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Hypofertilité masculine Yves J.R Ménézo, PhD, Dr Sci UNILABS, Paris Genève Marc Cohen, MD, Clinique Natecia , Lyon

Hypofertilité masculine Yves J.R Ménézo, PhD, Dr Sci UNILABS, Paris Genève Marc Cohen, MD, Clinique Natecia , Lyon. Several environmental Causes Chemicals (domestic use), electromagnetic fields, food. Une baisse de la fertilité masculine?.

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Hypofertilité masculine Yves J.R Ménézo, PhD, Dr Sci UNILABS, Paris Genève Marc Cohen, MD, Clinique Natecia , Lyon

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  1. Hypofertilité masculineYves J.R Ménézo, PhD, Dr Sci UNILABS, Paris GenèveMarc Cohen, MD, Clinique Natecia , Lyon

  2. Several environmental CausesChemicals (domestic use),electromagnetic fields, food

  3. Une baisse de la fertilité masculine?

  4. Electromagnetic radiations: Wifi, Mobile phones…. Mobilite et viabilite diminue significativement apres exposition aux ondes electro magnetiques Augmentation de 8-OH-dG, and DNA fragmentation biomarqueurs du stress oxydatif De Iuliis GN, Newey RJ, King BV, Aitken RJ.2008 Menezo and Cohen 2010

  5. Métaux lourds, stress oxydatif et spermatogénèse Des taux élevés de cadmium et de plomb sont associés a un niveau de stress oxydatif élevé et à des dommages associés dans la lignée germinale AR, Aydemir B, Onaran I, Alici B, Ozkara H, Gulyasar T, Akyolcu MC.2007 Le taux endogène de glutathion et de GST (glutathione S-transferase) sont inversement corrélés à la teneur en métaux lourds dans le sérum et le liquide séminal

  6. Drogues et medicaments Cannabis : THC et spermatogenese!!! Steroides anabolisants Regulateurs du cholesterol et de la pression sanguine antidepresseurs, cimetidine, phenitoine, Li+ Traitements contre la chute des cheveux!!! Minoxidil et Propecia: à deconseiller fortement si spermatogénèse defaillante

  7. estrogens : estrogens (USA), elevage Elimination dans l’eau : Pillule patch

  8. Endocrine disruptors Agriculture (water and food) : pesticides (lindane...) and herbicides Xeno-oestrogens : impact as early as fetal life, irreversible Testicular dis-genesis (malformation GO in males, apoptose germinal cells, Testicular cancer. Micro-penis Menezo et Cohen 2009

  9. Disrupteurs endocriniens DDT: reduces sperm count (estrogen like) Detergents: alkylphenol polyethoxylate Vinclozin (fungicide): estrogenic antiandrogenic L PE : Usage domestique: DHEP: Bis(2-ethylhexyl)phthalate) plastifiant Jouets pour 1er age, cosmetiques Plastic: Nonyphenol, Bisphenol, effet Disrupteur associe aà une augmentation du stress oxydatif

  10. Conséquences:décroissance de la qualité du sperme? Concentration, numeration mobilité? Oui mais controverses Danemark: Carlsen 1992 France: Auger et al 1995 Italie: Bilotta et al 1999

  11. Modification de la morphologie(Cytogramme) Augmentation du % de formes anormales* oui, consensus Benshushan 1997 Andolz 1999 *featuring ADN quality in sperm

  12. Acrosome Genetic material compacted Chromatine (protamines + DNA) Mid-piece Mitochondria « engine » « Propeller»

  13. FERTILIZATION • Intact sperm • Acrosome reaction (after the cell has bound to ZP). • After AR the acrosomal cap has disappeared

  14. MORPHOLOGICALLY NORMAL SPERMATOZOA A. Spermac stain: B. Papanicolaou stain: C. Diff-Quick stain:

  15. HEAD ABNORMALITIES A. Macrocephalic. Rounded head with various degrees of enlargement. The acrosome extends further than normal (we speculate that these cells are often diploid = spermatogenesis defect) B. Microcephalic with round head and absence of the acrosome (intense pink staining of the nucleus attribuable to a hypercondensation of sperm chromatin. Spermac stains the nuclear portion of the head pink, proportional to the degree of nuclear condensation)

  16. MIDPIECE ABNORMALITIES A. Pseudodroplet. Mitochondria (m) have loosened proximally and have clumped distally (arrow = proximally denuded axoneme) B. Mitochondrial loss. The mitochondria have detached completely (arrow) t = tail

  17. Oligo-asthénospermiePour IAC: au moins 106 spz mobiles

  18. Determination de la quantité de spermatozoïdes efficaces II Impact de la morphologie 1-Les anomalies du flagelles et de la pièce intermédiaire peu favorables à la remontée des spz: IAC à déconseiller, meme chose pour Acrosome 2-Anomalies de la têtes qualité de l’ADN Nécrospermie signe généralement une fragmentation élévée (pic de l’iceberg)

  19. MIDPIECE ABNORMALITIES A. Broken neck. Head still attached to the middle of the midpiece by means of a small cytoplasmic stalk (arrow) B. Broken midpiece. Midpiece folden back on itself in the middle (arrow). This defect is characterized by severe axonemal disruption including rupture abd disorganization of fibrils.

  20. TAIL ABNORMALITIES A. Minor « Dag » defect. Simple folding back of the tail arround a distal cytoplasmic droplet (dd) B. Dag defect. More severe form with tail coiled once arround the midpiece (arrows)

  21. ELONGATED HEAD FORMS A.Mild elongation with small lateral equatorial vacuole on the left side (acrosome intact) B. Mild elongation. Slightly more elongated cell with narrowing in the postacrosomal region (acrosome intact)

  22. SECONDARY ACROSOMAL ALTERATIONS A. Complete loss of acrosomal contents B. Acrosomal membrane damage shown by the fuzzy outline of the acrosome (arrows) with irregular loss of acrosomal contents. Dark-staining material (probably thickened midpiece and not cytoplasm). This damage is commonly seen after cryopreservation.

  23. PRIMARY ACROSOMAL ABNORMALITIES A. Acrosomal cyst (nipple acrosome) = large apical cyst within the acrosome, with empty center (arrow). The circular area just above the equatorial region is probably also a cyst within the acrosome. B. Acrosomal cyst. Severe central equatorial cyst with lighter center (arrow).

  24. Sperm DNA fragmentation: Definition ADN • DNA fragmentation = damages in the double strand due to « agression ». • Apoptosis,ROS, anomalies in topoisomerase activity, sperm antibodies Breaks

  25. Collecting Lens Photomultiplier Tube Stained Cells Excitation Source (Laser Beam) Flow Cytometry

  26. SCSA® - Acridine Orange Stained DNA 100 Native DNA Stainability (green fluorescence) 0 100 Fragmented DNA (red fluorescence)

  27. HDS 3% DFI: 68%,

  28. Dernier étape avant necrospermie? DFI Coeff. de corrélation de Spearman: rho = 0,65  p < 2,2 x 10-16 100 80 60 40 20 NECROSPERMIE 20 40 60 80 100 Menezo 2007 140 patients

  29. Age and DFI Age et fragmentation: Notre experience (Ménézo et al. 2005) P<0.0001: 83 patients Y= 0.098X + 34.4 Menezo 2008

  30. Apoptose, arret du cycle cellulaireReparation,Tolerance CDC25 TP 53 Menezo 2007 OK +++ Mutagenesis, carcinogenesis)

  31. ICSI avec sperm totalement fragmente 8modele souris)Fernandes-gonzales et al. Biol Reprod 2008 • Anatomopathological analysis of animals at 16 months of age showed some large organs and an increase in pathologies (33% of CD1 females produced with DFS presented some solid tumors: lungs and dermis of back or neck. • 20% of the B6D2F1 generated with DFS died during the first 5 months of age, 25% of the surviving animals showed premature ageing symptoms • 70% of mice died earlier than control with different kind of tumors. Menezo 2008

  32. Antioxidants and fertilityL’ovocyte a une capacité limitée à réparer l’ADNCette capacité diminue avec l’Age maternel Plusieurs centaines de milliers d’opérations de DNA repair pendant les premières 24 heures Post fecondation Ménézo and Cohen 2011

  33. Antioxidants and fertilityTenter de diminuer la charge de réparation de l’ovocyteTout dépassement de la capacité de reparation entrainera arrêt de développement embryonnaireouMutation

  34. Les Anti-oxydants habituellement utilisés. Basés sur le vieillissement The role of free radicals and antioxidants in reproduction. Agarwal A, Gupta S, Sikka S 2006 Ménézo 2009 Selenium Vitamine C (A+E) SOD Le vitamines du groupe B?

  35. Sperm DNA fragmentation Se, vit C, Vit A , VIT E treatments: Δ HDS, 57 patients Increase in HDS DFI HDS Decrease in HDS with treatment Menezo et al. 2007

  36. DFI / HDS and antioxidanttreatments : 57 patients 32.4 DFI p<0.0004 HDS p<0.0009 26.2 21,5 17.5 After treament Before treatment

  37. Acrosome Genetic material compacted Chromatine (protamines + DNA) Mid-piece Mitochondria « engine » « Propeller»

  38. Selenium • - Sodium selenite induit l’apoptose par generation de RLO° via les mitochondries (Wong 2009) • - “Mitochondries principale source de RLO dans le sperme ” (Aitken 2007) • - Jamais de déficience en selenium decrite dans la “Caucasian population “ • -Une interaction se produit entre certaines structures associees au selenium et les “Zinc finger proteines” impliquées dans la réparation de l’ADN • Se: anticarcinogénique à faible concentration mais • compromet la stabilité génétique à de fortes concentrations : il affecte la möthylation de l’ADN (Hartwig et al. 2003, Davis et al. 2000) Ménézo 2009

  39. Antioxydants et fertilitéNouvelles approches Menezo and Cohen 2010

  40. Glutathion: Sulfur Oxigen Nitrogen Carbone SH S-S

  41. Antioxydants: IQuercetine *Folliquelia, procrelia, condensyl, Alleviative effects of quercetin and onion on male reproductive toxicity induced by diesel exhaust particles.Izawa H et al. Biosci Biotechnol Biochem. 2008 Stimulating effects of quercetin on sperm quality and reproductive organs in adult male rats.Taepongsorat L, AsianJ Androl. 2008 Quercetin protects spermatogonial cells from 2,4-d-induced oxidative damage in embryonic chickens.Mi Y.J Reprod Dev. 2007

  42. Absorption des polyphenols:Ils stimulent la synthese endogene de Glutathion .Measurement of GSH in mouse gastrocnemius (muscle) 3-4wks intake P<0.01

  43. Betalaïnes (Betanine and Indicaxanthine) Indicaxanthine Betanine: purple red

  44. Betalaïnes: protection against lipid peroxidation Augmentation de la resistance à l’oxydation des LDL en présence de betalaînes Tesoriere L 2003 Betalains: elles se lie aux lipides des LDL ; eles s’oxydent et protege le cœur lipidique de l’oxydation Les LMDL enrichies en Indicaxanthin et betanin sont plus resistantes à la peroxydation induite par les ions cuivre Menezo and Cohen 2010

  45. Antioxydants: acide folique, Zn et TOUTES les vitamines du groupe B *Folliquelia, procrelia, condensyl, Fertibiol Association membrane methylation and motility Associations of MTHFR DNMT3b 4977 bp deletion in mtDNA and GSTM1 deletion, and aberrant CpG island hypermethylation of GSTM1 in non-obstructive infertility in Indian men. Dhillon VS, Mol Hum Reprod. 2007 Low seminal plasma folate concentrations are associated with low sperm density and count in male smokers and nonsmokers. Wallock LM et al 2001 Depression of human sperm motility by inhibition of enzymatic methylation. Sastry BV, Janson VE.2007 Menezo and Cohen 2010

  46. Empreinte, apoptose et defense contre le stress oxydatif Incorporation of uracyle in DNA (/thymidine) Defective methylation Hyperhomocysteinehemie et deficience en folate Oxidative Stress Me Diminution de la croissance cellulaire Diminution de L’expression génique Alteration de la synthese proteique Alteration des lipides membranaires Fragmentation DeL’ADN a Cellular dysfonctions Apoptosis Menezo and Cohen 2010

  47. Before any treatment Ménézo et Cohen 2009 Check Fragmentation Decondensation Various dosages of AO and folic acid according to the type of defects

  48. Mr C.M. 40 Years old, Naples Italydecondensation March 200845% July 2008 51% August 2008, did 90 days Folliquelia therapy Tosti 2009

  49. Mr C.M. 40 Years old, Naples Italy January 2009 13% February 2009 3%

  50. Intégrité de la structure nucléoprotéique (ADN Protéine) du spermatozoïde: therapeutique Différents types d’altération de cette structure Structure Iaire et IIaire Apoptose (# nécrose) Cassure de l’ADN en fragments de 200 paires de bases (chaîne des caspases) Cassure de la double hélice ADN Rupture des ponts disulfures Exploration par coloration au bleu d’aniline (agent intercalant) Exploration par technique TUNEL Valeur pathologique: frag > 35% Origine des cassures Décondensation: Altération de la structure tertiaire de l’ADN Condensyl Génétique : anomalies des enzymes de réparation de ADN (topoisomérases …) Action des ROS Radicaux libres oxygénés Utile si dispersion < 20% Procrelia Traitements par antioxydants Pas de traitement si dispersion > 20%

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