Alex Reichenbach

Romana Stark

Mathieu Mequinion

Sarah H Lockie

Moyra B Lemus

Randall L Mynatt

Serge Luquet

Zane B Andrews
email: zane.andrews@monash.edu

Carnitine acetyltransferase (Crat) in hunger-sensing AgRP neurons permits adaptation to calorie restriction

Keywords: rebound weight gain, body composition, RER, feeding behavior, metabolic flexibility

Hunger-sensing agouti-related peptide (AgRP) neurons ensure survival by adapting metabolism and behavior to low caloric environments. This adaption is accomplished by consolidating food intake, suppressing energy expenditure, and maximizing fat storage (nutrient partitioning) for energy preservation. The intracellular mechanisms responsible are unknown. Here we report that AgRP carnitine acetyltransferase (Crat) knockout (KO) mice exhibited increased fatty acid utilization and greater fat loss after 9 d of calorie restriction (CR). No differences were seen in mice with ad libitum food intake. Eleven days ad libitum feeding after CR resulted in greater food intake, rebound weight gain, and adiposity in AgRP Crat KO mice compared with wild-type controls, as KO mice act to restore pre-CR fat mass. Collectively, this study highlights the importance of Crat in AgRP neurons to regulate nutrient partitioning and fat mass during chronically reduced caloric intake. The increased food intake, body weight gain, and adiposity in KO mice after CR also highlights the detrimental and persistent metabolic consequence of impaired substrate utilization associated with CR. This finding may have significant implications for postdieting weight management in patients with metabolic diseases.-Reichenbach,

The neural processing of the peripheral energy state is indispensable for energy homeostasis, and disturbances in this process are linked to the emergence of metabolic disorders, including obesity and type 2 diabetes [START_REF] O'rahilly | Human obesity: a heritable neurobehavioral disorder that is highly sensitive to environmental conditions[END_REF]. Neuronal populations within the hypothalamus are all implicated in the control of energy homeostasis; however, agoutirelated peptide (AgRP) neurons in the ARC are arguably the most fundamentally important population to maintain body energy homeostasis.

AgRP neurons coexpress neuropeptide Y (NPY) and GABA, and together with AgRP, all 3 play an important role in regulating food intake and energy homeostasis [START_REF] Krashes | Rapid versus delayed stimulation of feeding by the endogenously released AgRP neuron mediators GABA, NPY, and AgRP[END_REF]. The ablation of AgRP neurons during adulthood, which avoids development compensation, causes a dramatic loss of body weight and food intake to the point of starvation [START_REF] Luquet | NPY/ AgRP neurons are essential for feeding in adult mice but can be ablated in neonates[END_REF]. Moreover, AgRP ablation during the early postnatal period permits survival but results in increased peripheral lipid utilization and lipolysis [START_REF] Joly-Amado | Hypothalamic AgRP-neurons control peripheral substrate utilization and nutrient partitioning[END_REF]. In particular, AgRP/NPY neurons conserve energy by suppressing thermogenesis in white adipose tissue [START_REF] Dodd | A hypothalamic phosphatase switch coordinates energy expenditure with feeding[END_REF] and brown adipose tissue (BAT) [START_REF] Ruan | O-GlcNAc transferase enables AgRP neurons to suppress browning of white fat[END_REF] and reducing energy expenditure [START_REF] Shi | Arcuate NPY controls sympathetic output and BAT function via a relay of tyrosine hydroxylase neurons in the PVN[END_REF] via the CNS and the sympathetic nervous system. These studies highlight the fundamental actions of AgRP neurons to signal hunger, as well as to preferentially store lipids, particularly during energy deficits to promote survival.

AgRP itself is an endogenous antagonist at the melanocortin 4 receptor (MC4R), and central AgRP antagonism of the MC4R increases BAT temperature and glucose oxidation as indicated by the respiratory exchange ratio (RER), whereas stimulating MC receptor signaling increases lipolysis [START_REF] Nogueiras | The central melanocortin system directly controls peripheral lipid metabolism[END_REF][START_REF] Brito | Differential activation of the sympathetic innervation of adipose tissues by melanocortin receptor stimulation[END_REF]. Moreover, NPY knockout (KO) mice have increased fat loss caused by lipolysis during calorie restriction (CR) [START_REF] Park | Neuropeptide Y resists excess loss of fat by lipolysis in calorie-restricted mice: a trait potential for the lifeextending effect of calorie restriction[END_REF]. Collectively, these results show that both AgRP and NPY peptides conserve energy by regulating nutrient partitioning and utilization in peripheral tissues.

AgRP neurons are most active in response to a metabolic signature of negative energy balance that includes ABBREVIATIONS: AgRP, agouti-related peptide; BAT, brown adipose tissue; CR, calorie restriction; Crat, carnitine acetyltransferase; FEO, foodentrainable oscillator; GFP, green fluorescent protein; KO, knockout; MC4R, melanocortin 4 receptor; NEFA, nonesterified fatty acid; NPY, neuropeptide Y; RER, respiratory exchange ratio; WT, wild type increased plasma ghrelin, free fatty acids, low glucose, low insulin, and low leptin [START_REF] Lockie | The hormonal signature of energy deficit: increasing the value of food reward[END_REF][START_REF] Mandelblat-Cerf | Arcuate hypothalamic AgRP and putative POMC neurons show opposite changes in spiking across multiple timescales[END_REF][START_REF] Joly-Amado | The hypothalamic arcuate nucleus and the control of peripheral substrates[END_REF][START_REF] Andermann | Toward a wiring diagram understanding of appetite control[END_REF], and they communicate with peripheral tissues through the sympathetic nervous system [START_REF] Dodd | A hypothalamic phosphatase switch coordinates energy expenditure with feeding[END_REF][START_REF] Ruan | O-GlcNAc transferase enables AgRP neurons to suppress browning of white fat[END_REF][START_REF] Nogueiras | Regulation of lipid metabolism by energy availability: a role for the central nervous system[END_REF][START_REF] Shi | Arcuate neuropeptide Y inhibits sympathetic nerve activity via multiple neuropathways[END_REF]. We recently discovered that carnitine acetyltransferase (Crat) in AgRP neurons is required to control food intake, feeding behavior, glucose clearance, lipid utilization, and AgRP protein expression during food withdrawal and refeeding. In particular, Crat in AgRP neurons regulated metabolic flexibility and facilitated a rapid switch from fatty acid to glucose oxidation upon nutrient replenishment after a single time of food withdrawal [START_REF] Reichenbach | AgRP neurons require carnitine acetyltransferase to regulate metabolic flexibility and peripheral nutrient partitioning[END_REF], indicating appropriate peripheral nutrient partitioning. These observations are consistent with studies deleting Crat in skeletal muscle, which increases fatty acid utilization and reduces metabolic flexibility, or the ability to switch metabolic substrates, during the transition from the unfed to fed state [START_REF] Muoio | Muscle-specific deletion of carnitine acetyltransferase compromises glucose tolerance and metabolic flexibility[END_REF]. This mechanism ensures that as glucose from food becomes available for oxidation, such as during refeeding, fatty acid utilization is acutely and rapidly switched off to conserve energy reserves.

Although AgRP neurons adapt to low-energy states by reducing thermogenesis and energy expenditure (4), the mechanisms responsible remain largely unknown. Research suggests that mitochondrial fission/fusion dynamics within AgRP may play a role [START_REF] Ruan | O-GlcNAc transferase enables AgRP neurons to suppress browning of white fat[END_REF], which indicates a broader role for mitochondrial metabolism within AgRP neurons as a means to detect changes in metabolic states. Indeed, our previous study showed that Crat in AgRP neurons regulates metabolic flexibility and peripheral nutrient partitioning in response to a single time of food withdrawal [START_REF] Reichenbach | AgRP neurons require carnitine acetyltransferase to regulate metabolic flexibility and peripheral nutrient partitioning[END_REF]; however, the response to chronic CR remains unknown. Because CR involves multiple transitions from an unfed to fed state for a chronic period of time, we hypothesized that Crat in AgRP neurons would be critical for the adaptation to chronic energy deficit during CR. Moreover, we predict that increased fatty acid utilization in a low caloric environment would increase fat loss in AgRP Crat KO mice. To test this hypothesis, we calorierestricted mice with an AgRP-specific deletion of Crat and analyzed changes in feeding behavior, energy expenditure, and body composition related to a low caloric environment.

MATERIALS AND METHODS

Animals

All experiments were conducted in compliance with the Monash University Animal Ethics Committee guidelines. AgRP-ires-cre mice were obtained from The Jackson Laboratory (Bar Harbor, ME, USA) AgRP tm1(cre)Low/J (stock no. 012899) and bred with NPY green fluorescent protein (GFP) mice (B6.FVB-Tg[Npy-hrGFP]1Lowl/J; stock number 006417; The Jackson Laboratory). AgRP-ires-cre::NPY GFP mice were then crossed with Crat lox/lox mice donated by Randall Mynatt (Pennington Biomedical Research Center, Baton Rouge, LA, USA) to delete Crat from AgRP neurons (AgRP crat KO). Before starting CR experiments, mice were kept at standard laboratory conditions with free access to food (chow diet, 8720610; Barastoc Stockfeeds, Pakenham, VIC, Australia) and water at 23°C in a 12-h light/dark cycle and were group-housed to prevent isolation stress.

Calorie restriction and ad libitum refeeding experiments

Male mice, 8-12 wk of age, were single-housed in specialized feeding cages (BioDaq; Research Diets, New Brunswick, NJ, USA), fitted with computer-controlled automatic gates to prevent access to feeding hoppers during CR experiments. This method also allows stress-free intervention without human interaction. After 12 d ad libitum feeding (inclusive acclimation period), mice were subjected to 60% CR of their average food intake during ad libitum conditions for 9 d. Mice gained access to food hoppers 1 h before onset of the dark phase, and individual gates closed after mice consumed 1.8 g of chow. Body weight and blood glucose levels (Accu-Chek Active; Roche Diagnostics, Tokyo, Japan) of mice were measured daily 1-2 h before refeeding. (Fig. 1A presents details of the experimental protocol.) After 9 d CR, mice were allowed access to ad libitum chow for 11 d to assess changes in feeding behavior.

Telemetry surgery

Mice were anesthetized with isoflurane, and anesthesia was maintained by constant nasal delivery of 2.5% isoflurane. Each animal received 50 ml Metacam (0.25 mg/ml meloxicam; Boehringer Ingelheim, Ingelheim am Rhein, Germany) before surgery to minimize postsurgery pain. Telemeter probes (Starr Life Science, Holliston, MA, USA) were implanted into the interscapular BAT, and mice were monitored for 48 h. After a 1 wk recovery period, temperature and activity were recorded every minute with Vital View 2.0 (Starr Life Science) and data averaged over 15 min.

Body composition

Body composition of mice was assessed through the EchoMRI-100H Body Composition Analyzer (EchoMRI LLC, Houston, TX, USA) by creating the average of 3 repeated scans. Body fat loss during CR was calculated as the difference of baseline (ad libitum fed) percent body fat and after 9 d CR. Loss and regain of body weight and lean and fat mass after 1, 4, 7, and 11 d ad libitum refeeding were calculated as percent change from baseline.

Metabolic phenotyping

Energy expenditure and respiratory quotient were measured with Promethion metabolic cages (Sable Systems International, North Las Vegas, NV, USA) from d 7 to 9 of CR and from d 1 to 11 during ad libitum refeeding. During calorie-restricted conditions, mice were subjected to the same protocol described earlier (access to 1.8 g chow commencing 1 h before onset of the dark phase).

Analysis of blood chemistry

Plasma insulin concentrations were determined through an inhouse ELISA assay. Plasma concentrations of acylated and desacylated ghrelin (Mitsubishi Chemical Medience, Tokyo, Japan), glucagon (Yanaihara Institute, Fujinomiya, Japan), and corticosterone (Abnova, Heidelberg, Germany) were measured with ELISA according to manufacturer's instructions.

Nonesterified fatty acid (NEFA) concentration, ketone bodies, and triglycerides levels in plasma were measured by using a NEFA C Assay Kit (Wako Pure Chemicals, Osaka, Japan), b-Hydroxybutyrate Colorimetric Assay Kit (Cayman Chemicals, Ann Arbor, MI, USA), or a Triglyceride Assay Kit (Roche/ Hitachi; Roche Diagnostics), respectively.

Tissue collection

After 9 d CR or 11 d ad libitum refeeding, animals were deeply anesthetized with isoflurane, and blood was collected via cardiac puncture, and hypothalamus, gastrocnemius and soleus muscle, and liver samples were collected. Plasma collected from blood samples was stored at 280°C, and samples collected were snap frozen in liquid nitrogen.

RT-PCR

Total RNA was extracted from tissue with Qiazol (Qiazol Sciences, Germantown, MD, USA) as previously described [START_REF] Reichenbach | AgRP neurons require carnitine acetyltransferase to regulate metabolic flexibility and peripheral nutrient partitioning[END_REF].

Primers were obtained from GeneWorks (Thebarton, SA, Australia): phosphoenolpyruvate carboxykinase 1 (cytosol) (59-TGCCCAAGGCAACTTAAGGG-39, 59-CAGTAAACAC-CCCCATCGCT-39); glucose-6-phosphatase, catalytic (59-AGTCTTGTCAGGCATTGCTGT-39, 59-AAAGTCCACAG-GAGGTCCAC-39); glycogen phosphorylase, liver (59-AA-GAAGGGGTATGAGGCCAAA-39, 59-GACACTTGACATAG-GCTTCGT-39); glycogen synthase 2, liver (59-AATGTGAG-CCCACCAACGAT-39, 59-CTTCCAAAATGCACCTGGCA-39); glycerol kinase (59-GCAACCAGAGGGAAACCACA-39, 59-TAGGTCAAGCCACACCACG-39); adipose triglyceride lipase (59-AGAGCCCATGGTCCTCCGA-39, 59-AGCAAAGG-GTTGGGTTGGTT-39); diacylglycerol acyltransferase (59-TAGAAGAGGACGAGGTGCGA-39, 59-GTCTTTGTCCC-GGGTATGGG-39); glycerol-3-phosphate acyltransferase, mitochondrial (59-CCAGTGAGGACTGGGTTGAC-39, 59-CTCTGTGGCGTGCAGGAATA-39); fatty acid synthase (59-TGGGTGTGGAAGTTCGTCAG-39, 59-CTGTCGTGTC-AGTAGCCGAG-39); carnitine palmitoyltransferase 1a Figure 1. Crat in AgRP neurons is required for adaption of feeding behavior in response to CR. Experimental timeline of CR in BioDaq feeding cages. A) Three cohorts were subjected to the same feeding schedule; BAT temperature and locomotor activity were measured in cohort 1, body composition was assessed in cohort 2, and metabolic phenotype was analyzed in cohort 3. B, C) There were no differences in body weight development (B) or blood glucose development (C ) between genotypes. D-H) Body composition examination was performed before and after the experiment, and revealed greater loss of relative fat mass (D) and less loss of lean mass (E ) in KO mice during CR. Food intake (F ), cumulative food intake during CR (G), and food intake under ad libitum fed and CR conditions shown as a heat map (H ). Each voxel represents food intake of 1 h, each column represents a day, the black bar on the left-hand side indicates dark phase, the green bar on the top of the heat map indicates ad libitum access to food, and the red bar indicates restricted access to 1.8 g/24 h starting at 11 zeitgeber time (ZT), indicated by the horizontal dotted line. I, J) Time spent in bouts (I ) and bouts (J ) averaged over 9 d of CR quantify the deficits in feeding behavior. All data are expressed as means 6 SEM; n = 9-11. P , 0.05 (ANOVA with Sidak post hoc analysis, 2-way repeated measures, where appropriate).

(59-ATCGCTTTGGGAGTCCACAT-39, 59-CCATCGTTAAG-GCACTGGGT-39); citrate synthase (59-TGTAGCTCTCTCC-CTTCGGT-39, 59-ACGAGGCAGGATGAGTTCTTG-39); and 18S ribosomal RNA (59-TTCCGATAACGAACGAGACTCT-39, 59-TGGCTGAACGCCACTTGTC-39). Real-time quantitative PCR tests were performed by using the Real Plex4 Mastercycler (Eppendorf, Hamburg, Germany).

Liver glycogen and triglycerides

Liver glycogen and triglyceride concentrations were measured as previously described [START_REF] Reichenbach | AgRP neurons require carnitine acetyltransferase to regulate metabolic flexibility and peripheral nutrient partitioning[END_REF] with a glucose oxidase assay kit (Mil-liporeSigma, Burlington, MA, USA) or a triglyceride assay kit (Roche/Hitachi; Roche Diagnostics), respectively.

Statistical analysis

Statistical analyses were performed by using GraphPad Prism (La Jolla, CA, USA) for MacOS X (v.7.0b) All data are represented as means 6 SEM. Two-way ANOVAs with post hoc tests were used to determine statistical significance between treatment and genotype. A 2-tailed, unpaired Student's t test was used when comparing genotype only. Linear regression analyses were performed to establish relationships between BAT temperature or locomotor activity and time. Correlation analysis was performed to determine relationships between 2 continuous variables. Values of P , 0.05 were considered statistically significant.

RESULTS

Crat in AgRP neurons is required for adaption of feeding behavior in response to CR Our previous study confirmed Crat deletion in AgRP neurons and showed that Crat in AgRP neurons is required to regulate food intake, feeding microstructure, fatty acid metabolism, and energy substrate utilization after food deprivation. To determine if this scenario predisposes to metabolic impairments in models of chronic energy deficits, we exposed AgRP Crat wild-type (WT) and KO mice to a CR schedule (Fig. 1A). Both AgRP Crat WT and KO mice were allowed to eat 1.8 g of chow, which was assessed as ;60% of ad libitum food intake during a baseline period.

After mice consumed 1.8 g of chow, the computercontrolled gates closed and prevented access to the food hopper for the remainder of the 24-h period. This approach allowed CR without human intervention and additional stressors unrelated to the experimental paradigm. Interestingly, CR for 9 d did not produce a difference in body weight or blood glucose level (Fig. 1B,C); however, body composition analysis before and after caloric restriction in the same mice revealed that AgRP Crat KO mice lost a significantly greater percentage of body fat (Fig. 1D) while preserving lean mass (Fig. 1E). There were no differences in body weight and body composition in AgRP Crat WT or KO mice on an ad libitum diet during the entire experiment (Supplemental Fig. 1).

Although all mice were allowed only to eat 1.8 g of chow, AgRP Crat KO mice displayed significant differences in meal feeding microstructure, consistent with results from food withdrawal and ad libitum feeding behavior [START_REF] Reichenbach | AgRP neurons require carnitine acetyltransferase to regulate metabolic flexibility and peripheral nutrient partitioning[END_REF]. All mice started to consume chow within a few minutes after gaining access; however, AgRP Crat KO mice took significantly longer (;8 vs. 4 h in WT mice) to consume most of the 1.8 g ration (Fig. 1F-H) and had fewer feeding bouts but spent more time in a feeding bout (Fig. 1I,J). Overall, WT mice adapted to the feeding regimen and changed feeding behavior accordingly by progressively consolidating their food intake into a smaller feeding window (Fig. 1H), whereas KO mice did not by the end of the 9 d CR.

Crat in AgRP neurons is needed to regulate nutrient partitioning and energy expenditure in response to CR Implanting temperature probes into the interscapular BAT of mice allowed us to record real-time BAT temperature changes as well as locomotor activity in response to CR. KO mice had a slightly lower average BAT temperature during the dark phase under ad libitum conditions (Fig. 2A,B) and failed to reduce average BAT temperature during the dark phase in response to CR (Fig. 2A,C). A similar effect was observed for locomotor activity in which WT mice exhibited greater locomotor activity during ad libitum conditions (Fig. 2D,E), with a 40% reduction from the first to last day of CR. KO mice showed a nonsignificant reduction in their locomotor activity by ~20% during this period (Fig. 2D,F), collectively suggesting greater energy conservation in WT mice during CR. To estimate energy expenditure under these advanced CR conditions, we placed mice in metabolic cages on d 7-9 of the CR protocol. The KO mice were found to have increased average energy expenditure during this period (Fig. 2G,H), matching the discrepancy observed in BAT temperature (Fig. 2C), and lower RER (Fig. 2I,J) compared with WT litter mates. The lower RER indicates that KO mice are preferentially utilizing fatty acids as an energy substrate, which accounts for the greater loss of fat mass during CR. This increase in fatty acid utilization during CR is consistent with results during acute food deprivation [START_REF] Reichenbach | AgRP neurons require carnitine acetyltransferase to regulate metabolic flexibility and peripheral nutrient partitioning[END_REF].

Crat in AgRP neurons affects hepatic function in response to CR

In previous food deprivation studies, we observed no differences measurable in blood glucose in WT and KO mice; however, there were significant adaptive changes in the liver of KO mice to support ongoing glucose production [START_REF] Reichenbach | AgRP neurons require carnitine acetyltransferase to regulate metabolic flexibility and peripheral nutrient partitioning[END_REF]. These changes included reduced liver glycogen and elevated liver triglyceride levels, as well as changes in gene expression and the substrate contributing to gluconeogenesis. To test if similar changes occur in the calorie-restricted state, liver glycogen and triglyceride levels were measured at the end of the 9 d restriction protocol. Liver analysis revealed lower glycogen (Fig. 3A) but equal triglyceride (Fig. 3B) concentrations in AgRP Crat KO mice relative to WT mice. Liver gene expression revealed an increase in gene transcripts involved in gluconeogenesis (Pck1, G6pc) and glycogen breakdown (Glycogen phosphorylase), as well as carnitine B, C) Average BAT temperature during the light and dark phases of ad libitum fed animals (B) and regression analysis of average BAT temperature during the dark phase of 9 consecutive days CR (C ). E, F) Total locomotor activity during the light and dark phases of ad libitum fed animals (E ) and regression analysis of total dark phase locomotor activity under CR conditions (F ). G-J) Average 24-h profile of energy expenditure (G) and respiratory quotient (RQ) (I ) of CR d 7-9, and light and dark phase average of energy expenditure (H ) and RQ (J ) of this period. All data are expressed as means 6 SEM; n = 5-7. P , 0.05 (ANOVA with Sidak post hoc analysis, 2-way repeated measures, where appropriate).

CRAT IN AGRP NEURONS DURING CALORIE RESTRICTION 5 palmitoyltransferase 1a (Cpt1a) and citrate synthase (Cs), suggesting increased liver fatty acid oxidation and mitochondrial biogenesis in KO mice relative to WT mice (Fig. 3C). Interestingly, we observed an increase in Fasn, suggesting a possible compensatory attempt to increase de novo lipid synthesis due to low liver triglyceride levels after 9 d of CR, as described elsewhere [START_REF] Bruss | Calorie restriction increases fatty acid synthesis and whole body fat oxidation rates[END_REF]. Plasma NEFAs (Fig. 3D) were significantly higher in AgRP Crat KO mice, reflecting greater lipolytic rates; plasma triglyceride levels and ketone bodies were not significantly increased, however (Fig. 3E,F). No differences were observed in corticosterone, acyl ghrelin, insulin, or glucagon between AgRP Crat WT and KO mice at the end of CR (Fig. 3G-J). However, leptin levels were significantly lower in calorie-restricted KO mice (Fig. 3K), reflecting reduced adiposity.

Crat in AgRP neurons influences rebound weight gain, food intake, and adiposity after cessation of CR At the end of CR, body weight and blood glucose levels did not differ between genotypes; however, due to changes in nutrient partitioning, KO mice had a significantly greater fat loss and less lean muscle loss. To determine if this change in body composition affected rebound weight gain, food intake, or adiposity, we observed mice for 11 d after the cessation of CR.

Compared with pre-CR body weight, WT and KO mice rapidly recovered body weight, reaching their baseline weight after 4 d refeeding, and continued to moderately gain weight (WT: 105%; KO: 109%) (Fig. 4A). However, weight gain after CR significantly increased in KO mice compared with body weight at the end of CR (Fig. 4B). Intriguingly, after the cessation of CR, both WT mice and KO mice dramatically increased their 24 h food intake to ;165 and 150% of baseline ad libitum food intake, respectively. With time, WT mice reduced their daily food intake to below baseline levels after 7 d ad libitum refeeding; however, KO mice continued to consume more compared with ad libitum food intake, resulting in significantly greater chow consumption at the end of the experimental period (Fig. 4C,D).

Body composition analysis with ECHO MRI dissociated weight gain in lean mass from that in fat mass (Fig. 4E). This analysis showed that the increase in body weight the first day after cessation of CR is due to complete restoration of pre-CR lean mass. Although WT mice had slightly reduced lean mass thereafter, KO mice maintained the increased lean mass to the end of the experiment (Fig. 4F). The restoration of fat mass after CR takes longer, with WT mice reaching pre-CR adiposity between 4 and 7 d ad libitum refeeding. Despite gaining more absolute fat mass (Fig. 4H), KO mice did not reach their pre-CR adiposity level until after 11 d ad libitum refeeding (Fig. 4G), due to the greater fat loss during CR (Fig. 4G, compared with Fig. 1D). Dulloo et al. [START_REF] Dulloo | How dieting makes the lean fatter: from a perspective of body composition autoregulation through adipostats and proteinstats awaiting discovery[END_REF] suggest that the interplay between mechanisms regulating fat and protein mass, including the ratio of secreted adipokines and myokines, may regulate body composition and determine food intake to regain fat and lean mass. In our studies, KO mice gained more relative fat and lean mass compared with WT controls and also consumed more food. Furthermore, the significant inverse correlation between the relative change in fat mass and change in food intake (Fig. 4I,J) suggests that elevated food intake in KO mice in the post-CR period is to restore adiposity levels to pre-CR levels and not body weight per se (Fig. 4K,L). This concept is in line with leptin from adipose tissue as an endocrine signal that communicates long-term energy availability to the brain [START_REF] Stern | Adiponectin, leptin, and fatty acids in the maintenance of metabolic homeostasis through adipose tissue crosstalk[END_REF][START_REF] Rosen | What we talk about when we talk about fat[END_REF].

We therefore analyzed circulating leptin from plasma of refed mice (Fig. 5A) and found no difference between genotypes for leptin levels or for the correlation between circulating leptin levels and fat mass (Fig. 5D,E). Measuring insulin (Fig. 5B) and NEFA (Fig. 5C) from the same animals also revealed no differences between genotypes. Metabolic analysis during ad libitum refeeding found that there are no genotype differences in energy expenditure (Fig. 5G). Furthermore, KO mice have lower RER compared with WT controls (Fig. 5F,H), showing a persistent increase in fatty acid utilization 11 d post-CR. Despite this persistent increase in lipid utilization, KO mice consume more food and gain fat mass compared with WT mice to restore pre-CR adiposity levels.

DISCUSSION

The metabolic flexibility to acutely switch fatty acid to glucose utilization during refeeding represents an important mechanism referred to as "nutrient partitioning" that helps store energy-rich fatty acids and maximizes energy conservation until required. Our previous studies showed that Crat in AgRP neurons is important to regulate metabolic flexibility and peripheral nutrient partitioning [START_REF] Reichenbach | AgRP neurons require carnitine acetyltransferase to regulate metabolic flexibility and peripheral nutrient partitioning[END_REF].

Here we show that Crat-mediated metabolic signaling in AgRP neurons regulates metabolic adaptation to a chronic state of low caloric intake (60% CR) by regulating meal feeding structure, BAT thermogenesis, peripheral energy expenditure, and energy substrate utilization.

Although Crat deletion from AgRP neurons does not affect body weight loss or blood glucose level during CR, it causes a greater loss of body fat. Intriguingly, rebound weight gain, lean and fat mass, and food intake all increased, and fatty acid utilization remained high after the cessation of CR in KO mice compared with WT controls. These studies suggest that Crat in AgRP neurons regulates peripheral nutrient partitioning to preserve lipid stores and suppress energy expenditure during the chronic nutrient shortfall associated with CR. It is important to note that we observed no differences in body weight, lean mass, or fat mass in AgRP Crat WT and KO mice on a normal ad libitum diet, indicating that the observed phenotype is not simply due to the deletion of Crat from AgRP neurons. The phenotype in KO mice only manifests once the mice are subjected to CR, at a time when AgRP neurons become more active to restore energy homeostasis.

These studies also highlight that chronic impairment in nutrient partitioning during CR can affect rebound weight gain, rebound food intake, and rebound adiposity. Such an observation may have implications for rebound weight gain after diet-restricted weight loss interventions in humans. This finding is especially important considering metabolic diseases such as diabetes, nonalcoholic fatty liver disease, and dyslipidemia can develop due to inappropriate nutrient "fate" without significant changes in body weight [START_REF] Darabian | Significance of epicardial and intrathoracic adipose tissue volume among type 1 diabetes patients in the DCCT/EDIC: a pilot study[END_REF][START_REF] Padwal | Relationship among body fat percentage, body mass index, and allcause mortality: a cohort study[END_REF][START_REF] Gastaldelli | Insulin resistance and reduced metabolic flexibility: cause or consequence of NAFLD?[END_REF][START_REF] Goodpaster | Metabolic flexibility in health and disease[END_REF].

CRAT IN AGRP NEURONS DURING CALORIE RESTRICTION

In terms of meal feeding structure, WT mice adapted over the course of the CR protocol to quickly consume food, which was always introduced at the same time of the day during CR, an effect that is supported by previous studies [START_REF] Acosta-Rodriguez | Mice under caloric restriction selfimpose a temporal restriction of food intake as revealed by an automated feeder system[END_REF]. The finding that AgRP Crat KO mice did not show the same temporal adaptation over the course of CR implies that Crat in AgRP neurons is crucial for assessment of food availability and may be involved in a foodentrainable oscillator (FEO) [START_REF] Carneiro | The food-entrainable oscillator: a network of interconnected brain structures entrained by humoral signals?[END_REF]. Tan et al. [START_REF] Tan | Ablation of AgRP neurons impairs adaption to restricted feeding[END_REF] showed that ablating AgRP neurons delays food intake under a daytime restricted feeding regimen, as well as abolishes the food-anticipatory activity, an important feature of the FEO. In support of this theory, KO mice did not exhibit a similar temporal adaptation in BAT temperature, locomotor activity, or energy expenditure, all of which are important functions controlled by AgRP neurons [START_REF] Ruan | O-GlcNAc transferase enables AgRP neurons to suppress browning of white fat[END_REF][START_REF] Shi | Arcuate NPY controls sympathetic output and BAT function via a relay of tyrosine hydroxylase neurons in the PVN[END_REF][START_REF] Brito | Differential activation of the sympathetic innervation of adipose tissues by melanocortin receptor stimulation[END_REF]. Given that Crat in AgRP neurons is required for metabolic flexibility and peripheral nutrient partitioning, it is tempting to speculate that the metabolic processing of incoming nutrients via Crat is a key component of the FEO. There is evidence to support this claim because feeding time influences metabolism [START_REF] Shamsi | Metabolic consequences of timed feeding in mice[END_REF][START_REF] Zwighaft | Circadian clock control by polyamine levels through a mechanism that declines with age[END_REF][START_REF] Mauvoisin | Circadian and feeding rhythms orchestrate the diurnal liver acetylome[END_REF] and circadian cycles influence mitochondrial rate-limiting enzymes and nutrient utilization [START_REF] Neufeld-Cohen | The FASEB Journal x www.fasebj.org sebj.org by[END_REF]. In a proteomic screen of AgRP neurons from WT and KO mice, we observed numerous differences in mitochondrial enzymes, NAD+-regulating enzymes, and differences in protein acetylation [START_REF] Reichenbach | AgRP neurons require carnitine acetyltransferase to regulate metabolic flexibility and peripheral nutrient partitioning[END_REF], all of which affect NAD+ bioavailability and modulate mitochondrial oxidative function across cycles of food deprivation and feeding [START_REF] Peek | Circadian clock NAD+ cycle drives mitochondrial oxidative metabolism in mice[END_REF]. Collectively, these results suggest that Crat in AgRP neurons may be vital for the accurate assessment of peripheral nutrient availability and subsequent nutrient partitioning, which enables behavioral and metabolic adaptation to chronic low caloric environments to ensure and maximize energy conservation.

In previous experiments, we showed that AgRP Crat KO mice exhibit a shift toward increased fatty acid utilization and an attenuated switch to glucose oxidation with nutrient replenishment after food withdrawal [START_REF] Reichenbach | AgRP neurons require carnitine acetyltransferase to regulate metabolic flexibility and peripheral nutrient partitioning[END_REF]. Our observations herein show that prolonged preferential fatty acid utilization and increased lipolysis, as indicated by lower RER and increased NEFAs, respectively, during chronic low caloric conditions result in increased body fat loss. This shift toward fatty acid utilization might be facilitated by reduced AgRP-mediated antagonism of MC4R, resulting in augmented MC4R signaling and increased lipolysis [START_REF] Nogueiras | The central melanocortin system directly controls peripheral lipid metabolism[END_REF][START_REF] Brito | Differential activation of the sympathetic innervation of adipose tissues by melanocortin receptor stimulation[END_REF]. Nevertheless, our results are consistent with the ablation of AgRP or NPY neurons on substrate utilization and nutrient partitioning [START_REF] Joly-Amado | Hypothalamic AgRP-neurons control peripheral substrate utilization and nutrient partitioning[END_REF][START_REF] Park | Neuropeptide Y resists excess loss of fat by lipolysis in calorie-restricted mice: a trait potential for the lifeextending effect of calorie restriction[END_REF].

Consistent with our previous report [START_REF] Reichenbach | AgRP neurons require carnitine acetyltransferase to regulate metabolic flexibility and peripheral nutrient partitioning[END_REF], we observed a hepatic mRNA profile with increased gluconeogenic and glycogenolytic genes, as well as genes suggesting increased liver fatty acid oxidation and mitochondrial biogenesis in KO mice to support gluconeogenesis [START_REF] Jackowski | Deregulated coenzyme A, loss of metabolic flexibility and diabetes[END_REF]. The differences in hepatic gene profile are supported by a lower glycogen concentration in KO mice. Furthermore, we observed an increase in Fasn that suggests an increase in de novo lipid synthesis. Indeed, it has been reported that de novo lipid synthesis is elevated to meet the lipid demand during CR [START_REF] Bruss | Calorie restriction increases fatty acid synthesis and whole body fat oxidation rates[END_REF].

Although the integrated neural circuit responsible for the effect of CR on lipolysis and fat utilization remains unknown, it presumably involves impaired sympathetic nervous system-mediated communication with peripheral tissues as seen in previous studies on AgRP function [START_REF] Ruan | O-GlcNAc transferase enables AgRP neurons to suppress browning of white fat[END_REF][START_REF] Shi | Arcuate neuropeptide Y inhibits sympathetic nerve activity via multiple neuropathways[END_REF][START_REF] Reichenbach | AgRP neurons require carnitine acetyltransferase to regulate metabolic flexibility and peripheral nutrient partitioning[END_REF][START_REF] Kuperman | CRFR1 in AgRP neurons modulates sympathetic nervous system activity to adapt to cold stress and fasting[END_REF]. Furthermore, an AgRP-.PVN pathway subpopulation is required to drive positive reinforcement after food intake in caloric-depleted mice [START_REF] Garfield | A neural basis for melanocortin-4 receptor-regulated appetite[END_REF][START_REF] Chen | Hunger neurons drive feeding through a sustained, positive reinforcement signal[END_REF]. Moreover, food ingestion also alleviates a negative valence signal conferred by persistent AgRP neuronal activity [START_REF] Betley | Neurons for hunger and thirst transmit a negative-valence teaching signal[END_REF]. Thus, these combined actions in WT mice presumably underlie the temporal adaptation to consume food quickly over the 9-d restriction protocol. The finding that KO mice take longer to consume the same amount suggests altered processing in response to these valence cues, which may affect motivational aspects of food intake during a state of homeostatic need. Indeed, fewer feeding bouts 4 h after food access and reduced food consumption despite more time in a given feeding bout suggests that deletion of Crat in AgRP neurons affects the motivation for food intake. Future studies are required to address this suggestion.

We propose that Crat may be a critical enzyme that enables AgRP neurons to act as energy calculators, as proposed by Beutler et al. [START_REF] Beutler | Dynamics of gut-brain communication underlying hunger[END_REF], integrating information on acute changes in energy balance and regulating substrate utilization in response. Indeed, we have previously shown that deleting Crat in AgRP neurons impairs the switch in substrate utilization upon refeeding but does not disturb energy homeostasis under ad libitum fed conditions [START_REF] Reichenbach | AgRP neurons require carnitine acetyltransferase to regulate metabolic flexibility and peripheral nutrient partitioning[END_REF]. Furthermore, we found no differences in the orexigenic hormone ghrelin and lower levels of leptin in calorierestricted animals, supporting the notion that AgRP neurons regulate energy homeostasis through multiple mechanisms on different time scales. In our view, Crat activity in AgRP neurons links food availability with peripheral energy metabolism [START_REF] Joly-Amado | Hypothalamic AgRP-neurons control peripheral substrate utilization and nutrient partitioning[END_REF][START_REF] Reichenbach | AgRP neurons require carnitine acetyltransferase to regulate metabolic flexibility and peripheral nutrient partitioning[END_REF], ensuring appropriate adaptative nutrient partititoning during CR. In that view perception of food quality and availability and later on calorie intake would have a dynamic action onto AgRP neuron activity through Crat activity allowing for adaptive nutrient partitioning.

Our post-CR ad libitum refeeding experiments found that AgRP Crat KO mice, despite losing more fat under CR conditions, are capable of regenerating fat mass by increasing food intake until prediet adiposity is reached, similar to WT controls. Surprisingly, despite the greater loss of body fat at the end of CR, KO mice had no difference in body weight due to a preservation of lean mass and even an increase in their relative lean mass during post-CR ad libitum feeding, leading to greater rebound body weight compared with WT mice. In support of this outcome, AgRP activation is associated with increased circulating plasma myostatin [START_REF] Steculorum | AgRP neurons control systemic insulin sensitivity via myostatin expression in brown adipose tissue[END_REF], which is a negative regulator of muscle mass. Because the inhibition of myostatin increases muscle mass in mice [START_REF] Whittemore | Inhibition of myostatin in adult mice increases skeletal muscle mass and strength[END_REF], any influence on the ability of AgRP neurons to regulate myostatin may affect the preservation or restoration of lean mass; we suggest that the deletion of Crat in AgRP neurons represents one such influence over AgRP neurons. This finding might have implications for muscle degenerative diseases such as cachexia or muscle dystrophy, but further research is needed, as CR has differential effects on muscles dependent on strain, sex, and time [START_REF] Boldrin | The effect of calorie restriction on mouse skeletal muscle is sex, strain and timedependent[END_REF]. Recent studies also suggest osteocytes act as a body weight sensor, regulating food intake independent of leptin to maintain body weight [START_REF] Jansson | Body weight homeostat that regulates fat mass independently of leptin in rats and mice[END_REF]. The relative increase in lean mass elevates basal metabolic rate [START_REF] Hopkins | Modelling the associations between fat-free mass, resting metabolic rate and energy intake in the context of total energy balance[END_REF][START_REF] Weise | A potential role for the midbrain in integrating fat-free mass determined energy needs: An H2 (15) O PET study[END_REF], which is known to influence food intake [START_REF] Kennedy | The role of depot fat in the hypothalamic control of food intake in the rat[END_REF], offering an explanation for increased food intake in AgRP Crat KO mice during ad libitum refeeding.

In summary, Crat deletion in AgRP neurons promoted a shift toward increased fatty acid utilization, which persisted during the post-CR feeding period. This chronic impairment in nutrient partitioning affected food intake, body composition, adiposity, and rebound weight gain after a period of CR, which has significant implications for people in weight loss programs, especially considering metabolic inflexibility in obesity [START_REF] Goodpaster | Metabolic flexibility in health and disease[END_REF]. Collectively, our results show that Crat in AgRP neurons is crucial for adaptation to low caloric environments. In particular, this goal is achieved by adjusting feeding behavior to consume food quickly, control nutrient partitioning and substrate selection to preserve fat stores, and regulate energy expenditure to conserve energy. Finally, beyond dieting, chronic CR has been repeatedly shown to enhance life span in several species [START_REF] Lin | Calorie restriction extends Saccharomyces cerevisiae lifespan by increasing respiration[END_REF][START_REF] Slack | Activation of AMPK by the putative dietary restriction mimetic metformin is insufficient to extend lifespan in Drosophila[END_REF][START_REF] Weindruch | The retardation of aging in mice by dietary restriction: longevity, cancer, immunity and lifetime energy intake[END_REF][START_REF] Colman | Caloric restriction delays disease onset and mortality in rhesus monkeys[END_REF]. It is tempting to speculate that cratmetabolism in AgRP neurons is an important mechanism through which CR facilitates a global shift in peripheral substrate utilization and a whole-body optimization of fuel partitioning allowing for reduction of oxidative damage. This outcome highlights the fact that Crat in AgRP neurons acts as an integrator of acute changes to adapt metabolism and behavior by regulating nutrient partitioning, and that defects of this mechanism affect body weight and adiposity with implications for metabolic diseases.

Figure 2 .

 2 Figure 2. A, D) Crat in AgRP neurons is needed to regulate nutrient partitioning and energy expenditure in response to CR. BAT temperature (A) and locomotor activity (D) under ad libitum fed and CR conditions shown as a heat map. Each voxel represents average BAT temperature or cumulative locomotor activity of 15-min intervals, each column represents a day, the black bar on the left-hand side indicates the dark phase, the green bar on top of the heat map indicates ad libitum access to food, and the red bar indicates restricted access to 1.8 g/24 h starting at 11 zeitgeber time (ZT), indicated by the horizontal dotted line.B, C) Average BAT temperature during the light and dark phases of ad libitum fed animals (B) and regression analysis of average BAT temperature during the dark phase of 9 consecutive days CR (C ). E, F) Total locomotor activity during the light and dark phases of ad libitum fed animals (E ) and regression analysis of total dark phase locomotor activity under CR conditions (F ). G-J) Average 24-h profile of energy expenditure (G) and respiratory quotient (RQ) (I ) of CR d 7-9, and light and dark phase average of energy expenditure (H ) and RQ (J ) of this period. All data are expressed as means 6 SEM; n = 5-7. P , 0.05 (ANOVA with Sidak post hoc analysis, 2-way repeated measures, where appropriate).

Figure 3 .

 3 Figure 3. Crat in AgRP neurons affects hepatic function in response to CR. A, B) Liver glycogen (A) and triglyceride (B) after 9 d of CR. C) Hepatic gene expression of enzymes involved in gluconeogenesis, glycogenolysis, lipogenesis, and lipolysis. H-K) Plasma profiles after CR of metabolites: NEFA (D), triglycerides (E ), and ketone bodies (F ), and hormones: corticosterone (G), insulin (H ), glucagon (I ), acyl-ghrelin (J ), and leptin (K ). All data are expressed as means 6 SEM; n = 6-9. P , 0.05 (2-tailed, unpaired Student's t test).

Figure 4 .

 4 Figure 4. Crat in AgRP neurons influences rebound weight gain, food intake, and adiposity after cessation of CR. A, B) Relative changes to baseline in body weight after CR and ad libitum refeeding (A) and rebound weight gain during ad libitum refeeding (B). C, D) Changes in average 24-h food intake relative to baseline ad libitum fed conditions (C ) and cumulative food intake during ad libitum refeeding (D). E-H) Changes relative to baseline in lean mass (E ) and fat mass (G) after CR and ad libitum refeeding and gain in lean mass (F ) and fat mass (H ) during ad libitum refeeding. I-L) Correlation between changes in food intake and changes in fat mass (I, J) and correlation between changes in food intake and changes in body weight (K, L). All data are expressed as means 6 SEM; n = 9-11. P , 0.05 (ANOVA with Sidak post hoc analysis, 2-way repeated measures, where appropriate).

Figure 5 .

 5 Figure 5. Crat in AgRP neurons regulates substrate selection during ad libitum refeeding. A-E) Plasma leptin (A), insulin (B), and free fatty acids (NEFA) (C ) during ad libitum refeeding, and correlation between leptin levels and fat mass (D, E). F) Heat map depicting temporal changes in respiratory quotient (RQ) during transition from calorie restricted to ad libitum refed conditions. Each voxel represents average RQ of 1 h, each column represents a day, the black bar on the left-hand side indicates the dark phase, the red bar on the top indicates restricted access to 1.8 g/24 h starting at 11 zeitgeber time (ZT), indicated by the horizontal white line, ad libitum access to food is indicated by the green bar on top of the heat map, and start of ad libitum refeeding is indicated by the white arrows. G, H) Average energy expenditure (G) and RQ (H ) of the light and dark phases during ad libitum refeeding. All data are expressed as means 6 SEM; n = 9-11. P , 0.05 (ANOVA with Sidak post hoc analysis, 2-way repeated measures, where appropriate).

Vol.

December 2018 REICHENBACH ET AL. The FASEB Journal x www.fasebj.org sebj.org by Nagoya Daigaku Fuzoku Toshokan Igaku-Bu Bunkan (133.6.82.173) on July 31, 2018. The FASEB Journal Vol. ${article.issue.getVolume()}, No. ${article.issue.getIssueNu

CRAT IN AGRP NEURONS DURING CALORIE RESTRICTION

Vol.[START_REF] Mauvoisin | Circadian and feeding rhythms orchestrate the diurnal liver acetylome[END_REF] December 2018 REICHENBACH ET AL. The FASEB Journal x www.fasebj.org

Vol.[START_REF] Mauvoisin | Circadian and feeding rhythms orchestrate the diurnal liver acetylome[END_REF] December 2018 REICHENBACH ET AL. The FASEB Journal x www.fasebj.org sebj.org by Nagoya Daigaku Fuzoku Toshokan Igaku-Bu Bunkan (133.6.82.173) on July 31, 2018. The FASEB Journal Vol. ${article.issue.getVolume()}, No. ${article.issue.getIssueNu

sebj.org by Nagoya Daigaku Fuzoku Toshokan Igaku-Bu Bunkan (133.6.82.173) on July 31, 2018. The FASEB Journal Vol. ${article.issue.getVolume()}, No. ${article.issue.getIssueNu

Vol.[START_REF] Mauvoisin | Circadian and feeding rhythms orchestrate the diurnal liver acetylome[END_REF] December 2018 REICHENBACH ET AL. The FASEB Journal x www.fasebj.org sebj.org by Nagoya Daigaku Fuzoku Toshokan Igaku-Bu Bunkan (133.6.82.173) on July 31, 2018. The FASEB Journal Vol. ${article.issue.getVolume()}, No. ${article.issue.getIssueNu

ACKNOWLEDGMENTS

The authors thank Doug Compton (Research Diets, New Brunswick, NJ, USA) for helping construct and establish the BioDaq feeding cages. This study was supported by an Australian National Health and Medical Research Council (NHMRC) grant and fellowship (1126724, 1084344) to Z.B.A. This work used the Pennington Biomedical Research Center CRAT IN AGRP NEURONS DURING CALORIE RESTRICTION


Chef Michaël Grynberg
email: michael.grynberg@aphp.fr

Claire Murphy

Carole Doré

Laurence Fresneau

Ségolène Paillet

Nicoleta Petrica

Frédérique Maurel

Harinala Ravonimbola

A cost-effectiveness analysis comparing the originator follitropin alfa to its biosimilars in patients undergoing a medically assisted reproduction program from a French perspective

Keywords: follitropin alfa, cost-effectiveness, infertility, Gonal-F, gonadotropin JEL codes: I10, I19

de niveau recherche, publiés ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

Introduction

Infertility is a medical condition which is recognized as a global public health issue by the World Health Organization (WHO) 1 . Infertility is a growing concern in many countries, including the European Union (EU) member states, and fertility rates are steadily declining from the mid-1960s through the turn of the century [START_REF] Macaluso | A public health focus on infertility prevention, detection, and management[END_REF] . The common risk factors include age, smoking, alcohol consumption, obesity, diabetes or thyroid disease, and other occupational and environmental risk factors. It is estimated that about 48.5 million couples worldwide experience infertility [START_REF] Agarwal | A unique view on male infertility around the globe[END_REF] . A total of 1.9% of females aging 20-44 years were found to have difficulty in conception worldwide in 2010 4 5 . The prevalence of infertility varies considerably across countries and there is a paucity of reliable data due to the presence of multiple factors which complicate any estimates. Among the EU member states, France recorded the highest fertility rates in 2015 [START_REF] Macaluso | A public health focus on infertility prevention, detection, and management[END_REF] . However, its fertility rates fell steadily from 2.01 children per woman in 2012 to 1.93 in 2015 4 5 . The decreasing trends in fertility can be related to an overall increase in the incidence of infertility due to social, lifestyle, biological, and environmental factors [START_REF] Mazuy | Recent Demographic Developments in France: A Decline in Fertility, an Increase in Mortality[END_REF] . Furthermore, it estimated that about 20% couples experiencing subfertility or infertility, only 10% seek specialist care [START_REF] Mazuy | Recent Demographic Developments in France: A Decline in Fertility, an Increase in Mortality[END_REF] .

However, various treatment options are available for female fertility in France. Assisted reproductive technologies (ART) comprise one of the most commonly used treatment options for women encountering fertility issues. In ART, gonadotropins are usually administered in order to stimulate the follicular development. Exogenous gonadotropins, including follicle-stimulating hormone (FSH), are universally recognized as the key driver of controlled ovarian stimulation and maturation. Available FSH products include purified urinary-derived human menopausal gonadotropin (hMG), highly purified urinary (HP-uFSH), and recombinant human FSH (r-hFSH).

Gonal-F ® (follitropin alfa for injection) was the first FSH preparation of human recombinant DNA origin marketed since 1997 in several indications, including the stimulation of mutifollicular development in women undergoing superovulation for ART [START_REF] Mazuy | Recent demographic trends in France: The number of marriages continues to decrease[END_REF] . Two biosimilars, namely Ovaleap ® (Teva, Castleford, UK), and Bemfola ® (Finox AG, Burgdorf, Switzerland), are now marketed in Europe. Ovaleap ® was approved by the European Medicine Agency (EMA) in 2013 and recommended by the European guidelines as a biological product containing r-hFSHα [START_REF] Ovaleap | EPAR Public Assessment Report[END_REF] . Bemfola ® was approved by EMA in 2015 and licensed for all indications of reference products 9 . The biosimilar FSH products have nonclinical pharmacological, pharmacokinetic, and toxicological profiles similar to those of the originator FSH. The clinical safety and efficacy of biosimilars is well documented in literature among women undergoing ART in European countries 10 8 . However, their clinical bioequivalence has been demonstrated on an intermediate criterion that is the number of oocytes retrieved but not on the ultimate objective of interest, which is the number of live births [START_REF]Avis de la commision de transparence-GONAL-F[END_REF] and the secondary end-point of these randomized controlled trials (RCTs) 12 13 . Comparing live birth rates for Gonal-F ® versus the biosimilar FSH products for the 1 st treatment cycle, results are in favor of Gonal-F ® , but with non-statistically significant as the RCTs were not powered for this endpoint. While there is to date no hard-clinical evidence of the superiority of Gonal-F ® versus the biosimilar on live birth rates, considering the results observed on existing RCTs and the need to optimize outcomes in relation to limited public resources it is of interest to compare Gonal-F ® and the biosimilar FSH products in a cost effectiveness (CE) analysis.

Indeed, currently, the CE of Gonal-F ® in comparison to its biosimilars is yet to be demonstrated, considering the lower cost of the biosimilars. Hence, the aim of our study was to perform a CE analysis (CEA) of Gonal-F ® in patients undergoing a medically assisted reproduction (MAR) program compared to its biosimilars: Bemfola ® and Ovaleap ® in a French context.

Material and Method

A c c e p t e d M a n u s c r i p t

The analysis was based on previously conducted studies 12 13 and did not involve any new studies with human or animal subjects performed by any of the authors. The study was performed in accordance with the International Society for Pharmacoeconomics and Outcomes Research (ISPOR) Good Practices for Outcomes Research consensus guidance [START_REF]ISPOR. Good Practices for Outcomes Research[END_REF] and relevant international and national guidelines for health economics studies. Ethics board approval and informed consent were not required because the study did not involve human or animal participants and the analysis only used publicly available anonymized data.

Model design

A Microsoft Excel-based pharmacoeconomic model was developed to evaluate the CE of Gonal-F ® versus its biosimilars in women undergoing ART, focusing on the 1 st treatment cycle. The number of cycles used in our model was decided based on the availabilities of the clinical data used to feed the model, knowing that only data for the 1st cycle were available for both treatment comparisons (Gonal-F ® versus Bemfola ®12 and Gonal-F ® versus Ovaleap ®13 ). The CEA was carried out from a National Health Service perspective. To delineate the cost and efficacy of Gonal-F ® in patients undergoing an MAR program compared to its biosimilars in a French context, a decision-tree was used. This decision-tree model depicted different relevant outcomes of fertility treatment with r-hFSHα over the 1 st cycle. The modeling of decision-tree encompassed the following steps: a) Treatment stimulation; b) Oocyte retrieval; c) Embryo transfer; d) Pregnancy; and e) Live delivery or miscarriage. The probability of having live births/miscarriage was taken as the final outcome of the model. At each step, the probability of succeeding or failing was calculated. Only one reimbursed IVF cycle was considered in the model.

Ovarian hyperstimulation syndrome (OHSS), the most important adverse event of gonadotropin use, was taken into account in the model.

Six treatment arms based on the following three pair-wise comparisons were considered: a) Gonal-F ® O versus Ovaleap ® using data reported by Strowitzki et al [START_REF] Strowitzki | Randomized, active-controlled, comparative phase 3 efficacy and safety equivalence trial of Ovaleap® (recombinant human follicle-stimulating hormone) in infertile women using assisted reproduction technology (ART)[END_REF] ; b) Gonal-F ® B compared to Bemfola ® using data reported by Rettenbacher et al [START_REF] Rettenbacher | A multi-centre phase 3 study comparing efficacy and safety of Bemfola(®) versus Gonal-f(®) in women undergoing ovarian stimulation for IVF[END_REF] ; c) Pooled Gonal-F ® (i.e. Gonal-F ® P) versus pooled biosimilars (i.e. BioS) using data from both the clinical trials. The model in Figure 1depicts main steps of ART on which clinical outcomes and costs are based and simulates a patient's journey from the start of IVF therapy through various treatment stages.

Sensitivity analyses were also performed to investigate the stability of CE for the treatment over a range of value variations. For each comparison, one-way sensitivity analysis was performed to assess the impact of different parameters on the ICER. These parameters included the percentage of live birth per woman, mild/moderate OHSS and severe, r-hFSHα dose and the price of the treatment is performed to test the robustness of the results. The variation of each parameter was determined arbitrarily [± 20%], except for r-hFSHα dose, where the confidence intervals of UI dose reported in the two clinical studies have been considered. A tornado diagram was used to analyze the parameters having impact on the ICER.

Clinical studies and evidence details

The choice of clinical outcomes in infertility trials has been debatable owing to the multistage nature of the treatment and a recent review of outcome measures in the in vitro fertilization (IVF) clinical trials has indicated very wide diversity in the selection of these outcomes [START_REF] Rettenbacher | A multi-centre phase 3 study comparing efficacy and safety of Bemfola(®) versus Gonal-f(®) in women undergoing ovarian stimulation for IVF[END_REF] . Live births are the ultimate endpoint of fertility tests and there is a consensus among experts in reproductive medicine to advocate the need to adopt live births as the preferred primary outcome in infertility trials [START_REF] Strowitzki | Randomized, active-controlled, comparative phase 3 efficacy and safety equivalence trial of Ovaleap® (recombinant human follicle-stimulating hormone) in infertile women using assisted reproduction technology (ART)[END_REF] . Hence, the live birth rate was considered as the criterion of interest to assess efficacy of each treatment in our CE model. The

A c c e p t e d M a n u s c r i p t

model developed is based on the clinical evidences from head-to-head EMA registration trials of Bemfola ® 12 and Ovaleap ® 13 which assessed the number of oocytes retrieved as primary end-point and the live-birth rate as secondary end-point: One of these studies was a multinational, multicenter, randomized, assessor-blind phase III study comparing the efficacy (in terms of number oocyte retrieved) and safety of Ovaleap ® to Gonal-F ® in 299 women undergoing controlled ovarian stimulation with ART [START_REF] Strowitzki | Randomized, active-controlled, comparative phase 3 efficacy and safety equivalence trial of Ovaleap® (recombinant human follicle-stimulating hormone) in infertile women using assisted reproduction technology (ART)[END_REF] . The other study was a multicenter phase III study that compared the efficacy (in terms of number oocyte retrieved) and safety of Gonal-F ® to Bemfola ® in 372 women undergoing ovarian stimulation for IVF for ART superovulation [START_REF] Rettenbacher | A multi-centre phase 3 study comparing efficacy and safety of Bemfola(®) versus Gonal-f(®) in women undergoing ovarian stimulation for IVF[END_REF] . We mention that the clinical trials used to feed the model included women according to different criteria (such as age, which range between 20 and 38 years in the Gonal-F ® versus Bemfola ® clinical trial [START_REF] Rettenbacher | A multi-centre phase 3 study comparing efficacy and safety of Bemfola(®) versus Gonal-f(®) in women undergoing ovarian stimulation for IVF[END_REF] and between 18 and 37 year for the Gonal-F ® versus Ovaleap ® clinical trial [START_REF] Strowitzki | Randomized, active-controlled, comparative phase 3 efficacy and safety equivalence trial of Ovaleap® (recombinant human follicle-stimulating hormone) in infertile women using assisted reproduction technology (ART)[END_REF] or body mass index). It is therefore evident that based on other populations the CE model could lead to different results.

Adverse events associated with the use of gonadotropins have been considered in the model. Ovarian hyperstimulation syndrome (OHSS) is one of the most important adverse events of gonadotropin and the severity, management, and costs of OHSS have been considered for all treatment options using data provided in European Public Assessment Reports of Bemfola ®9 and Ovaleap ®8 . No other publication comparing the biosimilars to the originator follitropin alfa was available at the model development date.

The transition probabilities of clinical outcomes, including OHSS rates used in the model were calculated based on the evidence submitted for biosimilars in EMA registrations considering the intent-to-treat (ITT) population data from the two clinical trials. Probability of each treatment arm derived from clinical efficacy outcomes are presented in Table 1.

Costs inputs

The resource pertaining costs associated for each step of the MAR process were retrieved from literature review, PMSI [START_REF] Atih | [END_REF] (Programme de médicalisation des systèmes d'information) analysis (a comprehensive French hospital database), and French clinical experts opinions through a management questionnaire [START_REF] Iqvia | Questionnaire de prise en charge des patientes ayant recours à une aide médicale à la procréation[END_REF] .

Cost assumptions taken for each step in the model for each treatment were based on a market study based on the responses from 30 gynecologists/endocrinologists and 300 patients using a questionnaire completed by French experts. The ART cost included cost of treatment induction which was separated in three steps: blocking phase, stimulation phase, and trigger phase. Nurse tariffs [START_REF] Ameli | Actes à domicile[END_REF] were applied for one subcutaneous injection each.

Cost analysis also considered monitoring visits, biological hormones dosages [START_REF] Ameli | Le codage des actes biologiques -NABM[END_REF] , and IVF based on French health insurance tariff [START_REF] Ameli | CCAM en ligne -CCAM[END_REF] . The IVF cost is weighted between standard IVF cost (40%) and IVF Intra Cytoplasmic Spermatozoid Injection (ICSI) cost (60%) [START_REF] Iqvia | Questionnaire de prise en charge des patientes ayant recours à une aide médicale à la procréation[END_REF] . Oocyte retrieval costs involved anesthetic visit (10% of patients based on French expert opinion), oocyte retrieval, spermatozoid retrieval (0.09%, based on French Diagnosis-Related Group (DRG) tariff [START_REF] Tarifs | [END_REF] ), and spermatozoid preparation.

Embryo transfer involved technical examination, adding a specific fee and one beta-human chorionic gonadotropin (b-HCG) dosage. The management costs were considered similar to normal pregnancy costs from this stage onwards. Pregnancy follow-up costs were based on mensural medical visit and quarterly monitoring. The weighted mean cost of DRG for natural delivery (75%) and caesarian delivery (25%) was applied in case of pregnancy leading to live birth, whereas DRG tariff was considered in case of miscarriage. Costs of mild, moderate, and severe OHSS were used in the model considering the monitoring costs, unless for the severe OHSS were the cost of the associated DRG ("Autres affections de l'appareil génital féminin" -GHM: 13M041, 13M042, 13M04T, 13M043 and "Interventions sur le système utéroannexiel pour des affections non malignes" _ GHM: 13C071) [START_REF] Tarifs | [END_REF] was added in addition to the monitoring costs. All estimated cost and assumptions made are presented in Table 2.

The total cost per patient was calculated for each treatment group by multiplying the probability of having one of the clinical efficacy outcomes presented in Table 1 with the related costs. The incremental CE ratio (ICER) was calculated by taking the difference in total costs divided by the difference in live birth rate of the two treatment groups in France. All resources are valued in 2017 Euros using official local tariffs sources/database.

Results

Detailed results are presented in Table 3 for each set of pairwise comparisons, presenting the ICER values, cost per live birth, incremental cost, and incremental efficacy either with or without OHSS cases.

In all the analyses it was observed that Gonal-F ® was found to be cost-effective over its biosimilars, irrespective of the consideration of OHSS.

Base-case analysis

When Gonal-F ® O was compared to Ovaleap ® after taking OHSS into account, the use of Gonal-F ® O resulted in an incremental cost of €259.17 and an incremental efficacy of 0.05 over Ovaleap ® . This translated into an ICER of €4,804, which is the additional cost required for Gonal-F ® O to gain an additional live birth in comparison with Ovaleap ® . The costs of treatment with Gonal-F ® O and Ovaleap ® , were €3,826 and €3,567, respectively. The cost per live birth was €5,799 with Gonal-F ® O and €5,682 with Ovaleap ® . Efficacy in terms of live-birth rate was 0.32 and 0.27 for Gonal-F ® O and Ovaleap ® , respectively, which indicates that there would be 32 live-born children per 100 women treated with the Gonal-F ® and 27 live-born children per 100 women treated with Ovaleap ® . Similar results were observed when Gonal-F ® O was compared to Ovaleap ® without taking OHSS into account, with an ICER of €4,878. There was no change in the incremental efficacy (0.05), while the incremental cost was relatively higher without taking OHSS into account (€263) as compared to analysis taking OHSS into account (€259).

For the second set of pairwise comparison between Gonal-F ® B and Bemfola ® , the incremental efficacy was 0.08 in favor of Gonal-F ® B, irrespective of OHSS consideration. The incremental cost of Gonal-F ® B over Bemfola ® was €279 with OHSS and €299 without OHSS. This resulted in an ICER of €3,275 with OHSS and €3,505 without OHSS.

The third set of pairwise comparison between pooled Gonal-F ® P and BioS also showed a similar trend which favored Gonal-F ® P over BioS in terms of incremental efficacy (0.06) irrespective of OHSS consideration. The incremental cost of Gonal-F ® P over BioS was €260 with OHSS and €278 without OHSS.

The observed ICER values are €4,352 with OHSS and €4,668 without OHSS.

Sensitivity analysis

The results of one-way sensitivity analysis are presented in Table 4, and the outcomes for the comparison of Gonal-F ® P to BioS are depicted as tornado diagram in Figure 2. This analysis indicates that uncertainty in probability of birth and dosages of r-hFSHα are the most sensitive variables and have the highest impact on ICER values. Gonal-F ® O versus Ovaleap ® (without OHSS) and Gonal-F ® P versus BioS (without OHSS) analyses indicated cost-saving with higher efficacy for Gonal-F ® .

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Nearly all sensitivity analyses support that Gonal-F ® is a cost-effective strategy, even cost-saving when the lower dosage limit is considered for Gonal-F ® compared to Ovaleap ® or BioS. In case of Gonal-F ® probability of live birth decreased by 20%, the Ovaleap ® and BioS are observed to be dominant.

Discussion

Cumulative potential savings to health systems in the European Union (EU) and the US, as a result of the use of biosimilars, could exceed €50 billion in aggregate by 2020 and reach as much as €100 billion [START_REF] Aitken | Delivering on the potential of biosimilar medicines: The role of functioning competitive markets introduction[END_REF] . The cumulative spending in the EU5 (France, Spain, Germany, Italy and United-Kingdom) alone is expected to reach €47 billion over the period 2016-2020 on different originator biologic medicines. In this regard, Germany and France are leading the addressable biosimilar medicines market in the EU5 with 17 billion euros and 9 billion euros of spending, respectively, for 2016-2020 [START_REF] Aitken | Delivering on the potential of biosimilar medicines: The role of functioning competitive markets introduction[END_REF] . France is the first European country to explicitly permit biosimilar substitution, and the drop in price of originator products ranges from 1%-33% in France 22 23 . While biosimilars may offer a less expensive alternative for patients, it is essential to perform CEA to evaluate the cost per course of treatment of biosimilar r-hFSHα with respect to the originator on the base of equivalent therapeutic. However, to the best of our knowledge, there is a lack of published literature on any CEA of the biosimilar gonadotropin in France, and our study is the first CEA comparing biosimilars Ovaleap ® and Bemfola ® to their originator r-hFSHα Gonal-F ® in women undergoing ovarian stimulation for IVF in France.

Previous CEA studies conducted in Italy, Spain, Germany and Portugal have indicated Gonal-F ® to be a cost-efficient treatment strategy compared to its biosimilars Bemfola ® 24 25 and Ovaleap ® 26 27 . The ICER obtained for Gonal-F ® versus Ovaleap ® was €1,517 per woman with a new-born child in Portugal [START_REF] Silverio | Cost-Effectiveness Of Recombinant Human Follicle Stimulating Hormone Biosimilars In Portugal[END_REF] and between 415.43€ and 2917.47€ for the others countries [START_REF] Gizzo | A cost-effectiveness modeling evaluation comparing a biosimilar follitropin alfa preparation with its reference product for live birth outcome in Germany, Italy and Spain[END_REF] . The ICER values for Gonal-F ® compared to Bemfola ® were €3,600 in Italy and €900 in Spain [START_REF] Gizzo | A cost-effectiveness evaluation comparing originator follitropin alfa to the biosimilar for the treatment of infertility[END_REF] . Yet another recent study in Italy also supported the fact that Gonal-F ® provided a lower average cost per live birth than Bemfola ® and an ICER of €1,210 [START_REF] Ripellino | A Cost-Effectiveness Evaluation Comparing Biosimilar Bemfola to Gonal-F for the Treatment of Infertility in an Italian Contest[END_REF] . This is despite the fact that Gonal-F ® had a higher acquisition cost when compared to its biosimilars Bemfola ® and Ovaleap ® .

In context of extending these CEA results to other countries, our study was able to show that Gonal-F ® could remain the cost-effective strategy compared to its biosimilars, owing to its incremental efficacy in terms of the number of live births.

The CE model was fed with data reported in clinical trials and cannot be substituted for the direct reallife comparisons. Also, it should be noted that the methods used in our study were slightly different than other CE models in Portugal, Spain, and Italy, owing to differences in the number of stimulation cycles, stimulation steps and/or costs considered. All patients included in the second cycle of treatment in the Ovaleap ® trial were treated with Ovaleap ® , irrespective of whether they received Gonal-F ® or Ovaleap ® during the first cycle of treatment [START_REF] Strowitzki | Randomized, active-controlled, comparative phase 3 efficacy and safety equivalence trial of Ovaleap® (recombinant human follicle-stimulating hormone) in infertile women using assisted reproduction technology (ART)[END_REF] . Furthermore, the objective of the second treatment cycle in the Bemfola ® trial was to assess the immunogenicity and safety of Bemfola ® . Hence, the CE model was based on only one stimulation cycle since the introduction of second cycle efficacy data could have raised bias not only due to the trial design of the study but also for the small number of patients who underwent the second treatment cycle [START_REF] Rettenbacher | A multi-centre phase 3 study comparing efficacy and safety of Bemfola(®) versus Gonal-f(®) in women undergoing ovarian stimulation for IVF[END_REF] .

The outcomes used to feed the model derive from clinical trials that were designed to compare the number of oocytes retrieved between the biosimilars and Gonal-F ® . In our analyze we used the second end-point, which is the live birth rate, to calculate the ICER. Even though the first end-point is the number of oocytes retrieved, the number of live birth represents the most meaningful and relevant clinical outcome for these types of treatments as shown in multiple studies from the literature. In the

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French Technology Assessment label [START_REF]Avis de la commision de transparence-GONAL-F[END_REF] for Gonal-F ® , multiple sources are presented, such as: the metaanalyses made by Gerli et al (2013) [START_REF] Gerli | Clinical efficacy and cost-effectiveness of HP-human FSH (Fostimon®) versus rFSH (Gonal-F®) in IVF-ICSI cycles: a meta-analysis[END_REF] and Al-Inany et al (2009) [START_REF] Al-Inany | Highly purified hMG achieves better pregnancy rates in IVF cycles but not ICSI cycles compared with recombinant FSH: a meta-analysis[END_REF] which involved studies having the live birth rate as first end-point or other study such as Gholami et al (2010) [START_REF] Gholami | Pregnancy outcome following in vitro fertilization-embryo transfer (IVF-ET) in women aged < 37, undergoing ovulation induction with human FSH compared with recombinant FSH: a randomised controlled study[END_REF] or Sagnella et al (2011) [START_REF] Sagnella | A prospective randomized noninferiority study comparing recombinant FSH and highly purified menotropin in intrauterine insemination cycles in couples with unexplained infertility and/or mild-moderate male factor[END_REF] , where the first end-point is the pregnancy rate. The recent cost-effectiveness modeling evaluations, such as Gizzo et al (2016) [START_REF] Gizzo | A cost-effectiveness evaluation comparing originator follitropin alfa to the biosimilar for the treatment of infertility[END_REF] and Gizzo et al (2018) [START_REF] Gizzo | A cost-effectiveness modeling evaluation comparing a biosimilar follitropin alfa preparation with its reference product for live birth outcome in Germany, Italy and Spain[END_REF] , the effectiveness outcome used was also the live birth. Moreover, in order to proof the significance of the used outcome, we analyzed the level where the difference between the two groups was significant for the clinical efficacy outcomes used to feed the model. We observed that the difference become significant at a p-value >25% going up to 50% for some outcomes. However, in our CE model the outcome is the recalculate live birth rate, presented differently than in the original paper. Using the same statistical proportion test (Z test) as above, this difference is significant between the groups at a p-value equal to 13%. By accepting a higher value for the risk of being wrong we can make the hypothesis that our live birth rate difference is significantly different from 0. As mentioned above the studies used for these analyses were not designed to compare the live birth rates and the clinical trials showed that the second end-point was not significantly different between treatment groups. Such results represent an uncertainty for the conclusion. If sufficient data were available with a study design powered to demonstrate the live-birth rate, it might be interesting to analyze the success rate of all the cycles needed to obtain a child and also to confirm our results.

Another potential limitation is that the biosimilar Bemfola ® is available only as single-use, fixed-dose, prefilled pens in contrast to Gonal-F ® , which is available as multi-dose vials and prefilled pens. As doses need to be individually tailored to response, it is not possible to determine the impact of potential dose wastage on costs arising from the use of the different presentations. Furthermore, dissimilarities in dose reduction were observed between the biosimilar and originator groups, which also could have resulted in a higher incidence of OHSS for the biosimilar [START_REF] Ameli | Présentation BdM_IT[END_REF] . Clinical outcomes data stratified by age groups and types of ART would have access to model sub-populations avoiding biases on treatment-related benefits and potential harms due to different population characteristics.

In our study, treating 100 women with Gonal-F ® resulted in nine, six, and five additional live births compared to Bemfola ® , pooled biosimilars, and Ovaleap ® , respectively, irrespective of OHSS occurrence. The ICER values were €4,804 per live birth for Ovaleap ® , €3,275 per live birth for Bemfola ® , and €4,353 per live birth when considering OHSS, and the respective ICERs were relatively higher when OHSS was taken into account.

Sensitivity analyses confirmed the robustness of base case model, and the probability of birth was the most sensitive variable followed by Gonal-F ® dosage. When the lower dosage limit of FSH was taken into account, Gonal-F ® was found to be cost-saving and the dominant strategy. The biosimilars are considered dominant only when Gonal-F ® probability of birth is decreased by 20%. Similar results were also observed in two CEA studies conducted in Portuguese women, wherein the probability of birth and lower dosage limits in the sensitivity analyses indicated originator FSH to be the dominant strategy 26 10 .

Our study was conducted with a National Health Service (NHS) perspective and eventually, the preferred strategy depends on the NHS willingness-to-pay threshold. However, no national or international thresholds have been defined regarding ICER per live-birth, and hence, no clear implication of our findings on the willingness-to-pay per live-birth can be derived at the moment. Theoretically, the biosimilar r-hFSHα will be preferred if the NHS is willing to pay less than the value of the ICER for one extra live-born child, while the originator Gonal-F ® will be preferred if it is willing to pay the value of the ICER or more per extra live-born child.

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In conclusion, the results of this CEA indicate that the originator r-hFSHα Gonal-F ® could be a costefficient treatment strategy from the perspective of French health services in the treatment of infertility, as compared to the biosimilars. Given the limitations of the model, reliability of CEA can be greatly improved over time as evidence continues to grow and long-term data, especially in real-life scenario, are available in the individual patient populations of interest for different geographies.

List of Tables

A

  cost-effectiveness analysis comparing the originator follitropin alfa to its biosimilars in patients undergoing a medically assisted reproduction program from a French perspective M Grynberg, C Murphy, C Dore, L Fresneau, S Paillet, N Petrica, F Maurel, H Ravonimbola To cite this version: M Grynberg, C Murphy, C Dore, L Fresneau, S Paillet, et al.. A cost-effectiveness analysis comparing the originator follitropin alfa to its biosimilars in patients undergoing a medically assisted reproduction program from a French perspective. Journal of Medical Economics, 2019, 22, pp.108-115. 10.1080/13696998.2018.1551226. hal-03105446

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Table 1 : Model input data: clinical background Clinical efficacy outcomes Gonal-F ® O a Gonal-F ® B b Gonal-F ® P ab Ovaleap ®a Bemfola ®b BioS ab

 1 Pooled biosimilar data from both clinical trials; Gonal-F ® B: Gonal-F ® data from clinical trial versus Bemfola ® ; Gonal-F ® O: Gonal-F ® data from clinical trial versus Ovaleap ® ; Gonal-F ® P: Gonal-F ® pooled data versus both biosimilars; OHSS: ovarian hyperstimulation syndrome; r-hFSHα: recombinant human follicle-stimulating hormone alpha a Source: 13 b Source:[START_REF] Rettenbacher | A multi-centre phase 3 study comparing efficacy and safety of Bemfola(®) versus Gonal-f(®) in women undergoing ovarian stimulation for IVF[END_REF]

	Oocytes retrieval	97.9%	100.0%	98.9%	99.3% 100.0%	99.8%
	Embryo transfer	93.7%	92.7%	93.2%	92.8%	90.0%	91.0%
	Pregnancy	36.6%	44.7%	40.3%	29.8%	37.5%	34.5%
	Miscarriage	4.1%	2.0%	3.0%	2.4%	4.8%	4.0%
	Birth	95.9%	98.0%	97.0%	97.6%	95.2%	96.0%
	Live birth rate per woman	32.0%	41.0%	36.0%	27.0%	32.0%	30.0%
	Mild/moderate OHSS	2.1%	12.2%	6.7%	3.9%	21.3%	14.7%
	Severe OHSS	0.7%	0.8%	0.7%	0.7%	0.8%	0.7%
	r-hFSH mean dose	1614.3	1569.2	1593.7	1535.8	1555.7	1548.1
	BioS:

Table 2 : All estimated costs with assumptions Phase Assumptions Costs (€) Sources Induction stage (without treatment induction)

 2

	484.91	Ameli French data base 19
		KOL

Table 3 : Results of the base case cost-effectiveness analysis

 3

	Strategy	Cost (€)	Incremental	Efficacy Incremental	ICER (€)	Cost per live
			Cost (€)		Efficacy		birth (€)
	Gonal-F ® versus Ovaleap ®
	With OHSS
	Gonal-F ®	3,825.90	259.17	0.32	0.05	4,804.41	1,866.82
	Ovaleap ®	3,566.71	-	0.27	-	-	1,522.53
	Without OHSS
	Gonal-F ®	3,811.51	263.13	0.32	0.05	4,877.76	1,862.19
	Ovaleap ®	3,548.39	-	0.27	-	-	1,517.62
	Gonal-F ® vs Bemfola ®
	With OHSS
	Gonal-F ®	4,121.81	279.07	0.40	0.08	3,274.80	2,367.76
	Bemfola ®	3,842.74	-	0.32	-	-	1,857.76
	Without OHSS
	Gonal-F ®	4,081.80	298.68	0.40	0.08	3,504.89	2,351.50
	Bemfola ®	3,783.12	-	0.32	-	-	1,838.60
	Gonal-F ® pooled versus BioS
	With OHSS
	Gonal-F ®	3961.50	259.56	0.36	0.06	4,352.17	2,095.56
	BioS	3701.94	-	0.30	-	-	1,718.15
	Without OHSS
	Gonal-F ®	3 935.40	278.39	0.36	0.06	4,667.90	2,086.15
	BioS	3 657.02	-	0.30	-	-	1,704.63

ICER: incremental cost-effectiveness ratio; OHSS: ovarian hyperstimulation syndrome

Table 4 : Results of one-way sensitivity analyses for the comparison of Gonal-F ® with its biosimilars

 4

Declaration of funding

This study has been funded by Merck Santé S.A.S, which is developing products related to research described in this publication.