to delay human vascular EC senescence induced by hydrogen peroxide of mitochondrial origin [START_REF] Ruan | Retarding the senescence of human vascular endothelial cells induced by hydrogen peroxide: effects of 17beta-estradiol (E2) mediated mitochondria protection[END_REF] , thus suggesting that estrogens could also prevent the loss of FMR with age, possibly through a reduction in oxidative stress and inflammation.

Vascular aging and female hormones

Numerous epidemiological studies suggest that women are better protected than men against cardiovascular diseases and that this effect is lost after menopause [START_REF] Lenfant | Timing of the vascular actions of estrogens in experimental and human studies: why protective early, and not when delayed?[END_REF] . Indeed, the decline in ovarian function is associated with a decrease in NO production [START_REF] Arnal | Membrane and Nuclear Estrogen Receptor Alpha Actions: From Tissue Specificity to Medical Implications[END_REF] . FMR has been shown to occur in 12-month-old female but not in male rats of the same age. Compared to females, the NO-dependent pathway was more efficient, and the levels of ERK1/2 phosphorylation, oxidative stress, and vascular contractility were higher in old male rats and in ovariectomized female rats, thus suggesting that excessive oxidative stress opposes NOdependent enlargement of the arterial diameter. Importantly, in male rats and in ovariectomized female rats, treatment with hydralazine, the antioxidant TEMPOL (4hydroxy-2,2,6,6-tetramethylpiperidine-N-oxyl), or candesartan (an inhibitor of the angiotensin II type 1 receptor) normalized FMR and reduced arterial contractility, thus suggesting that estrogens balance oxidative stress and vascular contractility to preserve FMR in mature female rats. [START_REF] Tarhouni | Role of estrogens and age in flow-mediated outward remodeling of rat mesenteric resistance arteries[END_REF][START_REF] Tarhouni | Determinants of flow-mediated outward remodeling in female rodents: respective roles of age, estrogens, and timing[END_REF] Indeed, estrogen maintain NO bioavailability and thus the capacity of arteries to maintain FMR in response to a chronic increase in blood flow. Estrogens also lower endothelin-1 production by the endothelial cells and thus its vasoconstrictor and proinflammatory effects. Indeed, circulating T lymphocytes are more abundant in premenopausal women than in men [START_REF] Somani | Aging women and their endothelium: probing the relative role of estrogen on vasodilator function[END_REF] . This role of E2 in FMR through AF2-ERα activation could explain the protective effect of E2 in ischemic disorders, as shown in myocardium, hind limb, brain, and skin 76, 98- 100 . The protective effect of E2 against damage from myocardial ischemia/reperfusion involves the endothelial ERα 101 , while activation of the non-nuclear ERα pathway decreases the damage from 102 and the severity of stroke in the mice 103 . Using ERα-AF2 0 and C451A-ERα mice, which have selective inactivation of nuclear (AF2) and membrane ERα, respectively, we have shown that nuclear ERα plays a prominent role in the prevention of the kidney damage induced by angiotensin II-dependent hypertension in old female mice 104 .

Interestingly, selective activation of nuclear ERα with estetrol, which is a fetal estrogen, prevents angiotensin II-induced hypertension and promotes FMR [START_REF] Guivarc'h E | Predominant Role of Nuclear Versus Membrane Estrogen Receptor alpha in Arterial Protection: Implications for Estrogen Receptor alpha Modulation in Cardiovascular Prevention/Safety[END_REF] . Similarly, E2 and the selective estrogen receptor modulator tamoxifen accelerate endothelial healing in a mouse model of vascular injury 105 . This suggests that pharmacological targeting of the AF2 function of ERα warrants further investigation.

Flow-mediated remodeling in aging associated with risk factors

Unfortunately, aging and risk factors go hand-in-hand, often with fatal consequences. Moreover, hypertension, obesity, and diabetes are increasingly affecting young people.

Damage to the vascular wall also accumulates over time, and the frequency of ischemic diseases increases with age. For example, peripheral arterial occlusive disease is mostly observed in the elderly, and its incidence is expected to increase dramatically as the population ages, as well as due to an increase in the prevalence of metabolic syndrome and type 2 diabetes. Similarly, coronary heart disease remains a major reason of death worldwide despite an improved medical management and a better quality of the treatments. The main cause of heart attack is atherosclerosis with plaques instability and rupture which induce in myocardial ischemia and then hypoxia and tissue necrosis. Besides the current therapy involving coronary artery intervention or bypass grafting it is necessary to increase to capacity of the ischemic areas to recruit new vessels and to increase arterioles diameter through activation of FMR thus improving revascularization in order to prevent the occurrence and progression of cardiac and peripheral arterial diseases 106 . As depicted in Figure 2, hypertension, diabetes and obesity increase superoxide production and cellular level leading to the disruption of the physiological process involved in FMR. Thus, therapeutic tools reducing oxidative stress are likely to be efficient. Flow-mediated outward remodeling is severely reduced in spontaneously hypertensive rats (SHRs) [START_REF] Tuttle | Impaired collateral development in mature rats[END_REF] . Indeed, the kinetics of remodeling in very young SHRs is slower than in normotensive WKY rats, and SHRs reach a similar level of FMR in the mesenteric artery after 3 weeks instead of 1 week in WKY rats [START_REF] Dumont | Time-related alteration in flow-(shear stress-) mediated remodeling in resistance arteries from spontaneously hypertensive rats[END_REF] . In 10-week-old SHRs, shortly after the onset of hypertension, the damage induced by high intraluminal pressure is minimal. Interestingly, the FMR amplitude decreases after reaching a plateau after 3 weeks, and this is accompanied by wall hypertrophy, hypercontractility, and excessive oxidative stress, which are thought to synergistically abrogate FMR [START_REF] Dumont | Time-related alteration in flow-(shear stress-) mediated remodeling in resistance arteries from spontaneously hypertensive rats[END_REF] . In the mesenteric arteries of SHRs, the baseline H2O2 and NO levels are higher than in WKY rats, whereas after an increase in chronic flow, only the level of H2O2 increased in SHRs, while the level of NO was elevated in WKY rats [START_REF] Zhou | NAD(P)H oxidase-derived peroxide mediates elevated basal and impaired flow-induced NO production in SHR mesenteric arteries in vivo[END_REF] . This shift of the response to flow toward the production of H2O2 and ROS could participate in the decline in revascularization that occurs with age in association with hypertension 107 .

Obesity also impairs outward remodeling of collateral arteries. This has been shown in obese mice subjected to femoral occlusion, and it is due to excessive NADPH-oxidase activity and ROS production without a reduction in monocyte mobilization 108 . Surprisingly, FMR in mesenteric arteries was not reduced in obese rats despite pronounced impairment of NOdependent relaxation 109 . However, COX2 inhibition suppressed FMR in old obese rats, thus suggesting that inflammation associated with obesity provides a pathway to compensate for the loss of NO-dependent dilation and to maintain FMR in the mesenteric bed 110 . This could be specific to the mesenteric circulation, which is overstimulated in rats with hyperphagia, and this could contribute to the weight gain in these animals.

Flow-mediated outward remodeling is severely reduced in a rat model of type 2 diabetes 111 . In this study, MMPs could not be activated in the arteries subjected to a chronic increase in blood flow, and this was due to the blocking effect of the advanced glycation endproducts (AGEs) generated by the hyperglycemia (Figure 2). The AGE-breaker drug ALT-711 reverses this effect in young diabetic rats, resulting in restoration of FMR. However, in old diabetic rats, this drug was ineffective, suggesting that long-term (1 year in the rat) diabetes induces irreversible AGE accumulation 112 and possibly irreversible tissue damage.

In this study, performed in 1-year-old diabetic (ZDF) rats, FMR was not only absent but endothelium-mediated dilation was further decreased, although an antioxidant treatment could partially restore it [START_REF] Vessieres | Flow (shear stress)mediated remodeling of resistance arteries in diabetes[END_REF]112 . In these old diabetic rats, vasoconstrictor prostanoids and oxidative stress both reduced endothelium-mediated relaxation 113 .

Finally, unbalanced oxidative stress with excessive superoxide and H2O2 production is a common feature of the various risk factors that impair FMR and collateral growth with age.

Indeed, H2O2 is a potential vasodilating agent that could, at least in part, compensate for the loss of NO. For example, in human coronary arteries from coronary artery disease patients with a median age of 67, flow-mediated dilation depended on the mitochondrial production of hydrogen peroxide instead of nitric oxide 114 . Although this production of hydrogen peroxide allows dilation to be maintained, it creates an excessively oxidative environment that can lead to extensive tissue damage. 115 Of note, 17-beta-estradiol has been shown to delay human vascular EC senescence induced by hydrogen peroxide of mitochondrial origin [START_REF] Ruan | Retarding the senescence of human vascular endothelial cells induced by hydrogen peroxide: effects of 17beta-estradiol (E2) mediated mitochondria protection[END_REF] . Indeed, estrogens interact not only with smooth muscle and endothelial cells but also with immune cells and thus reduced endothelial dysfunction due to inflammation, thus contributing to delay vascular or at least endothelial cells senescence [START_REF] Trenti | Estrogen, Angiogenesis, Immunity and Cell Metabolism: Solving the Puzzle[END_REF] .

Ischemic heart disease is a common endpoint of the risk factors listed above and it is a main cause of death which affects now more and more younger people and women. Besides the classical surgical procedures available and the pharmacological tools available, another approach would be to better stimulate coronary arteries collateral growth and thus, FMR 116 .

As discussed above, AMPK is a promising therapeutic target to reach this end [START_REF] Chen | The Roles of AMPK in Revascularization[END_REF] .

Nevertheless, despite a large number of candidates that activates coronary collateral growth in preclinical models involving usually young and healthy animals, clinical trials remain deceiving. As described in detail in a recent review 116 , cytokines, MCP-1, growth factors such as VEGF, stem cells or ivabradine has been tested without a clear success, so far, in promoting collateral growth in patients with ischemic heart diseases. Based on the observation that exercising improves collateral growth, at least in part through an increase in shear stress, external counter pulsation has been tested in humans with promising results 116 .

Therapeutic tools

Exercise has been clearly shown to increase blood flow and to activate FMR, with a positive impact on arterial structure and function [START_REF] Green | Vascular Adaptation to Exercise in Humans: Role of Hemodynamic Stimuli[END_REF] . A sedentary lifestyle is usually associated with other risk factors such as obesity, diabetes, and hypertension. Inactivity per se induces inward remodeling whereas exercise has the opposite effect 117 . Even short-term daily exercise has been shown to induce remodeling of rat intramural coronary arterioles and to improve coronary blood flow autoregulation 117 . Indeed, well-controlled exercise is recommended for patients with a risk of cardiovascular events [START_REF] Green | Vascular Adaptation to Exercise in Humans: Role of Hemodynamic Stimuli[END_REF] . Unfortunately, there is an increasing trend nowadays in sedentary behavior, with more than one-third of the World population currently below the minimum physical activity requirements needed to remain healthy 118 . Thus, alternative solutions are needed, including the use of medications. Aside from specific treatments for diabetes, obesity, and hypertension, which have been described in detail, antioxidant treatments (Figure 2) may have the potential to rescue FMR and accelerate revascularization more generally [START_REF] Ito | Differential impact of diabetes mellitus type II and arterial hypertension on collateral artery growth and concomitant macrophage accumulation[END_REF]108,112 . As described above (5.1), in healthy aging, increasing blood flow is sufficient to restore FMR, as shown using a non-hypotensive dose of hydralazine in healthy old rats [START_REF] Dumont | Alteration in flow (shear stress)-induced remodelling in rat resistance arteries with aging: improvement by a treatment with hydralazine[END_REF] . Naturally, healthy aging does not require any treatment, and in aging associated with one or more risk factors, the dilatory machinery is not always efficient. Whereas targeting the renin-angiotensin system with an AT1R blocker and ACE inhibitors is common in cardiovascular and metabolic diseases 119, 120 , the AT2R is emerging as a potential new therapeutic target.

Importantly, the AT2R is essential for FMR, as shown in mouse mesenteric and skin arteries, and its stimulation restored FMR in 1-year-old male mice [START_REF] Caillon | The angiotensin II type 2 receptor activates flow-mediated outward remodelling through T cells-dependent interleukin-17 production[END_REF] . Activation of the AT2R results in opposite and beneficial pharmacological outcomes compared to activation of the AT1R. These include pronounced vasodilator, anti-fibrotic, and anti-inflammatory effects 121 .

Stimulation of the AT2R could be an alternative to blockade of the renin-angiotensin system in some ischemic diseases that occur with age (Figure 2). Indeed, AT2R stimulation targets only the associated dilatory and anti-inflammatory pathways, without blocking the production of angiotensin II or inhibition of the AT1R, and this is now feasible in light of the development of efficient AT2R agonists 122 .

Finally, targeting estrogen receptors is potentially another new therapeutic option (Figure 2), as their numerous functions are now better described [START_REF] Arnal | Membrane and Nuclear Estrogen Receptor Alpha Actions: From Tissue Specificity to Medical Implications[END_REF] . Two pathways lead to immediate vasodilatation with the potential for activation of FMR. First, membraneassociated ERα activates NO-dependent dilation, and selective entities are available to target this function without also stimulating the nuclear functions 123,124 . The G protein-coupled estrogen receptor (GPER) also induces vasodilation without obvious nuclear effects, and a wide panel of agonists such as natural estrogens, raloxifene, fulvestrant, and a number of phytoestrogens are able to activate this receptor 125 . Alternatively, targeting the AF2 function of ERα, which is directly involved in FMR [START_REF] Guivarc'h E | Predominant Role of Nuclear Versus Membrane Estrogen Receptor alpha in Arterial Protection: Implications for Estrogen Receptor alpha Modulation in Cardiovascular Prevention/Safety[END_REF] , is another option, and pharmacological entities such as estetrol (E4) hold considerable promise in this regard 126 .

Conclusion

FMR in collateral growth and arteriogenesis is a complex phenomenon that recruits endothelial and smooth muscle cells as well as the immune system. FMR is involved in postischemic revascularization, which often occurs in patients with one or several risk factors, including age. Activating FMR in older subjects requires increasing the stimulus on the endothelium by the addition of extra vasodilatation to the initial stimulus (the increase in shear stress). However, as the need for revascularization is greater in older individuals with risk factors, the therapy cannot merely amount to exercise with or without a mild vasodilating drug. Indeed, a key issue is the reversibility of the vascular damage induced over time, and thus promoting prevention and limiting exposure to the risk factors remain the best options in this regard. In resistance arteries, a chronic increase in blood flow (high shear stress) first activates the NO-pathway and thus the production of NO (within minutes). As the stimulus continues, after several hours a rise in oxidative stress occurs, with the production of superoxide (O2 ) and then the production of the monocyte chemo-attractant protein 1 (MCP1), which attracts macrophages to the perivascular space. The association of NO and O2 produces peroxynitrite ion (ONOO-), which triggers activation of matrix metalloproteinases (MMPs) and extracellular matrix (ECM) digestion. In this early phase, between days 1 and 3 after increasing blood flow, memory T cells and preferentially Th17 cells produce interleukin-17 (IL-17) upon stimulation of the angiotensin II type 2 receptor (AT2R). IL-17 further activates MMPs and also increases eNOS expression level in endothelial cells which thus produce more NO, thereby inducing outward remodeling as an increase in arterial diameter. At this stage, activation of the estrogen receptor alpha (ERα) participates in overactivation of the NO pathway. The enlargement in diameter allows normalization of the shear stress, and the increase in diameter then enhances the tensile stress (stretch) on the vascular wall, thereby leading to the stimulation of the angiotensin II type 1 receptor (AT1R) which induces hypertrophy and thus normalization of the tensile stress. Altogether, after 6 or 7 days the vessels become larger and thicker, with normalized shear and tensile stress. In blue, the simplified pathway involved in resistance arteries subjected to a chronic increase in blood flow (high shear stress), described in more detail in Figure 2. Hypertension (high blood pressure, HBP), obesity and diabetes, in addition to age, increases the production of superoxide (O2 ), especially of mitochondrial origin. This excessive superoxide production inhibits NO and peroxynitrite (ONOO-) production, matrix metalloproteinase (MMP) activation, and thus extracellular matrix (ECM) digestion. The latter is also inhibited by advanced glycation end products (AGEs) produced in diabetes. Memory T cells, which produce interleukin-17 (IL-17) that leads to activation of MMPs, can be stimulated by agonists of the angiotensin II type 2 receptor (AT2R). AGEs (and also hypertension through activation of superoxide production) also reduce eNOS activity. O2 also induces excessive hypertrophy. Antioxidant therapy can reduce most of these deleterious as long as it is applied early enough. Similarly, activation of the estrogen receptor alpha (ERα) induces NO production, which reduce inflammation and oxidative stress and induces expansion of the diameter.

Diameter enlargement + hypertrophy

Reduced outward remodeling + excessive hypertrophy

Figure 1 :

 1 Figure 1: Schematic representation of the vascular response to a chronic increase in blood flow.In resistance arteries, a chronic increase in blood flow (high shear stress) first activates the NO-pathway and thus the production of NO (within minutes). As the stimulus continues, after several hours a rise in oxidative stress occurs, with the production of superoxide (O2 ) and then the production of the monocyte chemo-attractant protein 1 (MCP1), which attracts macrophages to the perivascular space. The association of NO and O2 produces peroxynitrite ion (ONOO-), which triggers activation of matrix metalloproteinases (MMPs) and extracellular matrix (ECM) digestion. In this early phase, between days 1 and 3 after increasing blood flow, memory T cells and preferentially Th17 cells produce interleukin-17 (IL-17) upon stimulation of the angiotensin II type 2 receptor (AT2R). IL-17 further activates MMPs and also increases eNOS expression level in endothelial cells which thus produce more NO, thereby inducing outward remodeling as an increase in arterial diameter. At this stage, activation of the estrogen receptor alpha (ERα) participates in overactivation of the NO pathway. The enlargement in diameter allows normalization of the shear stress, and the increase in diameter then enhances the tensile stress (stretch) on the vascular wall, thereby leading to the stimulation of the angiotensin II type 1 receptor (AT1R) which induces hypertrophy and thus normalization of the tensile stress. Altogether, after 6 or 7 days the vessels become larger and thicker, with normalized shear and tensile stress.

Figure 2 :

 2 Figure 2: Risk factors (red) that reduce the vascular response to a chronic increase in blood flow and potential therapeutic agents (green).In blue, the simplified pathway involved in resistance arteries subjected to a chronic increase in blood flow (high shear stress), described in more detail in Figure2. Hypertension (high blood pressure, HBP), obesity and diabetes, in addition to age, increases the production of superoxide (O2 ), especially of mitochondrial origin. This excessive superoxide production inhibits NO and peroxynitrite (ONOO-) production, matrix metalloproteinase (MMP) activation, and thus extracellular matrix (ECM) digestion. The latter is also inhibited by advanced glycation end products (AGEs) produced in diabetes. Memory T cells, which produce interleukin-17 (IL-17) that leads to activation of MMPs, can be stimulated by agonists of the angiotensin II type 2 receptor (AT2R). AGEs (and also hypertension through activation of superoxide production) also reduce eNOS activity. O2 also induces excessive hypertrophy. Antioxidant therapy can reduce most of these deleterious as long as it is applied early enough. Similarly, activation of the estrogen receptor alpha (ERα) induces NO production, which reduce inflammation and oxidative stress and induces expansion of the diameter.

Funding

This work was supported in part by the foundation for Medical Research (Fondation pour la Recherche Médicale, contract FRM -DPC20171138957, Mitoshear project) and the National Agency for Research (Agence Nationale de le Recherche, contract # ANR-18-CE14-0016-01, Estroshear project)

Conflict of interest

The authors declare that there are no conflicts of interest.


Hélène Jourdain
email: helene.jourdain@chu-reunion.fr

H Flye

Sainte Marie

B Courbiere

G Lazaro

M Boukerrou

P L Tran

Evaluation of physicians' practice patterns in France concerning fertility preservation in women with endometriosis

Keywords: physicians' practice, fertility preservation, endometriosis American Fertility Society AMH: Anti-Mullerian Hormone

Objective: We aim to evaluate the knowledge and physicians' practices concerning fertility preservation in women with endometriosis.

Design: Descriptive, observational, national study using an online self-questionnaire, sent by email to French gynaecologists in October 2019 within 2 months.

Results: We obtained 110 analyzable responses from mainly surgeons (54%) and reproductive clinicians (19%) with a good experience (average 15 years of practice). Amongst these practitioners, 91% seemed aware of latest French recommendations on endometriosis issued in December 2017. The most commonly used surgical techniques for management of endometriomas were intra-peritoneal cystectomy (51%), vaporization by plasma energy (29%) and destruction by bipolar coagulation (8.5%). Preoperative AMH was systematically or often prescribed by 78% of the practitioners against 37.3% who did it postoperatively. Furthermore, 74% also considered and performed fertility preservation strategy to manage endometriosis. It was offered in situations of bilateral or recurrent endometrioma, but only 33% offered it in unilateral endometrioma cases. In the cases recorded, vitrification of mature oocytes appears to be the most common fertility preservation technique (used by 87% of the practitioners). Conclusion: We observed in our population of sensitized practitioners a good and adequate knowledge concerning endometriosis physiopathology and recommendations for its management, with good information delivery to women. Operating techniques are adapted although information and education concerning fertility preservation indications seem necessary. The place of multidisciplinary concertation meeting in endometriosis appears essential both for discussion of surgical indications and for fertility preservation possibilities. Creation of dedicated structures should be encouraged.

Introduction

According to French law, "any person whose medical care is likely to impair fertility, or whose fertility is likely to be prematurely impaired, may benefit from collection and conservation of gametes or germinal tissues, and may benefit from assisted reproductive technology for restoration of fertility". Initially developed to be performed prior to cancer treatment, fertility preservation (FP) has now been extended to benign indications: autoimmune diseases treated with gonadotoxic agents, risk of premature ovarian failure, etc… The most commonly used techniques are: vitrification of mature oocytes after controlled ovarian hyperstimulation and cryopreservation of ovarian tissue after oophorectomy by laparoscopy.

Endometriosis is a common benign gynecological condition affecting about one in ten women [START_REF] Viganò | Endometriosis: epidemiology and aetiological factors[END_REF]. It is often associated with infertility [START_REF] Ozkan | Endometriosis and infertility: epidemiology and evidencebased treatments[END_REF]. Though the cause-effect relationship remains controversial, the etiology of infertility may lay within the manifestation of endometriosis itself; tubal obstruction secondary to adhesions, chronic pelvic inflammation responsible for impairment of tubal ciliary action [START_REF] Lyons | Peritoneal fluid, endometriosis, and ciliary beat frequency in the human fallopian tube[END_REF], endometrial receptivity disorder, reduction in ovarian reserve [START_REF] Kitajima | Enhanced follicular recruitment and atresia in cortex derived from ovaries with endometriomas[END_REF]; and surgical treatment of endometriosis is furthermore likely to have a negative impact on the follicular capital. Affected patients are therefore potential candidates for fertility preservation.

The aim of our study was to evaluate the knowledge and physicians' practices of French gynaecologists concerning FP in women with endometriosis.

Materials and Methods

We carried out a descriptive, observational, national study. An evaluation by self- December 2017, were used as reference to evaluate the clinical practices [START_REF] Has/Cngof | Prise en charge de l'endométriose[END_REF]. The data was collected using Google Form and statistical analysis performed by Google Sheet. Qualitative variables were expressed as numbers and percentages, and quantitative variables as means ± standard deviation.

Results

We collected 110 analyzable responses. The socio-demographic characteristics of our respondents are presented in Table 1.

Clinical practice and training:

Almost all of our population of gynecologists (98%, n=108) had patients with endometriosis with an estimated number of consultations of 122 ± 161 [2-800] per year. Among those who answered our questionnaire, 91% (n=100) seemed aware of latest recommendations. We have highlighted that more than a third of the practitioners (37%, n=41) had never received specific training on FP in endometriosis.

Knowledge of endometriosis:

Global prevalence of endometriosis (10%) amongst women of reproduction age was correctly estimated by 70% (n=77) of our sampled practitioners. All (100%) considered endometriosis to be a factor of infertility and 91% (n=100) knew that it was not systematically pathological. Practitioners stated that diagnosis was mainly clinical (68%, n=75) and/or histological (70%, n=77).

Practices concerning fertility: Our findings revealed that 75% (n=83) reported that patients discussion about their fertility occurred "systematically" and 22% (n=24) described these to occur "often". In addition, 29% (n=32) of the practitioners also reported that women spontaneously and frequently discussed the impact and consideration for FP.

Surgical management:

Figure 1 shows the most commonly used surgical techniques for management of endometriosis cysts: 51% performed intra-peritoneal cystectomy by "stripping" or divergent traction, 29% vaporization by plasma energy and 8.5% destruction by bipolar coagulation.

Surgeons in our cohort operated about 57 ± 69 [0-300] patients with endometriosis every year. Amongst our cohort, 64% (n=38) systematically indicated an AFS (American Fertility Society) score in their operation report. For 47.5% (n=28), prescription of Anti-Mullerian Hormone (AMH) dosage in preoperative care was systematic whilst 30.5% (n=18) often prescribed it. Only 37.3% (n=22) prescribed it systematically or often in a postoperative setting: 70% prescribed AMH at 3 months and 22% at 6 months. A medical treatment in post-operative period to suppress ovarian activity was prescribed by 88% (n=52) of the surgeons, of which oestroprogestative contraception was the preferred option for 59% (n=35) of them.

Fertility preservation practices:

Practitioners worked in collaboration with a dedicated center for 90% (n=99) of them. Almost 75% (n=81) had already performed FP for endometriosis. The situations in which they offered it are presented in Table 2.

The main inclusion parameter for FP decision was age (98%, n=85). The most commonly performed technique was vitrification of mature oocytes in 87% (n=58).

Discussion

To the best of our knowledge, this is the first study about physicians' practice patterns in the diagnosis and management of endometriosis.

The results show that the physiopathology of endometriosis and recommendations for its management, including FP were well known in the population of 110 practitioners who participated in our study. They often communicate the management strategies and address patients' expectations. They seem to deliver clear information about the opportunity of FP in most cases (75%), facing a population particularly wellaware and sensitized.

Preferentially performed operative techniques for the management of endometriomas by our sub-population of surgeons were: intra-peritoneal cystectomy, which is the reference treatment [START_REF] Has/Cngof | Prise en charge de l'endométriose[END_REF] and vaporization by plasma energy, the latter being potentially less harmful for the ovarian reserve (OR) at the cost of a higher number of recurrences of endometriomas [START_REF] Dan | Laparoscopic ovarian cystectomy versus fenestration/coagulation or laser vaporization for the treatment of endometriomas: a meta-analysis of randomized controlled trials[END_REF][START_REF] Mircea | Fertility Outcomes After Ablation Using Plasma Energy Versus Cystectomy in Infertile Women With Ovarian Endometrioma: A Multicentric Comparative Study[END_REF][START_REF] Roman | Ovarian endometrioma ablation using plasma energy versus cystectomy: a step toward better preservation of the ovarian parenchyma in women wishing to conceive[END_REF]. Destruction by bipolar coagulation was still used, although it is no longer recommended [START_REF] Has/Cngof | Prise en charge de l'endométriose[END_REF].

An average of 57 patients per year was operated by each surgeon. The surgical level of expertise has previously been inversely correlated with the amount of healthy ovarian tissue removed during cystectomy [START_REF] Muzii | Histologic analysis of specimens from laparoscopic endometrioma excision performed by different surgeons: does the surgeon matter?[END_REF][START_REF] Yu | Laparoscopic ovarian cystectomy of endometriomas: surgeons' experience may affect ovarian reserve and live-born rate in infertile patients with in vitro fertilization-intracytoplasmic sperm injection[END_REF]. Therefore "expert surgeons" in regional reference center [START_REF] Golfier | The definition of Endometriosis Expert Centres[END_REF] may be preferred for operations of patients with endometriosis. A national working group is currently dedicated to the creation of such structures on French territory.

Staging of rAFS [START_REF]Revised American Society for Reproductive Medicine classification of endometriosis[END_REF] is not compulsory, and a third of our surgeons did not notify it in their operative report. Its use is nevertheless encouraged in order to facilitate exchanges between professionals. AFS score can be integrated into the EFI score (endometriosis fertility index) [START_REF] Adamson | Endometriosis fertility index: the new, validated endometriosis staging system[END_REF], in order to adapt the treatment.

The adverse effects of removal of endometriomas are increasingly clear and AMH pre and post-surgery is a useful marker in assessing the degree of damage to the ovary.

In our analysis, it emerged that an evaluation of the OR, reliably predicted by a preoperative AMH measurement, was prescribed by only half of our surgeons.

Preoperative measurements can detect any alteration of OR which may be attributable to the disease. Moreover, ovarian cystectomy presents a risk, adversely impacting the follicular capital by removal or destruction of the healthy parenchyma surrounding the cyst, resulting in a significant reduction in post-operative AMH [START_REF] Streuli | In women with endometriosis anti-Müllerian hormone levels are decreased only in those with previous endometrioma surgery[END_REF][START_REF] Raffi | The impact of excision of ovarian endometrioma on ovarian reserve: a systematic review and meta-analysis[END_REF][START_REF] Somigliana | Surgical excision of endometriomas and ovarian reserve: a systematic review on serum antimüllerian hormone level modifications[END_REF]. This risk seems to be higher in cases of endometriomas compared with other benign cysts [START_REF] Rustamov | Effect of salpingectomy, ovarian cystectomy and unilateral salpingo-oopherectomy on ovarian reserve[END_REF], as well as in patients undergoing iterative interventions [START_REF] Muzii | Second surgery for recurrent endometriomas is more harmful to healthy ovarian tissue and ovarian reserve than first surgery[END_REF].

Bilateral surgery in patients may also trigger earlier menopause (42 years) and a higher incidence of premature ovarian failure (PFO) [START_REF] Coccia | Ovarian surgery for bilateral endometriomas influences age at menopause[END_REF]. Finally, when facing an operation for severe endometriosis without planned cystectomy, knowledge of low preoperative AMH could guide the surgeon who will adapt his gesture in order to limit excessively large peri-ovarian dissections.

A limited number of physicians (37.3%) integrated AMH dosage in post-operative care. Although it is not part of the recommendations, it is interesting to assess the impact of the intervention on OR. There is a significant reduction in OR during the immediate post-cystectomy period [START_REF] Streuli | In women with endometriosis anti-Müllerian hormone levels are decreased only in those with previous endometrioma surgery[END_REF][START_REF] Raffi | The impact of excision of ovarian endometrioma on ovarian reserve: a systematic review and meta-analysis[END_REF][START_REF] Somigliana | Surgical excision of endometriomas and ovarian reserve: a systematic review on serum antimüllerian hormone level modifications[END_REF], which could be explained by the unintentional removal of ovarian cortex surrounding the cyst. Medium-term influence of cystectomy on OR might be attributed to vascular compromise resulting from excessive electrosurgical coagulation and postsurgical inflammation. A recovery of OR is possible with a variable capacity according to studies (Table 3). It can be explained by a favorable healing process with ovarian reperfusion [START_REF] Alborzi | The impact of laparoscopic cystectomy on ovarian reserve in patients with unilateral and bilateral endometriomas[END_REF] or a follicular cohort rearrangement [START_REF] Vignali | Surgical excision of ovarian endometriomas: Does it truly impair ovarian reserve? Long term anti-Müllerian hormone (AMH) changes after surgery[END_REF]. It is thus difficult to predict the proper time to offer this control but it could be advisable not to dose AMH too early after cystectomy, not before 3 months at least. The majority (70%) of our surgeons prescribed it at that time.

Our cohort (88%) applies the prescription of a medical treatment in post-operative period, such as oestroprogestative contraception, in accordance with the recommendations [START_REF] Has/Cngof | Prise en charge de l'endométriose[END_REF], in order to reduce the risk of recurrence of endometriomas.

Our sample of experienced physicians appropriately applies recommendations for fertility preservation [START_REF] Decanter | Endométriose et préservation de la fertilité, RPC Endométriose[END_REF] in situations in which a systematic proposal should be made, such as bilateral endometriomas (77%) or recurrent endometriomas (96%) but only a few do so in cases of unilateral endometrioma (33%). Currently, fertility preservation is recommended facing endometriomas measuring ≥ 5 cm. Therefore efforts to increase awareness needs to be implemented.

Gynecologists also include FP in the management of isolated deep endometriosis in patients with reduced AMH (87%). However, there is no real framework for this situation and it should be discussed on a case-by-case basis according to age, marital status, OR, progressive nature of the lesions and the likelihood of using IVF techniques [START_REF] Decanter | Endométriose et préservation de la fertilité, RPC Endométriose[END_REF].

Although FP is included in endometriosis management, there is no consensus in the international literature on strategies for FP in this context, and some indications may be very close to societal demands. In this sense, it seems essential to discuss the demands for FP during multidisciplinary, multisite, specialized consultation meetings dedicated to endometriosis with the presence of a reproductive physician.

Cryopreservation of mature oocytes, mostly performed (87%) by our cohort, is the reference technique [START_REF]Practice Committees of American Society for Reproductive Medicine, Society for Assisted Reproductive Technology. Mature oocyte cryopreservation: a guideline[END_REF], since it is reliable. Cryopreservation of embryos or ovarian tissue remains a possible alternative.

We still need to define the best timing to offer this FP program to our patients. A critical factor is age, correlated with oocyte quality. It remains to be the main parameter involved in the decisions made by our cohort of physicians (98%).

Importantly, age also correlates with the rate of embryonic aneuploidy wherein it appears to be increasing (>40%) at both extremes: before 23 and after 36 years old in general population [START_REF] Franasiak | The nature of aneuploidy with increasing age of the female partner: a review of 15,169 consecutive trophectoderm biopsies evaluated with comprehensive chromosomal screening[END_REF]. The clinical significance of aneuploidy is undoubted. Cobo et al [START_REF] Cobo | Oocyte vitrification as an efficient option for elective fertility preservation[END_REF] found that the probability of live birth for patients >36 years are halved compared to patients ≤35 years (19.9% vs 40.8%) when eight oocytes were used.

As such, evaluation of the benefit/risk ratio of this FP procedure should be extensively discussed beforehand. There is an inherent risk related to anesthesia, regardless of its mode during ovarian puncture but also a risk of post-puncture abscess that can reach 1.7% [START_REF] Benaglia | Endometrioma and oocyte retrieval-induced pelvic abscess: a clinical concern or an exceptional complication?[END_REF]. Encouragingly, the risk of a possible recurrence or increase in endometriosis-related pain by ovarian stimulation does not appear to be increased [START_REF] Coccia | Does controlled ovarian hyperstimulation in women with a history of endometriosis influence recurrence rate?[END_REF]. Also, the risk of ovarian hyperstimulation is normally ruled out by carrying out antagonist protocols, allowing for ovulation to be triggered by a GnRH agonist. Nevertheless, the patient must be informed about risks, as well as the challenging pathway of the procedure, the possibility that several stimulation cycles may be necessary to achieve satisfactory egg accumulation and the low chance of reusing vitrified oocytes (9% in Cobo's study [START_REF] Cobo | Oocyte vitrification as an efficient option for elective fertility preservation[END_REF]).

One limitation of this study is the very low participation rate. Indeed, among 7865 gynecologists listed in France, we obtained only 110 answers. Practitioners who answered the questionnaire were those who were interested in endometriosis and who were dealing with the pathology for a long time (57 patients operated every year and 15 years of practice). This may explain their good knowledge and their sensitivity towards FP. Due to this major selection bias, our results cannot be extrapolated to the overall population of gynecologists in France.

Conclusion

In this study, among physicians responding to the questionnaire, we underlined good knowledge concerning endometriosis physiopathology and the associated risk of impaired fertility. Surgeons used appropriate operating techniques to preserve patients' ovarian prognosis but information and education concerning FP indications seemed necessary.

The place of multidisciplinary consultation meeting in endometriosis appears essential both for discussion of surgical indications and for FP possibilities. Creation of dedicated structures should be encouraged.

Fertility preservation must be carried out "neither too early nor too late" in order to achieve a significant chance of pregnancy for the patient later on. Nevertheless, it remains difficult to predict the risk of infertility in these patients with endometriosis, with sometimes difficult situations close to societal FP. Finally, the possible cost to society is also an important parameter to take into account when setting up such a program, especially regarding the high frequency of endometriosis in general population.

Figure 1 :

 1 Figure 1: Most commonly used surgical technique for the management of

Table 1 :

 1 Socio-demographic characteristics of gynecologists answering questionnaire

	Characteristics	N=110	%
		n
	Sex
	-male	48	44
	-female	62	56
	Mean age (years)	45.4 ± 11 [28-67]
	Main activity
	-surgical	59	54
	-reproductive medicine	21	19
	-medical gynecology	22	20
	-obstetrics	8	7
	Practice
	-public hospital	61	55
	-private clinic	49	45
	Average number of years of practice
	(years)

Acknowledgements

None

Funding sources

This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.