Giada Zurlo

Jérôme Piquereau

Maryline Moulin

Julie Pires Da Silva

Mélanie Gressette

Benoît Ranchoux

Benoît RANCHOUX b Anne Garnier

Renée Ventura-Clapier

Elie Fadel

Marc Humbert

Julie Pires

D A Silva

Renée Ventura- Clapier

Christophe Lemaire

Frédéric PERROS b Vladimir Veksler
email: vladimir.veksler@u-psud.fr

Vladimir Pr

Veksler

Sirtuin 1 regulates pulmonary artery smooth muscle cell proliferation: role in pulmonary arterial hypertension

Keywords: pulmonary artery smooth muscle cells, proliferation, oxidative metabolism, mitochondrial biogenesis, sirtuin 1, protein acetylation

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

Introduction

Pulmonary arterial hypertension (PAH) is a rare but deadly cardiopulmonary disorder characterized by an extensive remodeling of small-and medium-sized pulmonary arteries, mainly due to the hyperproliferation of pulmonary artery smooth muscle cells (PASMCs). Such vascular remodelling increases pulmonary vascular resistance and pulmonary arterial pressure, ultimately leading to right-sided heart failure and death [START_REF] Humbert | Advances in Therapeutic Interventions for Patients With Pulmonary Arterial Hypertension[END_REF]. The causes that initiate and perpetuate pulmonary vascular cell proliferation in PAH remain unknown, which explains the absence of curative therapies and, thus, the poor prognosis for this disease. PAH PASMCs share some common features with cancer such as growth dysregulation, resistance to apoptosis, and a metabolic remodeling [START_REF] Paulin | The metabolic theory of pulmonary arterial hypertension[END_REF]. Similar to cancer cells, PASMCs display a shift from oxidative phosphorylation towards glycolysis even in the presence of oxygen, condition known as the Warburg effect. This metabolic alteration is sustained by the pathological activation of transcription factors such as c-Myc and the hypoxia-inducible factor-1 α (HIF-1α) [START_REF] Bonnet | An abnormal mitochondrial-hypoxia inducible factor-1alpha-Kv channel pathway disrupts oxygen sensing and triggers pulmonary arterial hypertension in fawn hooded rats: similarities to human pulmonary arterial hypertension[END_REF], as well as the inhibition of the transcription factor forkhead box protein O1 (FOXO1) [START_REF] Savai | Pro-proliferative and inflammatory signaling converge on FoxO1 transcription factor in pulmonary hypertension[END_REF], contributing to PAH PASMC mitochondrial dysfunction. Indeed, PAH PASMC mitochondria present numerous abnormalities (for review, see [START_REF] Ryan | Emerging Concepts in the Molecular Basis of Pulmonary Arterial Hypertension Part I: Metabolic Plasticity and Mitochondrial Dynamics in the Pulmonary Circulation and Right Ventricle in Pulmonary Arterial Hypertension[END_REF]) including lowered oxidative capacity and altered mitochondrial dynamics leading to mitochondrial fragmentation. The recovery of mitochondrial function via the increase in oxidative capacity [START_REF] Mcmurtry | Dichloroacetate prevents and reverses pulmonary hypertension by inducing pulmonary artery smooth muscle cell apoptosis[END_REF][START_REF] Michelakis | Dichloroacetate, a metabolic modulator, prevents and reverses chronic hypoxic pulmonary hypertension in rats: role of increased expression and activity of voltage-gated potassium channels[END_REF][START_REF] Sutendra | Fatty Acid Oxidation and Malonyl-CoA Decarboxylase in the Vascular Remodeling of Pulmonary Hypertension[END_REF] and/or the restoration of fusion/fission balance [START_REF] Marsboom | Dynamin-Related Protein 1-Mediated Mitochondrial Mitotic Fission Permits Hyperproliferation of Vascular Smooth Muscle Cells and Offers a Novel Therapeutic Target in Pulmonary Hypertension[END_REF][START_REF] Ryan | PGC1αmediated mitofusin-2 deficiency in female rats and humans with pulmonary arterial hypertension[END_REF] proved beneficial for the treatment of PAH in animal models, thus appearing as a promising therapeutic approach.

Cellular acetylation/deacetylation balance is an important regulator of energy metabolism including mitochondrial function. Particularly, sirtuin 1 (SIRT1), which is a NAD + -dependent deacetylase, has been found to augment mitochondrial function via several mechanisms. In various tissues including liver and skeletal muscle, SIRT1 enhances mitochondrial biogenesis and fatty acid oxidation by deacetylating and activating one of the master regulators of mitochondrial biogenesis, PGC-1α (reviewed in [START_REF] Houtkooper | Sirtuins as regulators of metabolism and healthspan[END_REF]). SIRT1 is able to promote mitochondrial gene expression also via a PGC-1α independent pathway. By destabilizing HIF-1α, it prevents HIF-1α-mediated inhibition of c-Myc, activator of the transcription of mitochondrial transcription factor A (TFAM), thus increasing the expression of mitochondrial-encoded respiratory genes [START_REF] Gomes | Declining NAD(+) induces a pseudohypoxic state disrupting nuclear-mitochondrial communication during aging[END_REF]. Finally, SIRT1 can improve mitochondrial activity by activating stress signaling pathways via the mitochondrial unfolded protein response and the nuclear translocation and activation of one of FOXO transcription factors as shown in worms [START_REF] Mouchiroud | The NAD+/Sirtuin Pathway Modulates Longevity through Activation of Mitochondrial UPR and FOXO Signaling[END_REF].

Therefore, one could hypothesize that SIRT1 is affected in PAH and that its activation could have beneficial effects in this disease. However, neither the expression/activity of SIRT1 nor the acetylation/deacetylation balance in PAH pulmonary vessels of patients or in experimental models has been investigated. Of note, another enzyme belonging to this family, sirtuin3, which localizes in the mitochondria to activate metabolic pathways including fatty acid oxidation [START_REF] Hirschey | SIRT3 regulates fatty acid oxidation via reversible enzyme deacetylation[END_REF] and oxidative phosphorylation [START_REF] Ahn | A role for the mitochondrial deacetylase Sirt3 in regulating energy homeostasis[END_REF][START_REF] Cimen | Regulation of Succinate Dehydrogenase Activity by SIRT3 in Mammalian Mitochondria[END_REF], is downregulated in human PAH [START_REF] Paulin | Sirtuin 3 Deficiency Is Associated with Inhibited Mitochondrial Function and Pulmonary Arterial Hypertension in Rodents and Humans[END_REF]. Importantly, some studies pointed out a potential protective role of SIRT1 in PAH. Indeed, resveratrol, a non-specific SIRT1 activator, has been found to prevent, and even reverse, [START_REF] Paffett | Resveratrol reverses monocrotaline-induced pulmonary vascular and cardiac dysfunction: A potential role for atrogin-1 in smooth muscle[END_REF] monocrotaline- [START_REF] Csiszar | Resveratrol prevents monocrotaline-induced pulmonary hypertension in rats[END_REF] and chronic hypoxia- [START_REF] Chen | Resveratrol prevents hypoxia-induced arginase II expression and proliferation of human pulmonary artery smooth muscle cells via Akt-dependent signaling[END_REF] induced PAH, as well as PAH-associated right ventricle hypertrophy [START_REF] Yang | Resveratrol inhibits right ventricular hypertrophy induced by monocrotaline in rats[END_REF]. Furthermore, Ding et al. [START_REF] Ding | Calorie Restriction Attenuates Monocrotaline-induced Pulmonary Arterial Hypertension in Rats[END_REF] have recently shown that calorie restriction activates SIRT1 and SIRT1 overexpression can also ameliorate monocrotaline-induced PAH. However, all these studies used non-specific SIRT1 activators, resveratrol and calorie restriction, all of which affect a large range of other targets [START_REF] Bitterman | Metabolic effects of resveratrol: addressing the controversies[END_REF][START_REF] Testa | Calorie restriction and dietary restriction mimetics: a strategy for improving healthy aging and longevity[END_REF]. Moreover, SIRT1-triggered metabolic signaling pathways (which could be involved in resveratrol or calorie restriction beneficial effects on PAH) were not explored.

Here, we investigate the state of SIRT1 signaling pathway in human PAH PASMCs as well as in rat PASMCs stimulated with platelet-derived growth factor (PDGF). We demonstrate that the acetylation/deacetylation balance is shifted towards acetylation in PAH and PDGF-treated PASMCs. We show that SIRT1 is a negative regulator of PASMC growth: its pharmacological inhibition, and specific downregulation enhance human and rat PASMC proliferation. Moreover, we provide evidence for SIRT1 role in vascular remodeling, since its genetic deletion aggravates chronic hypoxia-induced pulmonary artery muscularization in mice. We then prove that SIRT1 can have beneficial effect in PAH treatment by using the SIRT1 specific activator Stac-3, which completely prevents PDGF-induced proliferation and, importantly, abolishes almost completely the proliferation of PASMCs from PAH patients without effect on the cell mortality. Finally, we show that SIRT1 activation is associated with the increase in the expression of genes implicated in mitochondrial function and the augmentation of mitochondrial biogenesis, thus providing a mechanistic explanation for the role of acetylation/deacetylation balance.

Material and Methods

Rat and human PASMCs

Primary culture of rat PASMCs were derived from 13 250-300 g male Wistar rats. After anaesthesia via intraperitoneal injection of sodium pentobarbital (100 mg/kg), the trachea, heart and lungs were rapidly removed, and the pulmonary artery isolated and deprived of residual adventitial tissue using dissection forceps and tweezers. Pulmonary artery was then cut in small pieces and incubated in 1% antibiotic/antimycotic (a/a) Dulbecco's modified eagle medium (DMEM) containing 150 U/mL type I collagenase and 0.125 mg/mL elastase at 37°C for 90 minutes under shaking. Tissue lysate was then filtered through a 70 µm cell strainer. Cells were collected and cultivated in 15% fetal bovine serum (FBS) and 1% a/a DMEM at 37°C in a 95% air-5% CO2 atmosphere till subconfluence. Medium was changed every other day. Culture material was previously coated with 50 µg/mL collagene I. Experiments were performed between passages 2 and 6. All animal care and experimental procedures conformed to the European Community guiding principles in the care and use of animals (Directive 2010/63/EU of the European Parliament) and authorizations to perform animal experiments according to this decree were obtained from the French Ministry of Agriculture, Fisheries and Food (No. D-92-283, 13 December 2012). Human PASMCs were prepared from pulmonary artery explants (5-10 mm of diameter) obtained during lung transplantation from 5 patients with idiopathic PAH and during lobectomy or pneumonectomy for localized lung cancer from 7 control (CTL) subjects (Table 1). In control subjects, pulmonary arteries explants were isolated at a distance from tumor areas. Transthoracic echocardiography was performed preoperatively in the control subjects to rule out pulmonary hypertension. Patients studied were part of the French Network on Pulmonary Hypertension, a program approved by our institutional Ethics Committee, and had given written informed consent (Protocol N8CO-08-003, ID RCB: 2008-A00485-50, approved on June 18, 2008). The principles of the Declaration of Helsinki were followed. Human PASMCs were cultivated in DMEM containing glucose (4.5 g/L), FBS (15%), Lglutamine (2 mmol/L), HEPES (20 mmol/L), epidermal growth factor (EGF) (10 U/mL), insulin-transferrin-selenium (ITS) 1X, normocin (0.1 mg/mL), 1% a/a, and used until passage 6.

Treatments of rat and human PASMCs After 24-hour incubation in 0.5% FBS and 1% a/a supplemented medium, PASMCs were pre-incubated for 2 hours with one of the following compounds diluted in the same medium: 10µmol/L Stac-3 (Santa Cruz, Heidelberg, Germany, sc-222315), 5 µmol/L sirtinol (Santa Cruz, Heidelberg, Germany, sc-205976) and 1 µmol/L SRT1720 (APExBIO, Houston, TX, A4180). Control cells were treated with vehicle (DMSO). Rat PASMC proliferation was then induced with 10 ng/mL PDGF for 24 hours. Since PAH PASMCs maintain their hyperproliferative phenotype in culture, no compound was used to stimulate their growth.

Evaluation of PASMC proliferation and death PASMC proliferation was measured via four different approaches. BrdU incorporation was evaluated using a kit (Millipore, Molsheim, France), following the manufacturer instructions. WST-1 (Sima Aldrich, Saint Quentin Fallavier, France) cleavage was measured by a spectrophotometer. Cell counting was performed using a Malassez haemocytometer. The protein amount of proliferating cell nuclear antigen (PCNA) was detected via Western blotting (WB). For measuring PASMC death, cells were labeled with 0.1 µg/mL fluorescein diacetate (FDA), and FDA negative cells were detected by flow cytometry (FC500, Beckman Coulter, Villepinte, France). To analyze late apoptosis/necrosis, cells were also labeled with 10 µg/mL propidium iodide (PI). Only late apoptotic/necrotic cells, which have lost their membrane integrity, can be labeled by this nucleic acid intercalating fluorescent agent. For each sample, at least 7 500 cells were analyzed. Cell aggregates and debris were excluded from analysis.

PASMC transient transfection

Reverse short interference RNA transfection was performed to downregulate SIRT1 in rat and human PASMCs. Cells were seeded in a transfection medium (Santa Cruz) containing transfection reagent (Santa Cruz) and a mix of three short interfering RNA specifically directed against SIRT1 (siSIRT1, Santa Cruz) or having no known target (control siRNA-A, Santa Cruz). 24 hours after the transfection, cells were cultivated for additional 24 hours with 0.5% FBS containing medium, then treated. Transfection rate, evaluated by flow cytometry using a FITC-coupled siRNA (Santa Cruz), was about 60%. Transfection efficiency was confirmed by SIRT1 protein level. Immunohistochemistry 7-µm thicked slices were cut and stained with α smooth muscle actin (αSMA) (Sigma, F3777, 1:200), Von Willebrand endothelial factor (VWF) (DAKO, A0082, 1:600) and 4 µmol/L Hoechst for nuclear staining. For each mouse, 100 small vessels (with a diameter <50µm) were examined by a blinded observer. Each vessel was categorized as non muscular (vessels without smooth muscle), partially muscular (vessels with smooth muscle layer in only part of their wall) or totally muscular (smooth muscle is identifiable in the entire vessel circumference).

Immunoprecipitation

The lysates of PASMCs, including 500 μg protein, were precleaned with protein A magnetic beads (Millipore) at 4°C for 1 hour, and then supernatants were collected. The lysates were incubated with anti-acetylated lysine antibody (Cell Signalling, 9681, 1:20) or with anti-mouse IgG antibodies (Santa Cruz, sc-2025, 1:20) at 4°C overnight. Immune complexes were collected then resuspended in denaturing lysis buffer and heated at 70°C for 10 minutes. The immunoprecipitated proteins were subjected to Western blot analysis for PGC-1α detection. Immunoblot 30 to 50 µg of protein extract were loaded onto 8-15% polyacrylamide gel, and then transferred onto a PVDF membrane (Immobilon, Millipore). Primary antibody (see supplementary Table S1) incubation was performed either overnight at 4°C or for 2 hours at room temperature. The incubation of peroxydasecoupled secondary antibody was realized for 1 hour at room temperature. Proteins were visualized using Luminata Crescendo or Forte (Millipore) peroxydase substrates using a CCD camera (ChemiDoc XRS+Bio-rad). Band density was quantified using Quantity One software (BioRad) and normalized to a housekeeping protein (β actin for human PASMCs or vinculin for rat PASMCs).

Quantitative RT-PCR Total RNAs were extracted from cells using TRIzol (Molecular Research Center). Briefly, chloroform was added to TRIzol, samples were centrifuged at 10000 g for 15 minutes at 4°C, and three phases were thus obtained: a protein containing phenolchloroform fraction, a DNA containing interphase and a RNA containing aqueous phase. The addition of isopropanol to the aqueous phase allowed RNA precipitation. Reverse transcription was performed from 1 µg RNA in 20µl using BioRad complementary DNA (cDNA) synthesis kit, following manufacturer instructions. Negative controls without reverse transcriptase were performed to verify the absence of genomic DNA in the samples. Gene expression was evaluated using a quantitative PCR system (BioRad) with SYBR-Green dye. PCR reaction was realized on 5 µl of the reverse transcription reaction product diluted by 20. Reaction mix contained 0.5 µmol/L sense and antisens primers (see supplementary Table S2). To confirm the absence of exogenous contaminations, a negative control without sample was realized. For each gene, a calibration curve was obtained analyzing cDNA series dilutions, and was used to calculate sample concentration and determine amplification efficiency. TBP (TATA-Binding Protein), RPL32 (Ribosomal Protein Large 32), RPLP2 (Ribosomal Protein Large P2) and 14-3-3 zeta/delta protein (YWHAZ) were used as reference genes for rat PASMCs to obtain a normalization factor calculated by GeNorm software. For human PASMCs, RPL32, TBP, RPLP0 (Ribosomal Protein Large P0) and RPL4 (Ribosomal Protein Large 4) were used as reference genes.

In vivo study

Inducible SIRT1 KO mice were kindly provided by Dr. D.A.Sinclair (Harvard Medical School, Boston, MA)1. 3-month old female mice (C57BL/6J carrying both the UBC-Cre-ERT2 and the SIRT1floxΔE4/floxΔE4 alleles) were subjected to an injection of tamoxifene (30mg/kg) per day for four consecutive days to induce SIRT1 deletion. WT mice were injected with the same concentration of vehicle (peanut oil). After three weeks, mice were exposed to chronic hypoxia (10% O2) in a ventilated chamber (Biospherix, New York, NY). A mixture of room air and nitrogen was flushed in the chamber to maintain the hypoxic environment. Normoxic mice were kept in the same room, with the same light/dark cycle. After three weeks of hypoxia exposure, mice were anesthetized with 4%, 3 L/min isoflurane. After skin incision of the sternum, direct transthoracic insertion of a catheter into the right ventricle was performed, and right ventricle pressure was measured via PowerLab 4/35 software. The thorax was opened, mice were then euthanatized via exsanguination from the aorta, and the lungs and heart rapidly removed. The right ventricle (RV) was dissected from the left ventricle plus septum (LV+S), and then the samples were weighted for determining the Fulton's index (RV/(LV+S)). Right lungs were injected with a solution 1:1 of OCT (optimal cutting temperature) and physiological serum, and then frozen for subsequent immunohistochemical analysis, and left lungs were frozen for WB.

Mitochondrial network analysis

Cells were incubated for 15 minutes at 37°C with 500 nmol/L MitoTracker Orange MTO for mitochondrial staining. Cells were then fixed with 4% PFA for 10 min at 37°C, auto-fluorescence was quenched via 5-minute incubation with 50 mmol/L NH4Cl and were mounted using mowiol mounting medium. 3D confocal images were acquired collecting a Z-series stack with 63x objective by an inverted Laser Scanning Microscope (Zeiss LSM 510). MTO was excited by 543 nm helium neon laser. 3D images were deconvoluted using AutoDeblur AutoVisualize software (MediaCybernetics) to increase confocal system resolution, and then analyzed using Imaris software (Bitplane Company) to provide information on mitochondrial volume.

Statistical analysis

The data are presented as mean±SEM. For cell culture experiments, n means number of batches each being derived from one patient/animal. For Stac-3 and SRT1720 treatment of rat PASMC, differences were determined using one-way analysis of variance followed by Newman-Keuls post-hoc test. For short interference experiments, sirtinol treatment and in vivo study, differences were evaluated using a two-way analysis of variance followed by Newman-Keuls post-hoc test. Human CTL and PAH PASMCs were compared using Mann-Whitney U test. Differences were considered as significant when p-value <0.05.

Results

Metabolic Profile in Human PAH PASMCs

In PASMCs from idiopathic PAH patients (PAH PASMCs), mitochondrial mass was decreased compared with control PASMCs (CTL PASMCs) as evidenced by reduced mitochondrial voltage-dependent anion channel (VDAC) and citrate synthase (CS) protein content (Fig. 1A). Consistently, the protein level of mitochondrial biogenesis master regulator PGC-1α was reduced in PAH PASMCs, and this was associated with a downregulation of its targets including the transcription factor peroxisome proliferator-activated receptor α (PPARα) and the estrogen-related receptor α (ERRα) as well as the antioxidant enzyme superoxide dismutase 2 (SOD2) (Fig. 1B). In parallel, the glucose transporter 1 (GLUT1) and lactate dehydrogenase (LDH) protein levels were increased (Fig. 1C). Thus, these results suggest an energy metabolism shift from mitochondrial oxidative phosphorylation towards glycolytic pathway in human PAH.

Acetylation/deacetylation balance in PAH PASMCs and PDGF-treated rat PASMCs

Deacetylation of PGC-1α is one of main regulators of its activity, therefore we hypothesized that a downregulation of mitochondrial biogenesis cascade in PAH PASMCs controlled by PGC-1α could be linked to altered acetylation/deacetylation balance. The deacetylase SIRT1 gene expression (Fig. 2A) and protein content of SIRT1 were unaffected in PAH PASMCs, as well as the protein content of nicotinamide phosphoribosyltransferase (NAMPT) (Fig. 2B), an enzyme that produces the SIRT1 cofactor NAD + [START_REF] Revollo | The NAD biosynthesis pathway mediated by nicotinamide phosphoribosyltransferase regulates Sir2 activity in mammalian cells[END_REF] . However, the balance between the acetylated and total forms of the SIRT1 targets PGC-1α and histone H1 (Fig. 2C) was shifted towards higher acetylation in PAH PASMCs. Thus, this shift towards acetylation suggests that the SIRT1 signaling pathway is impaired in PAH PASMCs. Qualitatively similar results were obtained in rat PASMCs, where PDGF-induced proliferation (Fig. 2D) was associated with increased content of the acetylated forms of PGC-1α and histone H1 as well as that of FOXO1, another SIRT1 target [START_REF] Motta | Mammalian SIRT1 Represses Forkhead Transcription Factors[END_REF] (Fig. 2E) .

Effect of SIRT1 downregulation or inhibition on human and rat PASMC proliferation

Next, we investigated whether altered acetylation/deacetylation balance could play a causative role in PASMC hyperproliferation. We downregulated SIRT1 expression by 85% via siRNA in control human PASMCs (Fig. 3A). The decline in SIRT1 protein level boosted CTL PASMC proliferation, as shown by the 50% increase in the incorporation of the thymidine analog BrdU by replicating cells (Fig. 3A). In order to confirm this finding, we silenced SIRT1 also in rat PASMCs. Again, the reduced level of SIRT1 (by almost 70%, Fig. 3B) enhanced PDGF-induced proliferation, as evidenced by the increase in cell number quantified by WST-1 cleavage by viable cells and BrdU incorporation (Fig. 3B). We further corroborated these observations by showing a facilitation of PDGF-induced proliferation also via SIRT1 pharmacological inhibition with sirtinol (Fig. 3C). Thus, these results indicate that SIRT1 downregulation could be a causative factor for PASMC hyperproliferation.

Impact of SIRT1 genetic deletion on pulmonary vascular remodeling

We then investigated the potential role of SIRT1 downregulation in vascular remodeling induced by chronic hypoxia exposure known to trigger SMC proliferation. Thus, SIRT1 inducible knock out (SIRT1 KO) mice and their wild type (WT) littermates were exposed to either chronic hypoxia or normoxia for three weeks. As represented in Fig. 4A, the deletion of SIRT1 exon 4 which encodes the catalytic domain led to the production of a non-functional protein. SIRT1 wild-type protein was significantly reduced in SIRT1 KO lungs both in normoxia and hypoxia, respectively by 67% and 80%. In control lungs, chronic hypoxia exposure induced a slight increase in PGC-1α protein level, which was completely absent in SIRT1 KO lungs. Interestingly, hypoxia-induced increase of the transcription factor HIF-1α was less pronounced in SIRT1 KO compared to wild-type (WT) mice, whereas GLUT1 induction after chronic hypoxia was higher in SIRT1 deficient mice. A deeper vascular remodeling, with an increase in the percentage of completely muscular pulmonary arteries, paralleled by a decrease in the percentage of nonmuscular arteries, was observed in mutant lungs. The percentage of partially muscular vessels was also slightly increased in KO compared to WT mice lungs (Fig. 4B). This was associated with an exacerbated increase in the right ventricle pressure and hypertrophy (Fig. 4C).Thus, the inactivation of SIRT1 aggravates vascular and cardiac remodeling in vivo.

Inhibition of PASMC hyperproliferation via SIRT1 pharmacological activation

To test whether correction of the acetylation/deacetylation balance could have beneficial effect on PASMC hyperproliferation, we investigated the effects of the pharmacological activator of SIRT1 Stac-3. As expected, Stac-3 significantly decreased the content of acetylated SIRT1 targets histone H1 and FOXO1 in rat PASMCs as well as PGC-1α (Fig. 5A). Since SIRT1 seemed to be also involved in the expression of NAMPT [START_REF] Nakahata | Circadian control of the NAD+ salvage pathway by CLOCK-SIRT1[END_REF][START_REF] Bellet | Pharmacological modulation of circadian rhythms by synthetic activators of the deacetylase SIRT1[END_REF], the rate-limiting enzyme in the NAD + biosynthetic pathway, we studied this expression and found that Stac-3 considerable increased NAMPT mRNA level (supplemental Fig. I), thus favoring the synthesis of the SIRT1 cofactor. Next, we studied the effect of Stac-3 on PDGF-induced proliferation in rat PASMCs. This treatment completely abolished PDGF proliferative effect (Fig. 5B). Moreover, the protein level of the proliferation marker PCNA was reduced when Stac-3 was added to PDGF-treated PASMCs (Fig. 5B). To further verify this observation, we used another SIRT1 activator, SRT1720. This compound also stunted PASMC proliferation but to a lesser extent (Fig. 5C). Importantly, the anti-proliferative effect of Stac-3 was not associated with an increase in cell death. Indeed, Stac-3 increased neither the percentage of cells unable to cleave the viability marker FDA nor the percentage of propidium iodide permeable necrotic cells (Fig. 5D). Given the inhibitory effect of SIRT1 activation on rat PASMC proliferation, we tested Stac-3 in PAH PASMCs. The proliferation of these cells was almost completely abolished, with a decrease by more than 50% of WST-1 cleavage, a reduction of BrdU incorporation by 80% and the diminution of the protein content of the proliferation marker PCNA by almost 50% (Fig. 5E).

The state of PGC-1α downstream cascade and mitochondrial biogenesis upon SIRT1 activation

To provide mechanistic insights into the pathways potentially implicated in Stac-3 effect on PASMC proliferation, mRNA and protein levels of several actors of mitochondrial bioenergetics were analyzed. Stac-3-mediated SIRT1 activation increased PAH PASMC mitochondrial mass, as evidenced by the elevation of VDAC and CS protein level (Fig. 6A). This augmentation can be explained by activation of mitochondrial biogenesis as Stac-3 increased the expression of PGC-1α molecular partners PPAR and ERRα as well as the expression of the antioxidant enzyme SOD2, known to be regulated by PGC-1α [START_REF] Valle | PGC-1alpha regulates the mitochondrial antioxidant defense system in vascular endothelial[END_REF] (Fig. 6B). Similar results were obtained in rat PASMCs, where Stac-3 also promoted mitochondrial biogenesis, as indicated by the increase of VDAC and CS protein level, as well as of TFAM (Fig. 6C). In rat cells, Stac-3 also activated the expression of PGC-1α partners (PPARα, ERRα and NRF2), and the elements of PGC-1α-triggered cascade (TFAM, antioxidant enzyme glutathione peroxidase 1, and mitochondrial deacetylase sirtuin 3) (Fig. 6D). Therefore, SIRT1 activation is clearly associated with the concerted upregulation of mitochondrial biogenesis pathway.

Effects of SIRT1 activation on mitochondrial morphology

Since mitochondrial fragmentation can favor the hyperproliferation, we also studied SIRT1 effect on mitochondrial morphology. Stimulation with PDGF induced mitochondrial fragmentation, which was abolished by Stac-3 treatment (Fig. 7A andB). SIRT1 activation thus normalizes mitochondrial morphology by inhibiting hyperproliferation-associated excessive fragmentation.

Discussion

The present study has highlighted a potential role of the deacetylase SIRT1, an important modulator of cell metabolism, in PASMC hyperproliferation, SIRT1 is a powerful negative regulator of rat and human PASMC proliferation and a deficiency in SIRT1 can be a factor favoring vascular remodeling. Conversely, SIRT1 activation could be considered as an effective tool to inhibit PASMC proliferation. PAH PASMCs exhibited reduced expression of VDAC and CS, mitochondrial mass markers, confirming the rarefaction of the mitochondrial network observed in patients' cells in an earlier study [START_REF] Bonnet | An abnormal mitochondrial-hypoxia inducible factor-1alpha-Kv channel pathway disrupts oxygen sensing and triggers pulmonary arterial hypertension in fawn hooded rats: similarities to human pulmonary arterial hypertension[END_REF]. This was associated with a negative regulation of mitochondrial biogenesis, as indicated by the decreased protein level of PGC-1α, also shown by Ryan et al. [START_REF] Ryan | PGC1αmediated mitofusin-2 deficiency in female rats and humans with pulmonary arterial hypertension[END_REF], as well as with the reduced expression of its targets, the transcription factors PPARα and ERRα and the antioxidant enzyme SOD2. The partial loss of mitochondrial mass by PAH PASMCs was counterbalanced by a considerable increase in GLUT1 expression, which agrees with the increase in glycolytic flux determined by the uptake of glucose analogue FDG by PAH patients' lungs [START_REF] Zhao | Heterogeneity in lung (18)FDG uptake in pulmonary arterial hypertension: potential of dynamic (18)FDG positron emission tomography with kinetic analysis as a bridging biomarker for pulmonary vascular remodeling targeted treatments[END_REF]. The LDH expression was also increased, thus favoring the uncoupling of glycolysis from glucose oxidation. Such a remodeling could contribute to the increased plasma lactate levels found in Chuvash PAH [START_REF] Formenti | Regulation of human metabolism by hypoxia-inducible factor[END_REF], as well as in idiopathic PAH patients [START_REF] Hu E-C | High levels of serum lactate dehydrogenase correlate with the severity and mortality of idiopathic pulmonary arterial hypertension[END_REF]. The observed loss of mitochondrial mass in PAH PASMCs suggests a negative regulation of mitochondrial biogenesis probably at least in part due to downregulation of the PGC-1α-regulated cascade. Indeed, protein content of this master regulator of mitochondrial biogenesis is lowered. Besides, its activity is known to be modulated by post-translational modifications, such as the acetylation/deacetylation balance. Regardless of the activity of cell acetylases and deacetylases, the domination of acetylated forms of various cell targets (histone H1, FOXO1, PGC-1α) in PAH and PDGF-stimulated rat PASMCs shows a clear shift of the balance towards acetylation. The causal link between weakened deacetylation activity and PASMC proliferation has been provided by experiments using SIRT1 silencing or pharmacological inhibition by sirtinol. Indeed, both interventions increased rat and human PASMC proliferation, providing the first evidence of pathogenic role of acetylation in PASMC hyperproliferation, which could be overcome by SIRT1 activity. The positive anti-proliferative role of SIRT1 was further confirmed in the experiments where SIRT1 deletion in mice significantly aggravated vascular remodeling induced by chronic hypoxia exposure. This effect was accompanied by an increase in right ventricle pressure and hypertrophy. Interestingly, SIRT1 deficiency abolished hypoxia-induced increase in PGC-1α protein level seen in WT lungs and this could attenuate PGC-1α-controlled pathways including mitochondrial biogenesis. We and others [START_REF] Chen | Hypoxia Increases Sirtuin 1 Expression in a Hypoxia-inducible Factor-dependent Manner[END_REF] have shown that hypoxia increases SIRT1 expression which is able to regulate PGC-1α transcription [START_REF] Amat | SIRT1 controls the transcription of the peroxisome proliferator-activated receptor-gamma Coactivator-1alpha (PGC-1alpha) gene in skeletal muscle through the PGC-1alpha autoregulatory loop and interaction with MyoD[END_REF]. In SIRT1 KO lungs, hypoxia-mediated increase in SIRT1 expression was blocked and this could abolish the augmentation of PGC-1α expression. A marked rise in GLUT1 expression in SIRT1 KO lungs argues that under hypoxic conditions, SIRT1 deficiency induces an imbalance in favor of glycolysis at the expense of oxidative phosphorylation.

Recently it has been demonstrated that SIRT1 can maintain mitochondrial biogenesis also via a PGC-1α-independent pathway [START_REF] Gomes | Declining NAD(+) induces a pseudohypoxic state disrupting nuclear-mitochondrial communication during aging[END_REF]. SIRT1 stabilizes the ubiquitin ligase Von Hippel-Lindau (VHL), which recognizes HIF-1α hydroxylated prolines, thus leading to the degradation of this transcription factor. Since HIF-1α inhibits c-myc, transcription factor activator of TFAM expression, SIRT1-mediated HIF-1α downregulation positively modulates TFAM and, consequently, mitochondrial gene expression [START_REF] Gomes | Declining NAD(+) induces a pseudohypoxic state disrupting nuclear-mitochondrial communication during aging[END_REF]. However, in our experiments, HIF-1α protein level was unaltered by SIRT1 deficiency under normoxia. In chronic hypoxia, SIRT1 KO lungs show a reduced HIF-1α response. Therefore, PAH aggravation in SIRT1 KO mice seems to be independent of this transcription factor. It has been proposed that the stimulation of mitochondrial metabolism could be a promising therapeutic approach to proliferative disorders such as cancer [START_REF] Galluzzi | Metabolic targets for cancer therapy[END_REF]. Reversing the metabolic shift from oxidative metabolism to glycolysis has proved to be efficient to treat PAH in animal models, and is currently the subject of clinical trials [START_REF] Ryan | Emerging Concepts in the Molecular Basis of Pulmonary Arterial Hypertension Part I: Metabolic Plasticity and Mitochondrial Dynamics in the Pulmonary Circulation and Right Ventricle in Pulmonary Arterial Hypertension[END_REF]. Here, we show that the pharmacological activation of SIRT1 by Stac-3 leads to a strong inhibition of PAH PASMCs proliferation without effect on cell viability. An increase in the expression of transcription factors involved in mitochondrial biogenesis was observed, with a subsequent increase in mitochondrial mass. These effects of Stac-3 seem to be a consequence of direct SIRT1 activation because Stac-3 has no effect on mitochondrial mass in cells reconstituted with activation-defective SIRT1 [START_REF] Hubbard | Evidence for a Common Mechanism of SIRT1 Regulation by Allosteric Activators[END_REF]. More detailed investigation carried out on rat PASMCs has shown that Stac-3 increases the expression of different factors maintaining mitochondrial biogenesis (PPARα, ERRα, NRF2, TFAM). Moreover, SIRT1 activation prevents PDGF-induced mitochondrial fragmentation. Interestingly, a recent study has demonstrated that mitochondrial fission is essential for PAH PASMC proliferation; its inhibition causes cell cycle arrest and ameliorates PAH experimental models [START_REF] Marsboom | Dynamin-Related Protein 1-Mediated Mitochondrial Mitotic Fission Permits Hyperproliferation of Vascular Smooth Muscle Cells and Offers a Novel Therapeutic Target in Pulmonary Hypertension[END_REF]. In parallel, mitofusin 2, a protein involved in mitochondrial fusion, is downregulated in PAH PASMCs, and the restoration of its expression inhibits PAH PASMC hyperproliferation [START_REF] Ryan | PGC1αmediated mitofusin-2 deficiency in female rats and humans with pulmonary arterial hypertension[END_REF]. Thus, prevention of proliferation-induced mitochondrial fission by Stac-3 could also contribute to inhibition of PASMC proliferation. Finally, Stac-3 treatment leads to the increase of SOD2 expression, whose downregulation has been shown to contribute to PAH PASMC hyperproliferation. Indeed, SOD2 promoter and enhancer were found to be hypermethylated leading to a decrease in its expression [START_REF] Archer | Epigenetic attenuation of mitochondrial superoxide dismutase 2 in pulmonary arterial hypertension: a basis for excessive cell proliferation and a new therapeutic target[END_REF]. This in turn led to a decrease in H2O2 production, thus allowing HIF-1α stabilization and KV potassium channel inhibition, both pro-proliferative events [START_REF] Archer | Epigenetic attenuation of mitochondrial superoxide dismutase 2 in pulmonary arterial hypertension: a basis for excessive cell proliferation and a new therapeutic target[END_REF]. The decrease in SOD2 expression could thus be due to a combination of epigenetic inhibition and downregulation of the co-activator controlling its transcription. Therefore, its induction upon SIRT1 activation could be partly responsible for Stac-3 anti-proliferative effects. Such a concerted upregulation of various factors controlled by PGC-1 supports the hypothesis that the metabolic remodeling associated with the hyperproliferation inhibition induced by Stac-3 involves PGC-1α activation via deacetylation. SIRT1mediated deacetylation of other targets may also play a role. Indeed, SIRT1 catalyses the deacetylation of FOXO1 thus potentiating its transcriptional activity [START_REF] Daitoku | Silent information regulator 2 potentiates Foxo1-mediated transcription through its deacetylase activity[END_REF]. Yet, PASMC FOXO1 is a critical integrator of multiple signaling pathways driving PAH, and reconstitution of FOXO1 activity is able to restore the physiologically quiescent PASMC phenotype. [START_REF] Savai | Pro-proliferative and inflammatory signaling converge on FoxO1 transcription factor in pulmonary hypertension[END_REF] Regardless of the relative contribution of PGC-1α or FOXO1 deacetylation to the inhibition of PASMC hyperproliferation, the activation of SIRT1 leads to the same endpoint.

In summary, our results demonstrate the causative link between imbalance of the acetylated/deacetylated state in PASMC associated with weakening of the oxidative phosphorylation machinery and PASMC hyperproliferation, a PAH hallmark. As activation of the deacetylase SIRT1 restores mitochondrial biogenesis and normalizes the PASMC phenotype, targeting SIRT1 activity is potentially a novel therapy to inhibit PASMC proliferation and thus delay the progression of PAH. Such a new strategy for PAH treatment, which reinforces cell oxidative capacities via correction of the acetylation/deacetylation balance may also be beneficial for the failing right ventricle overloaded in PAH. Given that SIRT1 upregulation seems to be also beneficial for the systemic arteries and for the heart (see [START_REF] Tanno | Emerging beneficial roles of sirtuins in heart failure[END_REF][START_REF] Liao | Let-7g improves multiple endothelial functions through targeting transforming growth factor-beta and SIRT-1 signaling[END_REF] and references therein), effects of specific SIRT1 activation on the entire cardiovascular system should also be evaluated.

(

  A) PAH PASMCs display a decreased protein level of VDAC and CS, two markers of mitochondrial mass (n=4). (B) The expression of the transcriptional co-activator PGC-1α is reduced in PAH PASMCs, characterized also by a decrease in the mRNA content of PPARα, ERRα and SOD2 (n=4). (C) The expression of glucose transporter GLUT1 and LDH is increased in PAH PASMCs compared to CTL PASMCs (n=4). * p<0.05 versus control.

Figure 2 .

 2 Figure 2. Hyperproliferation of PASMCs is associated with altered

Figure 3 .

 3 Figure 3. SIRT1 downregulation favors PASMC hyperproliferation.

Figure 4 .

 4 Figure 4. SIRT1 downregulation aggravates chronic hypoxia-induced vascular

Figure 5 .

 5 Figure 5. SIRT1 activator Stac-3 decreases cell acetylation state and abolishes rat and

Figure 6 .

 6 Figure 6. SIRT1 activation is associated with a mitochondrial biogenesis enhancement in

Figure 7 .

 7 Figure 7. SIRT1 activation inhibits PDGF-induced mitochondrial fragmentation.

Table 1 : Characteristics of PAH and control patients.

 1

	Patient Age	Gender	NYHA	mPAP,	Cardiac index,	Specific
		yr		class	mm Hg	L.min-1 .m-²	medication
	PAH	41	F	4	50	3.4	PDE5i, ERA
	PAH	44	F	4	50	N/C
	PAH	35	F	3	53	2.15	PDE5i, ERA, PC
	PAH	26	F	4	94	1.7	ERA, PC
	PAH	44	M	4	N/A	N/C	LTRA
	CTR	43	F
	CTR	56	F
	CTR	56	M
	CTR	64	M
	CTR	65	M
	CTR	78	M
	CTR	61	F
	PDE5i: phosphodiesterase class 5 inhibitors, ERA: endothelin receptor antagonists; PC:
	prostacyclin; LTRA: leukotriene receptor antagonists; N/C: non-communicated

Captions Figure 1. PAH PASMC metabolic profile is shifted from oxidative phopshorylation towards glycolysis.

Table S1 . List of antibodies.

 S1

	Antibody Antibody	Dilution Dilution	Manufacturer Manufacturer	Reference Reference
	Ac-FOXO Ac-FOXO	1:500 1:500	Santa Cruz, Heidelberg, Santa Cruz, Heidelberg,	sc-49437 sc-49437
			Germany Germany
	Ac-H1 Ac-H1	1:1000 1:1000	Sigma Aldrich, Saint Sigma Aldrich, Saint	H7789 H7789
			Quentin Fallavier, France Quentin Fallavier, France
	CS CS	1:500 1:500	Abcam, Cambridge, Abcam, Cambridge,	ab96600 ab96600
			United Kingdom United Kingdom
	GLUT1 GLUT1	1:500 1:500	Abcam, Cambridge, Abcam, Cambridge,	ab32551 ab32551
			United Kingdom United Kingdom
	FOXO FOXO	1:1000 1:1000	Ozyme, Saint Quentin en Ozyme, Saint Quentin en	2880S 2880S
			Yvelines, France Yvelines, France
	HIF-1α HIF-1α	1:500 1:500	Santa Cruz, Heidelberg, Santa Cruz, Heidelberg,	sc-10790 sc-10790
			Germany Germany
	H1 H1	1:500 1:500	Santa Cruz, Heidelberg, Santa Cruz, Heidelberg,	sc-10806 sc-10806
			Germany Germany
	NAMPT NAMPT	1:250 1:250	Abcam, Cambridge, Abcam, Cambridge,	ab45890 ab45890
			United Kingdom United Kingdom
	PCNA PCNA	1:1000 1:1000	Santa Cruz, Heidelberg, Santa Cruz, Heidelberg,	sc-56 sc-56
			Germany Germany
	PGC-1α PGC-1α	1:500 1:500	Santa Cruz, Heidelberg, Santa Cruz, Heidelberg,	sc-13067 sc-13067
			Germany Germany
	SIRT1 SIRT1	1:4000 1:4000	Abcam, Cambridge, Abcam, Cambridge,	sc-13067 sc-13067
			United Kingdom United Kingdom
	Tfam Tfam	1:500 1:500	Santa Cruz, Heidelberg, Santa Cruz, Heidelberg,	sc-23588 sc-23588
			Germany Germany
	VDAC VDAC	1:1000 1:1000	Home made Home made
	Vinculine Vinculine	1:2000 1:2000	Sigma Aldrich, Saint Sigma Aldrich, Saint	V9131 V9131
			Quentin Fallavier, France Quentin Fallavier, France
	β actine β actine	1:10000 1:10000	Santa Cruz, Heidelberg, Santa Cruz, Heidelberg,	sc-47778 sc-47778
			Germany Germany

Acknowledgments

We thank Dr. V. Nicolas (Imaging facility of IFR141), Dr. V. Domergue (Animal facility of IFR141) and Dr. C. Deloménie (Transcriptomic platform of IFR141) for technical help. The authors would also like to thank Dr.R. Fischmeister, Dr. A.-M. Gomez, Dr. S. Cohen-Kaminsky for continuous support and A. Truong for his kind proofreading.

This manuscript, or part of it, never has been published. This work was supported by INSERM (Institut national de la santé et de la recherche médicale, France) as well as by grants from Laboratory of Excellence LERMIT, FRM (Fondation pour la Recherche Médicale) to AG (#DPM20121125546) and Région Ile de France CODDIM to RVC (#cod110153).

Abbreviations

Table S2. Primer sequences.


Rossella Cannarella

Rupin Shah

Ramadan Saleh

Florence Boitrelle

Taha Abo-Almagd

Abdel-Meguid Hamoda

Rajender Singh

Gianmaria Salvio

Tuncay Toprak

Marco Falcone

Murat Gul

Ashok Agarwal

Taha Abo-Almag

Abde-Meguid Hamo

Fotios Dimitriad

Amarnath Rambhatla

Giorgio I Russo

Edmund Ko

Arma Zini

Parviz Kavou

Vinh Phuoc

Hussein Kand

Ramy Abo Ghayd

Ponco Birowo

Naz Gherabi

Erm Ceyhan

Jie Dong

Vin Malhotra

Damayant Durairajanayagam

Bircan Kolbasi

Fahm Bahar

Gokhan Calik

Selahit Çayan

Germ-Michael Pingg

Aldo E Caloger

Osvaldo Rajmil

Taymour Mostafa

Widi Atmoko

Ahme M Harraz

Tan V Le

Jean De La Rose

Lukman Hak

Edoardo Pescatori

Oleg Sergeyev

Ayman Rashed

Pallavi Saini

Effects of Varicocele Repair on Sperm DNA Fragmentation and Seminal Malondialdehyde Levels in Infertile Men with Clinical Varicocele: A Systematic Review and Meta-Analysis

Keywords: Infertility, male, Malondialdehyde, DNA fragmentation, Varicocele

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INTRODUCTION

Infertility is defined as the inability of a couple to conceive a child after one year of regular, unprotected sexual intercourse [START_REF]National public health action plan for the detection, prevention, and management of infertility[END_REF]. It has been recognized as a universal public health issue by the World Health Organization (WHO), with current evidence indicating 9% prevalence and 56% of couples seeking medical care [START_REF] Boivin | International estimates of infertility prevalence and treatment-seeking: potential need and demand for infertility medical care[END_REF]. An estimated 48.5 million couples that have unprotected sexual intercourse suffer from infertility worldwide [START_REF] Martinez | Fertility of men and women aged 15-44 years in the United States: national survey of family growth, 2006-2010[END_REF], with a male factor solely contributing to approximately 2.5% to 12% of couple infertility around the world [START_REF] Agarwal | A unique view on male infertility around the globe[END_REF].

Although semen analysis represents a crucial investigation in the infertile male, it remains sub-optimal in the evaluation and management of male infertility, with serious concerns regarding its validity in discerning between fertile and infertile men [START_REF] Snow-Lisy | What does the clinician need from an andrology laboratory?[END_REF][START_REF] Barbăroșie | Diagnostic value of advanced semen analysis in evaluation of male infertility[END_REF]. Sperm DNA fragmentation (SDF) has emerged as a useful tool in offering a more functional assessment of male fertility [START_REF] Santi | Sperm DNA fragmentation index as a promising predictive tool for male infertility diagnosis and treatment management -meta-analyses[END_REF][START_REF] Lewis | Sperm DNA tests as useful adjuncts to semen analysis[END_REF]. SDF is a marker of damaged chromatin in spermatozoa. Chromatin damage may include a broad spectrum of DNA defects such as single or double DNA strand breaks, base deletions or modifications, inter/intra-strand DNA cross-linkage, or protamine deficiency [START_REF] Esteves | A translational medicine appraisal of specialized andrology testing in unexplained male infertility[END_REF]. The rationale for assessing DNA integrity resides in the crucial role of chromatin compaction and structural stability in producing a fully functional sperm cell [START_REF] García-Peiró | Protamine 1 to protamine 2 ratio correlates with dynamic aspects of DNA fragmentation in human sperm[END_REF][START_REF] Rogenhofer | The sperm protamine mRNA ratio as a clinical parameter to estimate the fertilizing potential of men taking part in an ART programme[END_REF][START_REF] Ni | The impact of sperm protamine deficiency and sperm DNA damage on human male fertility: a systematic review and meta-analysis[END_REF][START_REF] Spanò | Sperm chromatin damage impairs human fertility. The Danish First Pregnancy Planner Study Team[END_REF]. The recent WHO Laboratory Manual for the Examination and Processing of Human Semen 6th edition has subsequently dedicated an extensive narrative highlighting the importance of SDF assessment in www.wjmh.org male infertility management [START_REF]WHO laboratory manual for the examination and processing of human semen[END_REF].

Several studies have demonstrated the association between elevated SDF and failure of natural pregnancy [START_REF] Spanò | Sperm chromatin damage impairs human fertility. The Danish First Pregnancy Planner Study Team[END_REF][START_REF] Evenson | Utility of the sperm chromatin structure assay as a diagnostic and prognostic tool in the human fertility clinic[END_REF][START_REF] Simon | A systematic review and meta-analysis to determine the effect of sperm DNA damage on in vitro fertilization and intracytoplasmic sperm injection outcome[END_REF][START_REF] Siddhartha | The effect of sperm DNA fragmentation index on the outcome of intrauterine insemination and intracytoplasmic sperm injection[END_REF]. In a systematic review and meta-analysis (SRMA) on males from couples suffering from recurrent idiopathic miscarriage, SDF levels were reported to be higher compared to that of fertile controls (mean difference [MD]=11.98%, 95% confidence interval [CI] 6.64-17.32 p<0.001) [START_REF] Tan | Association between sperm DNA fragmentation and idiopathic recurrent pregnancy loss: a systematic review and meta-analysis[END_REF]. Additionally, in the context of assisted reproductive technology (ART), SDF has been recognized as a factor that correlates with failed intrauterine insemination (IUI) [START_REF] Bungum | Sperm DNA integrity assessment in prediction of assisted reproduction technology outcome[END_REF][START_REF] Duran | Sperm DNA quality predicts intrauterine insemination outcome: a prospective cohort study[END_REF][START_REF] Rilcheva | Sperm DNA integrity test and assisted reproductive technology (Art) outcome[END_REF].

SDF is considered an indirect marker of oxidative stress (OS). OS is defined as the imbalance between the generation of reactive oxygen species (ROS) and the scavenger capacity. While low levels of ROS are needed for sperm development and for acquiring their fertilizing capacity, high levels or impairment of sperm antioxidant systems result in OS and infertility [START_REF] Agarwal | Male oxidative stress infertility (MOSI): proposed terminology and clinical practice guidelines for management of idiopathic male infertility[END_REF]. Spermatozoa are particularly vulnerable to OS, as ROS can disrupt the sperm cell membrane by lipoperoxidation and can lead to sperm protein and DNA damage. Seminal malondialdehyde (MDA) is a direct biomarker of OS, which reflects the degree of sperm cell lipoperoxidation by ROS and is paralleled with an increase of SDF and impairment of sperm motility [START_REF] Chen | Hydroxyl radicalinduced decline in motility and increase in lipid peroxidation and DNA modification in human sperm[END_REF]. Interestingly, sperm lipid peroxidation has been questioned as possibly involved in recurrent pregnancy loss (RPL). A study on 16 RPL cases indeed reported significantly higher levels of MDA in the patient group compared to fertile healthy controls [START_REF] Mohanty | Histone retention, protein carbonylation, and lipid peroxidation in spermatozoa: possible role in recurrent pregnancy loss[END_REF], thus configuring a possible future role of this test in the diagnosis of male infertility. Varicocele is one of the most common correctable causes of male infertility, which is found in around 15% of healthy men, 35%-44% of men with primary infertility, and up to 45%-80% of men with secondary infertility [START_REF] Clarke | Incidence of varicocele in normal men and among men of different ages[END_REF][START_REF] Gorelick | Loss of fertility in men with varicocele[END_REF][START_REF] Witt | Varicocele: a progressive or static lesion?[END_REF]. Its prevalence rises with age and most varicocele develops during puberty [START_REF] Oster | Varicocele in children and adolescents. An investigation of the incidence among Danish school children[END_REF][START_REF] Levinger | Is varicocele prevalence increasing with age?[END_REF]. Varicocele can cause male infertility through numerous mechanisms. Impairment of venous drainage may increase OS, increase scrotal temperature, induce testicular hypoxia, and cause abnormal reflux of metabolites [START_REF] Su | Pathophysiology and treatment options of varicocele: an overview[END_REF]. Varicocele can cause up to 2.6 °C temperature elevation in the scrotum [START_REF] Garolla | Twenty-four-hour monitoring of scrotal temperature in obese men and men with a varicocele as a mirror of spermatogenic function[END_REF]. Increasing scrotal temperature has a detrimental effect on spermatogenesis, including increased SDF and impaired sperm fertilization capability [START_REF] Agarwal | Role of oxidative stress in pathogenesis of varicocele and infertility[END_REF].

Varicoceles are associated with increased OS which can lead to DNA damage in sperm [START_REF] Agarwal | Insight into oxidative stress in varicocele-associated male infertility: part 1[END_REF]. Approximately 50% of men with clinically palpable varicocele have elevated SDF, and varicocele repair (VR) is associated with a decrease in SDF, especially in higher-grade varicoceles [START_REF] Smit | Decreased sperm DNA fragmentation after surgical varicocelectomy is associated with increased pregnancy rate[END_REF]. The reduction in SDF after VR may result in higher clinical pregnancy rates [START_REF] Ni | Sperm protamine mRNA ratio and DNA fragmentation index represent reliable clinical biomarkers for men with varicocele after microsurgical varicocele ligation[END_REF]. Despite evidence linking elevated SDF with varicoceles and improvement in SDF after VR, there is a paucity of recommendations in the major male infertility guidelines regarding VR in patients with a palpable varicocele and elevated SDF, as well as scenarios on when to obtain SDF testing.

Whether VR can influence seminal OS markersparticularly seminal MDA-is unknown. A better understanding of the impact of VR on SDF and OSbeyond the bulk semen parameters-can help guide the treatment of infertile men with varicoceles. The aim of this study is to conduct a SRMA to examine the impact of VR on SDF values as well as seminal MDA levels in infertile men.

MATERIALS AND METHODS

Search strategy

This meta-analysis was performed following the Meta-analysis Of Observational Studies in Epidemiology (MOOSE) guidelines [START_REF] Stroup | Meta-analysis of observational studies in epidemiology: a proposal for reporting. Meta-analysis of Observational Studies in Epidemiology (MOOSE) Group[END_REF] and the Preferred Reporting Items for SRMA Protocols (PRISMA-P) guidelines [START_REF] Page | PRISMA 2020 explanation and elaboration: updated guidance and exemplars for reporting systematic reviews[END_REF]. The data were extracted through extensive search in the Scopus, PubMed, Embase and Cochrane databases considering publications from the 1970s to December 2022. The search strategy included the combination of the following Medical Subjects Headings (MeSH) terms and keywords: varicoc*, management, embolization, mbolization, microsurg*, varicocelectomy, repair, correction, treatment, ligation, surg*, operation, radiolog*, sperm*, semen, seminal, ejaculate, asperm*, azoo*, azoosperm*, oligo*, oligosperm*, oligozoosperm*, astheno*, asthenosperm*, asthenozoosperm*, terato*, teratosperm*, teratozoosperm*, and necro*, necrosperm*, necrozoosperm*, sperm DNA fragmentation, SDF, DNA fragmentation index, DFI. The search was restricted to human studies and no language limitations were applied. The studies were initially assessed for inclusion by reading their titles and abstracts. When the abstract did not immediately reveal whether the study contained data relevant to our meta-analysis, the full text was read carefully. The evaluation for inclusion of the studies was performed by a team of 49 researchers selected after a training period, to allow an objective approach. In detail, each article was independently assessed for eligibility by two different researchers. Any disagreement was resolved by a third author. The protocol of this SRMA has been registered in the PROS-PERO database (CRD42022329848).

Selection criteria

This review included all studies on infertile males with clinical varicocele until December 2022 that measured SDF, and included data on seminal MDA levels when available, in varicocele treated patients, whose pre-treatment values are used as control. All the eligible studies were chosen following the Population, Intervention, Comparison/Comparator, Outcomes, and Study type (PICOS) model (Supplement Table 1) [START_REF] Methley | PICO, PICOS and SPIDER: a comparison study of specificity and sensitivity in three search tools for qualitative systematic reviews[END_REF]. Studies including adolescents, studies of subclinical varicocele, studies with no VR, in vitro studies, animal studies, case reports, case series, communications, and studies in which the control group received any treatment were excluded.

Data extraction

Data were extracted from the selected studies by 49 trained researchers distributed among four teams [START_REF] Cannarella | Need for training in research methodology prior to conducting systematic reviews and metaanalyses, and the effectiveness of an online training program: the Global Andrology Forum model[END_REF]. They worked in pairs, and a third senior author resolved disagreements. The following information was obtained from each study: authors, year of publication, study design, number of varicocele patients, assays used to assess sperm DNA integrity, type of VR, followup period, mean SDF value before treatment, mean SDF value after treatment, mean MDA seminal level before treatment and mean MDA seminal level after treatment. When all researchers had completed their extractions, we generated a combined database, and discrepancies were resolved by consensus among extractors under the control of the leader of each team.

Quality assessment

The quality of evidence (QoE) assessment aims to evaluate whether each article reports high-quality data. The QoE assessment of the articles was done by the Cambridge Quality Checklists [START_REF] Murray | Drawing conclusions about causes from systematic reviews of risk factors: the Cambridge quality checklists[END_REF]. In-depth, three checklists are devised for recognizing robust studies pertaining to associations, risk factors, and causal risk factors. The correlation checklist assesses sample size and outcome measurement quality. The risk factor checklist awards high scores to studies with suitable chronological data. The causal risk factor checklist evaluates study design. Strong inferences necessitate elevated scores across corresponding checklists: for associations, risk factors, and causal risk factors. Researchers double-checked the QoE assessment of all included articles. For each article, a screener made the initial assessment of QoE scores, a verifier checked the screener's assessment, and a third expert affirmed the final agreed score.

Statistical analysis 1) Quantitative data analysis

For the meta-analysis, the Comprehensive Meta-Analysis software (version-2; Biostat Inc) was used. Both the standard mean difference (SMD) and the MD were used as the measure of effect size for statistical comparison between preoperative and postoperative values. To check the heterogeneity across pooled studies, the heterogeneity index (I 2 ) was used. I 2 is the representation of heterogeneity and it generally lies between 0 and 100%, where <50% represents lowto-moderate heterogeneity and >50% suggests high heterogeneity. Both the fixed effect and random effects models were used to calculate the pooled effect size. In the case of low heterogeneity, the fixed effect model was used to draw the statistical inference. In the case of >50% heterogeneity, the random effects model was acquired. For generating high-resolution forest plots, the SMD and CI were considered for both the "all studies" and the pooled data [START_REF] Andrade | Understanding the difference between standard deviation and standard error of the mean, and knowing when to use which[END_REF]. Additionally, we performed a meta-regression analysis to evaluate the effect of preoperative SDF rates on SDF differences before and after VR.

2) Sensitivity analysis

The method of one study removal at a time was used for sensitivity analysis. One study was excluded at a time and their pooled effect size and CI were calculated to determine if that study was sensitive. If the exclusion of a particular study resulted in a change of the inference, it was labeled as a 'sensitive study'.

3) Publication bias analysis

Asymmetrical funnel plots were taken to qualitatively suggest the presence of publication bias. The presence of publication bias represents the missing studies www.wjmh.org from one side of the graph. We further used Egger's intercept test, for representing publication bias quantitatively.

RESULTS

Using the aforementioned search strategy, 1,632 abstracts were extracted. After the elimination of 665 duplicates, 967 abstracts were assessed. Of these, 96 articles were identified by title and abstract as review articles, case reports, book chapters, papers on different topics, and were deemed not eligible. Of the remaining 871 articles, 164 full-texts were not found, and after reading the full-text, an additional 678 papers were excluded because they contained non-extractable data, and 524 for reporting different outcomes. Finally, 29 studies assessing the impact of VR on SDF and MDA were included in the present analysis (Fig. 1) [START_REF] Smit | Decreased sperm DNA fragmentation after surgical varicocelectomy is associated with increased pregnancy rate[END_REF].

Among the 29 included studies, 23 reported data on SDF, 5 reported MDA levels and one study reported both SDF and MDA levels before and after VR (Table 1). In three studies [START_REF] Vahidi | Effects of microsurgical varicocelectomy on semen analysis and sperm function tests in patients with different grades of varicocele: role of sperm functional tests in evaluation of treatments outcome[END_REF][START_REF] Zaazaa | Effect of varicocelectomy and/or mast cells stabilizer on sperm DNA fragmentation in infertile patients with varicocele[END_REF][START_REF] Ni | A comprehensive investigation of sperm DNA damage and oxidative stress injury in infertile patients with subclinical, normozoospermic, and astheno/oligozoospermic clinical varicocoele[END_REF], the results were subdi-vided by grade of varicocele, in one study, by laterality of VR [START_REF] Sun | Bilateral is superior to unilateral varicocelectomy in infertile males with left clinical and right subclinical varicocele: a prospective randomized controlled study[END_REF], in one study by different time to evaluation [START_REF] Zini | Effect of microsur-gical varicocelectomy on human sperm chromatin and DNA integrity: a prospective trial[END_REF], and in one study the patients were subclassified after VR as responders or non-responders [START_REF] Smit | Decreased sperm DNA fragmentation after surgical varicocelectomy is associated with increased pregnancy rate[END_REF]. The main characteristics of the included studies are shown in Table 1. The QoE assessment is shown in Table 2.

Sperm DNA fragmentation

A total of 23 studies reported the SDF differences between before and after VR. Twenty-three studies showed that there was a significant change in SDF in varicocele patients after treatment. Overall, the random effect model (heterogeneity: I 2 =90.965%) revealed that the SDF was significantly reduced after VR (SMD: -1.256; 95% CI -1.529, -0.982; p<0.0001; MD: -6.924; 95% CI -8.438, -5.410; p<0.001) (Fig. 2).

Sensitivity analysis was performed, but no study was sensitive enough to change the conclusion that VR significantly decreases the SDF in the random model (Supplement Fig. 1). Publication bias was not apparent for all studies in the meta-analyses of SDF based on funnel plot and Egger's test (p=0.298) (Supplement Fig. 2).

Identification

1) Subgroup analysis: technique of varicocele repair

Subgroup analysis was performed based on the type of VR (including microsurgical, non-microsurgical inguinal, high retroperitoneal, laparoscopic, embolization, and others).

In 13 studies using microsurgical treatment, the random effect model (heterogeneity: I 2 =80.517%) showed that VR significantly decreases SDF (SMD: -1.014; 95% CI: -1.263, -0.765; p<0.0001) (Fig. 3).

Sensitivity analysis was performed but no study was sensitive enough to change the conclusion that microsurgical repair of varicocele significantly decreases SDF (Supplement Fig. 3). Publication bias was apparent in those studies according to the funnel plot and Egger's test (p=0.028). By bias correction, the adjusted effect value indicated that SDF in varicocele men was robustly reduced after microsurgical repair (Supplement Fig. 4).

In four studies using the non-microsurgical inguinal method, the random effect model (heterogeneity: 90%-50%) showed that VR significantly decreases SDF (SMD: -1.495; 95% CI: -2.116, -0.873; p<0.0001) (Fig. 4).

2) Subgroup analysis: technique of sperm DNA fragmentation analysis

Additionally, subgroup analysis was checked according to the techniques used in SDF estimation (including sperm terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate-nick end labeling [TUNEL], sperm chromatin structure assay [SCSA], chromatin dispersion assay [SCD] and others).

In four studies using SCD to measure SDF, the random effect model (heterogeneity: I 2 =94.958%), showed that VR significantly decreases the SDF (SMD: -2.197; 95% CI: -3.187, -1.207; p<0.0001) (Fig. 5). Sensitivity analysis was performed but no study was sensitive enough to change the main result (Supplement Fig. 5). Funnel plot and Egger's test (p=0.487) found that there was no publication bias in the four papers (Supplement Fig. 6).

In 6 studies using SCSA to analyze SDF, the random effect model (heterogeneity: I 2 =81.446%) showed there was a significant decrease in SDF (SMD: -0.857; 95% CI: -1.156, -0.559; p<0.0001) (Fig. 6). Sensitivity analysis was performed but did not reveal a change in the main meta-analysis result (Supplement Fig. 7). Publication bias was apparent in those studies according to the funnel plot and Egger's test (p=0.022). By bias adjustment, the corrected effect value indicated that SDF in varicocele men was robustly reduced after surgical repair when using SCSA testing (Supplement Fig. 8).

In five studies using TUNEL to measure SDF,

Model Study name

Kavoussi et al [START_REF] Kavoussi | The impact of ipsilateral testicular atrophy on semen quality and sperm DNA fragmentation response to varicocele repair[END_REF] Fathi et al [START_REF] Fathi | The impact of varicocelectomy on sperm DNA fragmentation and pregnancy rate in subfertile men with normal semen parameters: a pilot study[END_REF] Abbasi et al [START_REF] Abbasi | Alpha-lipoic acid improves sperm motility in infertile men after varicocelectomy: a triple-blind randomized controlled trial[END_REF] Camargo et al [START_REF] Camargo | Molecular pathways of varicocele and its repair -a paired labelled shotgun proteomics approach[END_REF] Afsin et al [START_REF] Afsin | An examination on composition of spermatozoa obtained from pre-operative and post-operative varicocele patients[END_REF] Vahidi et al [START_REF] Vahidi | Effects of microsurgical varicocelectomy on semen analysis and sperm function tests in patients with different grades of varicocele: role of sperm functional tests in evaluation of treatments outcome[END_REF] Sun et al [START_REF] Sun | Bilateral is superior to unilateral varicocelectomy in infertile males with left clinical and right subclinical varicocele: a prospective randomized controlled study[END_REF] (1)

Sun et al [START_REF] Sun | Bilateral is superior to unilateral varicocelectomy in infertile males with left clinical and right subclinical varicocele: a prospective randomized controlled study[END_REF] (2)

Zaazaa et al [START_REF] Zaazaa | Effect of varicocelectomy and/or mast cells stabilizer on sperm DNA fragmentation in infertile patients with varicocele[END_REF] Abdelbaki et al [START_REF] Abdelbaki | The impact of coexisting sperm DNA fragmentation and seminal oxidative stress on the outcome of varicocelectomy in infertile patients: a prospective controlled study[END_REF] Ni et al [START_REF] Ni | A comprehensive investigation of sperm DNA damage and oxidative stress injury in infertile patients with subclinical, normozoospermic, and astheno/oligozoospermic clinical varicocoele[END_REF] (1)

Ni et al [ ] (2) Ni et al [ ] (3)
Telli et al [START_REF] Telli | Does varicocelectomy affect DNA fragmentation in infertile patients?[END_REF] Tavalaee et al [START_REF] Tavalaee | Effect of varicocelectomy on sperm functional characteristics and DNA methylation[END_REF] Mohammed et al [START_REF] Mohammed | Acridine orange and flow cytometry: which is better to measure the effect of varicocele on sperm DNA integrity?[END_REF] Smit et al [START_REF] Smit | Decreased sperm DNA fragmentation after surgical varicocelectomy is associated with increased pregnancy rate[END_REF] Li et al [START_REF] Li | Significant improvement of sperm DNA quality after microsurgical repair of varicocele[END_REF] Gabriel et al [START_REF] Gabriel | Influence of microsurgical varicocelectomy on human sperm mitochondrial DNA copy number: a pilot study[END_REF] La Vignera et al [START_REF] Vignera | Effects of varicocelectomy on sperm DNA fragmentation, mitochondrial function, chromatin condensation, and apoptosis[END_REF] Sadek et al [START_REF] Sadek | Sperm chromatin condensation in infertile men with varicocele before and after surgical repair[END_REF] Zini et al [START_REF] Zini | Effect of microsur-gical varicocelectomy on human sperm chromatin and DNA integrity: a prospective trial[END_REF] (1)

Zini et al [START_REF] Zini | Effect of microsur-gical varicocelectomy on human sperm chromatin and DNA integrity: a prospective trial[END_REF] (2)

Zini et al [START_REF] Zini | Effect of microsur-gical varicocelectomy on human sperm chromatin and DNA integrity: a prospective trial[END_REF] (3)

Ghazi and Abdelfattah [START_REF] Ghazi | Effect of microsurgical varicocelectomy on sperm DNA fragmentation in infertile men[END_REF] Dada et al [START_REF] Dada | Attenuation of oxidative stress & DNA damage in varicocelectomy: implications in infertility management[END_REF] Nasr Esfahani et al [START_REF] Esfahani | Can altered expression of HSPA2 in varicocele patients lead to abnormal spermatogenesis?[END_REF] Smit et al [START_REF] Smit | Decreased sperm DNA fragmentation after surgical varicocelectomy is associated with increased pregnancy rate[END_REF] (1)

Smit et al [START_REF] Smit | Decreased sperm DNA fragmentation after surgical varicocelectomy is associated with increased pregnancy rate[END_REF] (2)

Zini et al the random effect model (heterogeneity: I 2 =90.588%) showed that VR significantly decreases SDF (SMD: -1.599; 95% CI: -2.478, -0.719; p<0.0001) (Fig. 7). Sensitivity analysis was performed but no study was sensitive enough to change the conclusion (Supplement Fig. 9). The funnel plot and Egger's test (p=0.116) found that there was no publication bias in the five papers (Supplement Fig. 10).

In the three studies which did not describe the spe-cific testing methods for SDF, the random effect model (heterogeneity: I 2 =95.361%) showed that VR significantly improved the SDF (SMD: -1.375; 95% CI: -2.483, -0.266; p=0.015) (Fig. 8). Sensitivity analysis was performed but no study was sensitive enough to change the conclusion (Supplement Fig. 11). The funnel plot and Egger's test (p=0.137) found that there was no publication bias in the three papers (Supplement Fig. 12).

Model Study name

Kavoussi et al [START_REF] Kavoussi | The impact of ipsilateral testicular atrophy on semen quality and sperm DNA fragmentation response to varicocele repair[END_REF] Fathi et al [START_REF] Fathi | The impact of varicocelectomy on sperm DNA fragmentation and pregnancy rate in subfertile men with normal semen parameters: a pilot study[END_REF] Abbasi et al [START_REF] Abbasi | Alpha-lipoic acid improves sperm motility in infertile men after varicocelectomy: a triple-blind randomized controlled trial[END_REF] Camargo et al [START_REF] Camargo | Molecular pathways of varicocele and its repair -a paired labelled shotgun proteomics approach[END_REF] Vahidi et al [START_REF] Vahidi | Effects of microsurgical varicocelectomy on semen analysis and sperm function tests in patients with different grades of varicocele: role of sperm functional tests in evaluation of treatments outcome[END_REF] Sun et al [START_REF] Sun | Bilateral is superior to unilateral varicocelectomy in infertile males with left clinical and right subclinical varicocele: a prospective randomized controlled study[END_REF] (1)

Sun et al [START_REF] Sun | Bilateral is superior to unilateral varicocelectomy in infertile males with left clinical and right subclinical varicocele: a prospective randomized controlled study[END_REF] (2)

Zaazaa et al [START_REF] Zaazaa | Effect of varicocelectomy and/or mast cells stabilizer on sperm DNA fragmentation in infertile patients with varicocele[END_REF] Ni et al [ ] (1)

Ni et al [ ] (2) Ni et al [ ] (3)
Li et al [START_REF] Li | Significant improvement of sperm DNA quality after microsurgical repair of varicocele[END_REF] Gabriel et al [START_REF] Gabriel | Influence of microsurgical varicocelectomy on human sperm mitochondrial DNA copy number: a pilot study[END_REF] La Vignera et al [START_REF] Vignera | Effects of varicocelectomy on sperm DNA fragmentation, mitochondrial function, chromatin condensation, and apoptosis[END_REF] Zini et al [START_REF] Zini | Beneficial effect of microsurgical varicocelectomy on human sperm DNA integrity[END_REF] Zini et al [START_REF] Zini | Effect of microsur-gical varicocelectomy on human sperm chromatin and DNA integrity: a prospective trial[END_REF] Ghazi and Abdelfattah Li et al [START_REF] Li | Significant improvement of sperm DNA quality after microsurgical repair of varicocele[END_REF] Gabriel et al [START_REF] Gabriel | Influence of microsurgical varicocelectomy on human sperm mitochondrial DNA copy number: a pilot study[END_REF] Sadek et al [START_REF] Sadek | Sperm chromatin condensation in infertile men with varicocele before and after surgical repair[END_REF] Smit et al [START_REF] Smit | Decreased sperm DNA fragmentation after surgical varicocelectomy is associated with increased pregnancy rate[END_REF] (1)

Smit et al [START_REF] Smit | Decreased sperm DNA fragmentation after surgical varicocelectomy is associated with increased pregnancy rate[END_REF] (2)

Model Study name

La Vignera et al [START_REF] Vignera | Effects of varicocelectomy on sperm DNA fragmentation, mitochondrial function, chromatin condensation, and apoptosis[END_REF] Tavalaee et al [START_REF] Tavalaee | Effect of varicocelectomy on sperm functional characteristics and DNA methylation[END_REF] Afsin et al [START_REF] Afsin | An examination on composition of spermatozoa obtained from pre-operative and post-operative varicocele patients[END_REF] Vahidi et al [START_REF] Vahidi | Effects of microsurgical varicocelectomy on semen analysis and sperm function tests in patients with different grades of varicocele: role of sperm functional tests in evaluation of treatments outcome[END_REF] Abbasi et al [START_REF] Abbasi | Alpha-lipoic acid improves sperm motility in infertile men after varicocelectomy: a triple-blind randomized controlled trial[END_REF] Std diff in means and 95% CI

Model Study name

Kavoussi et al [START_REF] Kavoussi | The impact of ipsilateral testicular atrophy on semen quality and sperm DNA fragmentation response to varicocele repair[END_REF] Fathi et al [START_REF] Fathi | The impact of varicocelectomy on sperm DNA fragmentation and pregnancy rate in subfertile men with normal semen parameters: a pilot study[END_REF] Zaazaa et al [START_REF] Zaazaa | Effect of varicocelectomy and/or mast cells stabilizer on sperm DNA fragmentation in infertile patients with varicocele[END_REF] Nasr Esfahani et al [START_REF] Esfahani | Can altered expression of HSPA2 in varicocele patients lead to abnormal spermatogenesis?[END_REF] Std diff in means and 95% CI www.wjmh.org

Malondialdehyde

A total of 6 studies analyzed seminal MDA as an outcome before and after VR in infertile men. The random effect model (heterogeneity: I 2 =97.3%) showed that VR significantly reduced seminal MDA levels (SMD: -2.450; 95% CI: -3.903, -0.997; p=0.001; MD: -2.394; 95% CI -2.524, -1.275; p<0.001) (Fig. 9). As an overall, there appeared to be no study that was sensitive enough to alter the findings that VR lowers seminal MDA levels (Supplement Fig. 13). Based on the symmetry of the standard error and precision Funnel plots respectively (Supplement Fig. 14), as well as the Egger's test (p>0.05), no publication bias was apparent.

Meta-regression analysis

The meta-regression analysis revealed that the SDF reduction after VR was independent of the preoperative SDF levels. In the present analysis, the magnitude of the effect size did not increase as a function of preoperative SDF levels (coefficient: -0.01; 95% CI: -0.03, 0.02; p=0.64) (Fig. 10).

DISCUSSION

Varicocele is linked with DNA damage of spermatozoa due to elevated OS and decreased antioxidant capacity. In this meta-analysis, we strove to evaluate the effect of VR on SDF and seminal MDA levels in infertile patients with clinical varicocele. For SDF, a total of 23 studies were found to investigate the SDF differences before and after VR and a significant reduction was seen in SDF levels after VR. The improvement in SDF levels after VR emerged regardless of the surgical technique and methods that were used in SDF estimation. For the MDA, a total of six studies were included in the analysis, finding a significant decrease after VR.

Model Study name

Telli et al [START_REF] Telli | Does varicocelectomy affect DNA fragmentation in infertile patients?[END_REF] Mohammed et al [START_REF] Mohammed | Acridine orange and flow cytometry: which is better to measure the effect of varicocele on sperm DNA integrity?[END_REF] Zini et al [START_REF] Zini | Beneficial effect of microsurgical varicocelectomy on human sperm DNA integrity[END_REF] Std diff in means and 95% CI

Model Study name

Kamal et al [START_REF] Kamal | Association between seminal granulysin and malondialdehyde in infertile men with varicocele and the potential effect of varicocelectomy[END_REF] Omar et al [START_REF] Omar | Relation of nitric oxide synthase gene (NOS3) polymorphisms to varicocele risk and post-varicocelectomy seminal oxidative stress reduction[END_REF] Mostafa et al [START_REF] Mostafa | Seminal SIRT1-oxidative stress relationship in infertile oligoasthenoteratozoospermic men with varicocele after its surgical repair[END_REF] Lu et al [START_REF] Lu | Melatonin therapy adds extra benefit to varicecelectomy in terms of sperm www.wjmh.org parameters, hormonal profile and total antioxidant capacity: a placebo-controlled, double-blind trial[END_REF] Ni The American Urological Association/American Society for Reproductive Medicine (AUA/ASRM) guideline on male infertility suggests performing VR in the setting of clinically palpable varicocele, infertility, and abnormal sperm parameters on semen analysis [START_REF] Schlegel | Diagnosis and treatment of infertility in men: AUA/ASRM guideline part I[END_REF]. This guideline does not specifically address the role of VR in men with abnormal SDF. The European Association of Urology (EAU) male infertility guideline states that varicoceles can be a source of elevated SDF, and VR can be considered in men with increased SDF with abnormal semen parameters [START_REF] Minhas | EAU Working Group on Male Sexual and Reproductive Health. European Association of Urology guidelines on male sexual and reproductive health: 2021 update on male infertility[END_REF]. It is unclear whether VR should be offered to men who have elevated SDF in the setting of normal semen parameters, unexplained infertility, or those who have suffered from failed ART or RPL [START_REF] Minhas | EAU Working Group on Male Sexual and Reproductive Health. European Association of Urology guidelines on male sexual and reproductive health: 2021 update on male infertility[END_REF].

The lack of specific guidelines concerning the benefits and indications of VR for male infertility may raise doubts as to the implementation of this therapy in daily clinical practice [START_REF] Minhas | EAU Working Group on Male Sexual and Reproductive Health. European Association of Urology guidelines on male sexual and reproductive health: 2021 update on male infertility[END_REF]. Several studies have shown that there was an increase in SDF in varicocele cases and that VR could reduce these SDF levels [START_REF] Smit | Decreased sperm DNA fragmentation after surgical varicocelectomy is associated with increased pregnancy rate[END_REF]. Furthermore, increased levels of MDA can also be used as an indicator of high OS which is also one of the proposed effects of varicocele. Additionally, significant improvement was seen in MDA levels after VR, indicating a reduction in OS.

Of the 6 studies examined in the SRMA on MDA, all were found to show a significant reduction after VR. Previous studies have shown that MDA levels are increased in men with varicocele, and it has been shown that VR reduces MDA levels [START_REF] Kamal | Association between seminal granulysin and malondialdehyde in infertile men with varicocele and the potential effect of varicocelectomy[END_REF]. When calculated cumulatively with this SRMA, the increase in MDA levels in varicocele patients and the decrease after VR support the concept that VR decreases the levels of sperm lipoperoxidation, which can concur with loss of pregnancy [START_REF] Mohanty | Histone retention, protein carbonylation, and lipid peroxidation in spermatozoa: possible role in recurrent pregnancy loss[END_REF].

We examined the strengths of our MA compared to previously published SRMA (Table 3 and Fig. 11). Baazeem et al [START_REF] Baazeem | Varicocele and male factor infertility treatment: a new meta-analysis and review of the role of varicocele repair[END_REF] in their meta-analysis took into account only 3 prospective studies about sperm DNA damage. In those studies, all the investigators recorded pre-and post-VR sperm DFI (DNA fragmentation index). Two out of 3 studies found a statistically significant decrease in DFI postoperatively, however, in the remaining study, only 10 out of 25 patients completed the follow-up so the improvement in DNA fragmentation was not significant. In our opinion, the strength of this meta-analysis is that all the articles involved used the same method (SCSA) to measure the DFI for better comparison. Another recent meta-analysis involved 11 prospective studies with various methods of DNA damage measurement [START_REF] Qiu | Efficacy of varicocelectomy for sperm DNA integrity improvement: a meta-analysis[END_REF]. In the present meta-analysis, patients served as their controls, as preoperative parameters were used as control values. On average, the DFI % of clinical varicocele patients decreased by 5.79 (95% CI, -7.39 to-4.19) after VR. Furthermore, in the fixed effect model for 10 studies (number of par-ticipants=364, heterogeneity chi-squared=15.15, df=9, p=0.087, I 2 =40.6%), DFI% decreased by 6.14 (95% CI, -6.90 to -5.37) on average.

In their 2012 MA, Wang and colleagues reported that patients with varicoceles had significantly higher sperm DNA damage than controls, with a MD of 9.84% (95% CI 9.19-10.49; p<0.00001). Authors stated that a VR performed with either microsurgical or an unspecified method can improve sperm DNA integrity, with a MD of 3.37% (95% CI -4.09 to -2.65; p<0.00001) [START_REF] Wang | Relationship between varicocele and sperm DNA damage and the effect of varicocele repair: a meta-analysis[END_REF].

In another recent meta-analysis [START_REF] Birowo | The effects of varicocelectomy on the DNA fragmentation index and other sperm parameters: a meta-analysis[END_REF], the sperm DFI decreased by 6.86% after VR (MD: -6.86; 95% CI: -10.04, -3.69; p<0.00001). SCSA was the preferred method in most of the included studies, with the exception of two studies that used the TUNEL assay.

The most recent and largest MA on this topic was reported by Lira Neto et al [START_REF] Neto | Effect of varicocelectomy on sperm deoxyribonucleic acid fragmentation rates in infertile men with clinical varicocele: a systematic review and meta-analysis[END_REF] in 2021. VR was associated with postoperative decreased SDF rates (weighted mean difference -7.23%; 95% CI: -8.86 to -5.59; I 2 =91%). The pooled results were consistent for studies using SCSA, TUNEL, SCD, Comet, and microsurgical VR which in our opinion is a strength of this SRMA. Subgroup analyses in the latter study demonstrated a more pronounced treatment effect in men with elevated preoperative SDF levels. Indeed, their metaregression analysis showed that SDF decreased after VR as a function of preoperative SDF levels (coefficient: 0.23; 95% CI 0.07-0.39). Surprisingly, the present study found that SDF reduction after VR was independent of the preoperative SDF levels. In other words, this indicates that the extent of SDF improvement after VR is similar in both patients with either high and low SDF. The reasons for the finding of opposing results are not immediately clear. The methods used in this study are very similar to those used in the study by Lira Neto et al [START_REF] Neto | Effect of varicocelectomy on sperm deoxyribonucleic acid fragmentation rates in infertile men with clinical varicocele: a systematic review and meta-analysis[END_REF]. However, having been conducted more recently, it could be supposed that the increase in the size of the population examined in the present study (which is the largest examined so far) could represent one of the main factors responsible for the results.

Till now, five meta-analyses were conducted with a total of 2,014 patients investigating the effect of VR on SDF. Four meta-analyses included prospective studies and one meta-analysis included both prospective and retrospective studies. All meta-analyses indicate that sperm DNA damage is reduced after VR. Our current meta-analysis carried out on 1,523 patients (considering only studies on SDF), confirms and supports the current literature, adding 453 patients.

Up until now, there was no meta-analysis in the literature investigating the effect of VR on seminal levels of MDA in infertile patients with clinical varicocele. An interesting aspect of our meta-analysis is that it reports, for the first time, a reduction in MDA after VR.

Before concluding we would like to underline the strength and limitations of this study using a SWOT (strengths, weaknesses, opportunities, and threats) analysis (Fig. 11). The strengths of the present study were the inclusion of 29 articles that collected a total of 1491 patients. Additionally, we conducted subgroup analyses to assess heterogeneity and conducted the sensitivity analysis. Finally, our SRMA is the first one to have meta-analyzed the effects of VR on MDA. One limitation was the fact that our meta-analysis did not compare the impact of one VR technique to another. Furthermore, we did not evaluate the impact of different degrees of varicocele on SDF improvement after VR. Finally, we were unable to assess the impact of VR on the SDF rate as evaluated by the 8-hydroxydeoxyguanosine or Comet assays. The results of this SRMA should be a guide for professional organizations connected with the field of male infertility to further guide on the need for VR in men with not only quantitative indicators of male infertility in the conventional semen analysis but qualitative indicators of sperm dysfunction via SDF and MDA.

Fig. 1 .

 1 Fig. 1. Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA-P) flow-chart for inclusion/exclusion of studies.

Fig. 6 .

 6 Fig.6. Forest plot of the sperm DNA fragmentation in infertile patients after varicocele repair compared to before varicocele repair using SCSA. 95% CI: 95% confidence interval.

Fig. 7 .

 7 Fig. 7. Forest plot of the sperm DNA fragmentation in infertile patients after varicocele repair compared to before varicocele repair using TUNEL. 95% CI: 95% confidence interval.

Fig. 5 .

 5 Fig. 5. Forest plot of the sperm DNA fragmentation in infertile patients after varicocele repair compared to before varicocele repair using SCD. 95% CI: 95% confidence interval.

Fig. 8 .

 8 Fig.8. Forest plot of the sperm DNA fragmentation (SDF) in infertile patients after varicocele repair compared to before varicocele repair using a not specified methods for analysis of SDF. 95% CI: 95% confidence interval.

Fig. 9 .

 9 Fig. 9. Forest plot of seminal malondialdehyde level in infertile patients after varicocele repair compared to before varicocele repair. 95% CI: 95% confidence interval.

Fig. 10 .

 10 Fig. 10. Metaregression showing the influence of pre-treatment sperm DNA fragmentation (SDF) levels (moderator) on the standard difference of the mean of SDF after varicocelectomy. The size of the circles indicates the studies'sample sizes. The red line represents linear predictions for the standard mean difference of SDF change after varicocelectomy as a function of the mean absolute increase in preoperative SDF levels. The curved lines indicate the 95% confidence interval lines around the regression line.

Fig. 11 .

 11 Fig. 11. Strengths, Weaknesses, Opportunities, and Threats (SWOT) Analysis. SRMA: systematic review and metaanalysis, SDF: sperm DNA fragmentation, MDA: malondialdehyde, VR: varicocele repair, 8OHDG: 8-hydroxydeoxyguanosine.

Table 1 .

 1 Main characteristics of the included studies

	First author	Year Study design	Varicocele Side Grade Type Side Varicocele repair	Time to evaluation (mo)	Patients (n)	SDF (%) Before	After	MDA (nmol/mL) Before After
	Kavoussi [42]	2021 Observational	-	-	MS	-	3	121	35.3±11.6 19.6±5.3
	Fathi [43]	2021 Observational	-	-	MS	-	6	45 34.93±5.56 25.75±5.15
	Abbasi [44]	2020 Observational	-	-	MS	-	3	22 13.65±11.44 10.38±4.64
	Kamal [45]	2020 Observational	-	-	NMI	-	6	34			1.9±0.6	1.1±0.3
	Omar [46]	2020 Observational	-	-	MS	-	3	100			7.7±1.1	2.2±0.6
	Mostafa [47]	2020 Observational	-	-	NMI	-	3	30			3.4±1.3	1.9±0.7
	Camargo [48]	2019 Observational	-	-	MS	-	12	25	43.4±22.62 32.9±8.87
	Afsin [49]	2018 Observational	-	-	-	-	12	40 20.57±4.6	15.3±3.63
	Vahidi [50]	2018 Observational Left	-	MS	-	3	30 15.93±4.96 10.86±4.44
				1	MS	-	3	5	15.4±9.42	5.2±1.09
				2	MS	-	3	13	16±3.39 12.07±4.71
				3	MS	-	3	12 16.08±4.46 11.91±3.14
	Sun [51]	2018 Observational Bilateral	-	MS Bilateral	12	179	21.6±7.1	11.8±6
						Left	12	179	23±8.1	12.1±6.8
	Lu [52]	2018 Observational Left	-	MS Left	6	27			1.75±0.22 0.58±0.06
	Zaazaa [53]	2018 RCT	-	-	MS	-	3	40	34.6±4.1	28.3±5.2
				2	MS	-	3	14	32.7±1.6	28±2.3
				3	MS	-	3	26	35.5±4.7	28.5±6.3
	Abdelbaki [54]	2017 Observational	-	-	NMI	-	6	55 29.49±8.58 18.78±7.23
	Ni [55]	2016 Observational	-	1	MS	-	6	19 23.56±7.55 19.54±5.48 12.18±4.86 8.76±2.73
				2	MS	-	6	18 27.75±9.05 22.38±4.54 14.12±5.42 9.71±2.83
				3	MS	-	6	14 30.03±8.27 21.82±5.95 15.86±6.78 9.5±3.28
	Telli [56]	2015 Observational	-	-	NMI	-	3	72	34.5±3.3	28.2±3.5
	Tavalaee [57]	2015 Observational	1	-	-Left	3	23	15.9±1.2	10.8±1.1
	Mohammed [58] 2015 Observational	-	-	NMI	-	6	75	32.4±7.4	20±4.1
	Smit [59]	2013 Observational	-	-	-	-	3	49	35.2±13.1 30.2±14.7
	Li [60]	2012 Observational	-	-	MS	-	3	19	28.4±15.6 22.4±12.9
	Gabriel [61]	2012 Observational	-	-	MS	-	4	14	22.4±11.9 12.6±6
	La Vignera [62]	2012 Observational Left	3	MS Left	4	30	5±3	2.1±0.4
	Sadek [63]	2011 Observational Bilateral	-	NMI	-	3	72 65.15±20.97 51.9±15.2
	Zini [64]	2011 Observational	-	-	MS	-	4-6	25	18±11	11±6
							4	19	18±11	10±5
							6	19	18±11		7±3
	Ghazi [65]	2011 Observational Bilateral	-	MS Bilateral	6	82	21.5±11.2	13±14.3
	Dada [66]	2010 Observational	-	-	-	-	6	11 60.82±7.12 32.58±6.12
	Nasr Esfahani [67] 2010 Observational	-	3	-	-	6	70 45.69±2.43 35.9±2.4
	Smit [34]	2010 Observational	-	-	-	-	3	31 a 35.3±14.3 28.6±14.7
								18 b	35±11.2	33±14.8
	Zini [68]	2005 Observational	-	-	MS	-	6	37	27.7±17.64 24.6±16.42
	Yeşilli [69]	2005 Observational	-	-	MS	-	6	26			0.61±0.09 0.58±0.06
	Values are presented as mean±standard deviation.

MDA: malondialdehyde, MS: microsurgical varicocele repair, NMI: non-microsurgical inguinal varicocele repair, RCT: randomized controlled trial, SDF: sperm DNA fragmentation. a Responders to varicocele repair (improvement >50% in sperm concentration), b Non-responders to varicocele repair. www.wjmh.org

Table 2 .

 2 Quality of evidence assessment (results of the Cambridge Quality Checklist[START_REF] Murray | Drawing conclusions about causes from systematic reviews of risk factors: the Cambridge quality checklists[END_REF])

	First author	Year	Cambridge Quality Checklist	Total Quality score (3

-15) Checklist for correlates (1-5) Checklist for risk factors (1-3) Checklist for causal risk factor (1-7)

	Kavoussi [42]	2021	1	2	7
	Fathi [43]	2021	2	3	9
	Abbasi [44]	2020	1	2	6
	Kamal [45]	2020	2	3	11
	Omar [46]	2020	1	3	7
	Mostafa [47]	2020	2	3	8
	Camargo [48]	2019	1	3	7
	Afsin [49]	2018	3	3	9
	Vahidi [50]	2018	2	3	8
	Sun [51]	2018	2	3	8
	Zaazaa [53]	2018	2	3	12
	Lu [52]	2018	2	3	8
	Abdelbaki [54]	2017	2	3	9
	Ni [55]	2016	3	3	10
	Telli [56]	2015	3	3	9
	Tavalaee [57]	2015	2	3	8
	Mohammed [58] 2015	3	3	9
	Smit [59]	2013	3	3	9
	Li [60]	2012	1	3	7
	Gabriel [61]	2012	3	3	9
	La Vignera [62]	2012	3	3	9
	Sadek [63]	2011	3	3	10
	Zini [64]	2011	3	3	9
	Ghazi [65]	2011	2	3	8
	Dada [66]	2010	0	3	8
	Nasr Esfahani [67] 2010	2	3	11
	Smit [34]	2010	3	3	9
	Zini [68]	2005	4	2	9
	Yeşilli [69]	2005	1	3

Table 3 .

 3 Comparison with previous meta-

analyses Study (publication year) No. of studies/ patients (total) Study designs included in SRMA The measure of sperm DNA damage Outcomes

	Present study	29/1,523	Prospective, retrospective,	SCSA, SCD, TUNEL, and others	Varicocele repair reduces sperm
		6/268 a	and unspecified	8OHDG: Didn't qualify for meta-analysis	DNA damage and MDA levels.
				Comet: Didn't qualify for meta-analysis
	Lira Neto et al	19/1,070	Prospective and retrospective SCSA, TUNEL, SCD, Comet	Varicocele repair reduces sperm
	(2021) [76]				DNA damage.
	Birowo et al	7/289	Prospective	SCSA, TUNEL	Varicocele repair reduces sperm
	(2020) [75]				DNA damage.
	Qiu et al	11/394	Prospective	SCSA, TUNEL, SCD, Comet, AOT	Varicocele repair reduces sperm
	(2021) [73]				DNA damage.
	Wang et al	6/177	Prospective, retrospective,	SCSA, TUNEL, Comet	Varicocele repair reduces sperm
	(2012) [74]		and unspecified		DNA damage.
	Baazeem et al	3/84	Prospective	SCSA	Varicocele repair reduces sperm
	(2011) [72]				DNA damage.

SRMA: systematic review and meta-analysis, SCSA: sperm chromatin structure assay, SCD: Sperm Chromatin Dispersion, TUNEL: terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate-nick end labeling, 8OHDG: 8-hydroxydeoxyguanosine, AOT: acridine orange test. a This number represents MDA studies only.

Acknowledgements

The authors express gratitude to Gianmaria Salvio, MD (Catania, Italy) for his support in graphics and artwork, and to Damayanthi Durairajanayagam, PhD (Selangor, Malaysia) for assistance with scientific editing of this manuscript.

Funding

None.

Model Study name

Sadek et al [START_REF] Sadek | Sperm chromatin condensation in infertile men with varicocele before and after surgical repair[END_REF] Telli et al [START_REF] Telli | Does varicocelectomy affect DNA fragmentation in infertile patients?[END_REF] Mohammed et al [START_REF] Mohammed | Acridine orange and flow cytometry: which is better to measure the effect of varicocele on sperm DNA integrity?[END_REF] Abdelbaki et al [START_REF] Abdelbaki | The impact of coexisting sperm DNA fragmentation and seminal oxidative stress on the outcome of varicocelectomy in infertile patients: a prospective controlled study[END_REF] Std diff in means and 95% CI

CONCLUSIONS

By analyzing the largest sample so far, this SRMA provides additional evidence that VR significantly improves SDF. Reductions of SDF were observed independent of the surgical technique, type of SDF testing assay, or preoperative SDF values. To the best of our knowledge, the present study is the first SRMA evaluating the effect of VR on seminal MDA levels and demonstrating significant decrease of this OS biomarker following repair. Our findings further strengthen currently available evidence and could potentially serve to upgrade the professional societies' practice recommendations favoring VR to improve SDF and to ameliorate seminal OS in infertile men.

Conflict of Interest

The authors have nothing to disclose.

Author Contribution

Supplementary Materials

Supplementary materials can be found via https://doi. org/10.5534/wjmh.230235.