Female infertility: Role of Anti-Oxidants Female infertility: Role of Anti-Oxidants

1. Female infertility: Role of Anti-OxidantsFemale infertility: Role of Anti-Oxidants 4th International Congress of the Collège Marocain de Fertilité RABAT 15-16 March 2019 Prof Mohamed Ashraf Virmani (NL) Vice President European Mind and Metabolism Association, Rome, Italy and Utrecht, The NetherlandsR & D, AlfaSigma (Sigma Tau), Utrecht, The Netherlands

2. OVERVIEW Female infertility can result from a number of risk factors. L'infertilité féminine peut résulter d'un certain nombre de facteurs de risque. Risk factors related to metabolic dysfunction as a result of age and/or inadequate nutrition. Facteurs de risque liés à un dysfonctionnement métabolique dû à l'âge et / ou à une nutrition inadéquate. Strategies to improving oocyte quality and IVF success rates Stratégies visant à améliorer la qualité des ovocytes et les taux de réussite de la FIV

3. OVERVIEW • Female fertility, oocyte quality and IVF • Fertilité féminine, qualité des ovocytes et FIV • Risk factors – Hormonal, Metabolic and Psychological • Facteurs de risque - hormonaux, métaboliques et psychologiques • Role of Mitochondria and Carnitines on Oocytes Quality • Rôle des mitochondries et des carnitines sur la qualité des ovocytes • Carnitines and Ovulation (animal and human studies) • Carnitines et ovulation (études animales et humaines) • Carnitines and PCOS • Carnitines et SOPK • Genomics and DNA stability • Génomique et stabilité de l'ADN

4. Female fertility, oocyte quality and IVF • Fertilité féminine, qualité des ovocytes et FIV

5. 1. Female fertility, oocyte quality and IVF • Fertilité féminine, qualité des ovocytes et FIV • The pregnancy or IVF (ICSI) success rate will depend on oocyte quality as well as the sperm quality. • Le taux de succès de la FIV (ICSI) dépendra de la qualité des ovocytes ainsi que de la qualité du sperme.

6. Oocyte Quality Qualité des ovocytes • Quality-compromised oocytes are correlated with: • Les ovocytes compromis en qualité sont corrélés avec: • Infertility, • Infertilité • Developmental disorders, • Troubles du développement • Reduced blastocyst cell number • Nombre réduit de cellules blastocystiques • Embryo loss • Perte d'embryon

7. Oocyte Quality Qualité des ovocytes • Cellular mechanisms underlying oocyte quality still unclear • Les mécanismes cellulaires sous-jacents à la qualité des ovocytes ne sont toujours pas clairs • Bioenergetic capacity reduction during oocyte maturation have been suggested • La réduction de la capacité bioénergétique pendant la maturation des ovocytes a été suggérée • Reactive oxygen species (ROS) may produce oxidative stress (OS) and affect oocyte quality during its growth. • Les espèces réactives de l'oxygène (ROS) peuvent produire un stress oxydatif (OS) et affecter la qualité de l'ovocyte au cours de sa croissance. • Hormone and Redox-dependent signaling pathways may: • Les voies de signalisation redox-dépendantes peuvent: • Contribute to early embryo demise • Contribute to meiotic spindle disorders and chromosomal segregation defects (aneuploidies) • Epigenetic programming of growing oocyte may be affected by above and environmental factors • La programmation épigénétique de l'ovocyte en croissance peut être affectée par les facteurs ci-dessus et environnementaux

8. Factors affecting IVF and fertility successFacteurs affectant la FIV et le succès de la fécondité • Folliculogenesis– Oocyte development process takes approx 375 days. • Folliculogenèse - Le processus de développement des ovocytes prend env. 375 jours. • Quality, or developmental competence, is acquired during folliculogenesis(oocyte growth) and during oocyte maturation. • La qualité ou la compétence développementale est acquise pendant la folliculogenèse (croissance des ovocytes) et pendant la maturation des ovocytes. • Oocyte accumulates metabolites during growth allowing the nutrition of the embryo during the first four to six days of its development. • L'ovocyte accumule des métabolites pendant la croissance permettant la nutrition de l'embryon pendant les quatre à six premiers jours de son développement. • Assisted reproductive technologies involving ovarian hyperstimulation, or collection of immature oocytes followed by maturation in vitro, perturb this process and result in oocytes with reduced quality. • Les techniques de procréation assistée impliquant une hyperstimulation ovarienne, ou la collecte d'ovocytes immatures suivie d'une maturation in vitro, perturbent ce processus et conduisent à des ovocytes de qualité réduite

9. 2. Fertility Risk Factors Facteurs de risque - hormonaux, métaboliques et psychologiques Age Hormonal (Anovulation, Polycystic Ovary Syndrome (PCOS), Clomiphene resistance) Metabolic (Diabetes, Insulin Resistance, Anorexia, Over-exercise, Stress, Obesity, High Fat Diet) Infections, Drugs and Other (anatomical, chemo/radiotherapy) Psychological (Stress) Unexplained

10. 3. Role of Mitochondria and Carnitines on Oocytes Quality • Rôle des mitochondries et des carnitines sur la qualité des ovocytes

11. Mitochondria play central role in producing energy Adenosine Triphosphate (ATP) is a high-energy phosphate compound ISGE

12. Metabolic • (Diabetes, Insulin Resistance, Anorexia, Over-exercise, Stress, • Obesity, High Fat Diet) • Mitochondria, metabolic processes and free radicals play a central role in reproductive pathology. • Les mitochondries, les processus métaboliques et les radicaux libres jouent un rôle central dans la pathologie de la reproduction.

13. Role of Mitochondrial Complex in Neurodegeneration Alzheimer Parkinson Schizophrenia Rotenone MPP+ Coccaine Huntington 3-NPA Malonate Poliovirus Alzheimer Autism Ceramide ALS METH Cyanide Downs Oligomycin FCCP

14. 3. Role of Mitochondria and Carnitines on Oocytes Quality • Rôle des mitochondries et des carnitines sur la qualité des ovocytes Women affected by metabolic disorders - diabetes or obesity and oocyte aging seeking treatment in IVF clinics to overcome the effects of adverse metabolic conditions on mitochondrial functions. Femmes affectées par des troubles métaboliques - le diabète ou l'obésité et le vieillissement des ovocytes Transfer Mitochondria or Improve Mitochondria? Transférer les mitochondries ou améliorer les mitochondries? Schatten H, Sun QY, Prather R The impact of mitochondrial function/dysfunction on IVF and new treatment possibilities for infertility. ReprodBiol Endocrinol. 2014 Nov 24;12:111. doi: 10.1186/1477-7827-12-111. Turner N, Robker RL. Developmental programming of obesity and insulin resistance: does mitochondrial dysfunction in oocytes play a role? Mol Hum Reprod. 2015 Jan;21(1):23-30. MITOCHONDRIA

15. Mitochondrial dysfunction and other factors generate a lot of Reactive Oxygen Species La dysfonction mitochondriale et d'autres facteurs génèrent beaucoup d'espèces oxygénées réactives Alcohol Ionizing radiation Smoking Antioxidanttreatment ROS Free Radicals Infection Metabolicprocesses Physical activity IncreasedOxidative Stress DNA Damage, celldeath, mutagenesis Implantation or Loss of implantedembryo Fertilization, Oocyte penetration, oocyte function , viability

16. Fatty acid beta oxidation in mitochondria produces more energy 1 molecule of Glucose = 28 ATP 1 molecule of Fatty Acid = 106 ATP

17. Beta Oxidation needed for oocyte developmental and early embryo development Oxydation bêta nécessaire au développement de l'ovocyte et au développement embryonnaire précoce CPT1 CPT2 Free Fatty Acids ENERGY (ATP) + Carnitines Beta Oxidation inhibition Inhibition de l'oxydationbêta

18. Inhibition of FA Beta Oxidation (enzyme CPT1) by Etomoxir – Inhibits Embryo Development Inhibition de l'oxydation bêta de la FA (enzyme CPT1) par l'étomoxir - inhibe le développement de l'embryon Data mean % SEM, n 3; *P<0.05). Dunning KR, Cashman K, Russell DL, Thompson JG, Norman RJ, Robker RL. Beta-oxidation is essential for mouse oocyte developmental competence and early embryo development. Biol Reprod. 2010 Dec;83(6):909-18

19. Upregulation of Beta Oxidation with L-Carnitine and A. The effect of 1 mM L-carnitine on beta-oxidation in immature oocyte and during in vitro maturation of oocyte as measured by 3H20 production from [3H]palmitic acid • (B) Improved oocyte developmental competence with L-Carnitine Amélioration de la compétence développementale des ovocytes avec la L-Carnitine B. The effect of L-carnitine on oocyte developmental competence was assessed by the rate of embryo development following in vitro fertilization of oocyte matured in vitro in the presence or absence of 1 mM L-carnitine supplementation ***P , 0.0001, **P , 0.01, *P , 0.05). Dunning KR, Cashman K, Russell DL, Thompson JG, Norman RJ, Robker RL. Beta-oxidation is essential for mouse oocyte developmental competence and early embryo development. Biol Reprod. 2010 Dec;83(6):909-18

20. L-Carnitine/Acetyl-L-Carnitine protect and reduce cell damage • La L-Carnitine/Acetyl-L-Carnitine protège et réduit les dommages cellulaires • Increase mitochondrial energy production • Augmenter la production d'énergie mitochondriale • Increase antioxidant potential • Protect mitochondrial DNA (mtDNA) from free oxygen radicals • Stabilizecellular and mitochondrial membrane • Augmenter le potentielantioxydant • Carnitine may protects cell membrane directly against oxidative damage by • Capturing and removing toxic acyl groups. • Surface membrane effects • La carnitine peut protéger la membrane cellulaire directement contre les dommages oxydatifs • Disposal of CoA-activated xenobiotics i.e. pivalate, benzoate, valproate • Othersynergistic/unknownactions e.g. aiding membrane repair processes.

21. 4. Carnitines and ovulation (animal and human studies) • Carnitines et ovulation (études animales et humaines)

22. PROXEED WOMEN PRE-CLINICAL STUDY Carnitines increased oocyte number Carnitines a augmenté le nombre d'ovocytes * p<0.01 Values mean number of oocytes per mouse, n=10 SD were less than 10 percent of the mean). . Significance versus control untreated CD1 Mouse Oocyte Superovulation Number in Control (untreated) versus Group Treated with L-Carnitine plus Acetyl-L-carnitine and Group treated with L-C + ALC + Antioxidants Virmani M.A., Vitullo P. & Cossetti C. Effect of nutrients on ovulation and oocytes quality in mice. 11th Congress of the European Society of Gynecology, Prague Society October 21st to 24th 2015

23. PROXEED WOMEN PRE-CLINICAL STUDY Carnitines reduces oocyte degradation Les carnitines réduisent la dégradation des ovocytes * p<0.01 Percentage of oocytes degraded versus total per mouse, n=10. Significance versus control untreated CD1 Mouse Oocyte Percentage Degraded in Control (untreated) versus Group Treated with L-Carnitine plus Acetyl-L-carnitine and Group treated with L-C + ALC + Antioxidants Virmani M.A., Vitullo P. & Cossetti C. Effect of nutrients on ovulation and oocytes quality in mice. 11th Congress of the European Society of Gynecology, Prague Society October 21st to 24th 2015

24. PROXEED WOMEN PRE-CLINICAL STUDY Carnitines increased blastocyte number Les carnitines ont augmenté le nombre de blastocytes * p<0.01 Percentage of blastocyte versus oocyte in stage 2 total per mouse, n=10. CD1 Mouse Blastocyte: Percentage in Control (untreated) versus Group Treated with L-Carnitine plus Acetyl-L-carnitine Virmani M.A., Vitullo P. & Cossetti C. Effect of nutrients on ovulation and oocytes quality in mice. 11th Congress of the European Society of Gynecology, Prague Society October 21st to 24th 2015

25. OTHER PRE-CLINICAL STUDIES SHOW Carnitines reduces improve blastocyst development and reduce DNA damage Abdelrazik H, Sharma R, Mahfouz R, Agarwal A. L-carnitine decreases DNA damage and improves the in vitro blastocyst development rate in mouse embryos. FertilSteril. 2009 Feb;91(2):589-96 • Many studies show that carnitines increase oocyte and embryo development Abdelrazik H, et al. A L-carnitine decreases DNA damage and improves the in vitro blastocyst development rate in mouseFertil Steril. 2009 Feb;91(2):589-96. doi: 10.1016/j. Reader KL, et al. Effects of acetyl-L-carnitine on lamb oocyte blastocyst rate, ultrastructure, and mitochondrial DNA copy number. Theriogenology. 2015 Jun;83(9):1484-92. doi: 10.1016/j You J, et al. L-Carnitine treatment during oocyte maturation improves in vitro development of cloned pig embryos by influencing intracellular glutathione synthesis and embryonic gene expression. Theriogenology. 2012 Jul 15;78(2):235-43. doi: 10.1016/j.theriogenology.2012.02.027 Wu G.Q et al. L-Carnitine enhances oocyte maturation and development of parthenogenetic embryos in pigs. Volume 76, Issue 5, 15 September 2011, Pages 785–793 TamásSomfai et al Enhancement of lipidmetabolismwith L-carnitineduring in vitro maturationimprovesnuclearmaturation and cleavageability of follicularporcineoocytes. Reproduction, Fertility and Development 23(7) 912-920 Fathi M, El-Shahat KH. L-carnitineenhancesoocytematuration and improves in vitro development of embryos in dromedarycamels (Camelusdromedaries). Theriogenology. 2017 Aug 4;104:18-22. doi: 10.1016/j.theriogenology.2017.08.006. Phongnimitr T, et al. Effect of L-carnitine on maturation, cryo-tolerance and embryo developmental competence of bovine oocytes. AnimSci J. 2013 Nov;84(11):719-25. doi: 10.1111/asj.12067. Zare Z, et al. Effect of L-carnitine supplementation on maturation and early embryo development of immature mouse oocytes selected by brilliant cresyle blue staining. J Assist Reprod Genet. 2015 Apr;32(4):635-43..

26. Carnitines modulate the neuroendocrine and reproductive (Hypothalamo-Ovarian) axis • Les carnitines modulent l'axe neuroendocrinien et reproducteur (axe hypothalamo-ovarien)

27. GnRH release is increased in hypthalamicneurones and GT1 neuronal cell line by Acetyl-L-Carnitine La libération de GnRH est augmentée dans les neurones hypothalamiques et la lignée cellulaire neuronale GT1 par l'acétyl-L-carnitine • Effect over time in culture • Basal release from 6 dayold GT1 cell culture • Cell number in control and ALC treated 6 day old cultures • Significancemean SEM, n =12. * = p<0,01 Krsmanovic LZ, Virmani MA, Stojilkovic SS, Catt KJ. Stimulation of gonadotropin-releasing hormone secretion by acetyl-L-carnitine in hypothalamic neurons and GT1 neuronal cells. NeurosciLett. 1994 Jan 3;165(1-2):33-6.

28. PROXEED WOMEN CLINICAL STUDY 1 • Functional hypothalamic amenorrhea (FHA) is a relatively frequent disease due to the combination of metabolic, physical, or psychological stressors. • L'aménorrhéehypothalamiquefonctionnelle (FHA) • Study treatment for 12 weeks of ALC (250 mg/die) and L-carnitine (500mg/die) was effective in improving the endocrine and metabolic pathways in a group of patients (n=27) with FHA. • According to baseline LH levels, they were subdivided in 2 groups: hypoLH (< 3 mIU/ml, n=14) and normoLH (>3 mIU/ml, n=13)

29. Improved GnRH-induced LH response in hypo-LH FHA patients Amélioration de la réponse LH induite par la GnRH chez les patients hypo-LH FHA • Maximal response (∆max) of LH to GnRH bolus in the two groups of FHA patients. • Hypo-LH subjects showed the significant increase of the LH response under integrative treatment. No changes in normo-LH subjects.

30. PROXEED WOMEN CLINICAL STUDY 1 • After the treatment, plasma levels of cortisol and amylase decreased significantly • Data sustain the hypothesis that the integrative administration of ALC plus L-carnitine reduced both the metabolic and the neuroendocrine impairment of patients with FHA Après le traitement, les taux plasmatiques de cortisol et d'amylase ont diminué de manière significative

31. PROXEED WOMEN CLINICAL STUDY 2 In Vitro Fertilization and Embryo Transfer • Mounir AjinaComparison of ICSI results in a group of patients with and without oral L-carnitine, acetyl-L-carnitine and nutrients supplementation 17th World Congress of the Academy of Human Reproduction Rome 15-18 March 2017 Comparaison des résultats d'ICSI dans un groupe de patients avec et sans supplément oral de L-carnitine, d'acétyl-L-carnitine et de nutriments

32. Patients and Methods • 56 Infertile couples. • The mean age of women was 33 ± 2.3 years. • Each patient received oral L-carnitine, acetyl-L-carnitine and nutrients supplementation during the two months preceding controlled ovarian stimulation. • In their antecedents each couple had an ICSI cycle without oral L-carnitine, acetyl-L-carnitine and nutrients supplementation.

33. The effects of oral L-carnitine, acetyl-L-carnitine and nutrients supplementation on the ICSI outcomes Les effets de la supplémentation en L-carnitine, en acétyl-L-carnitine et en nutriments par voie orale sur les résultats de l'ICSI

34. 5. Carnitines and PCOS

35. PCOS as cause of Infertility 85% of anovulatory women have PCOS.

36. Polycystic Ovarian Syndrome (PCOS) Syndrome des ovaires polykystiques (SOPK) • PCOS is common, affecting as many as 1 out of 15 women. • SOPK est commun, affectant jusqu'à 1 femme sur 15. • 90% of women with PCOS are unable to conceive.90% des femmes atteintes de SOPK sont incapables de concevoir

37. PCOS and Metabolic Disorders SOPK et troubles métaboliques Women with PCOS have: • Increased oxidative stressanddecreased antioxidant capacity Augmentation du stress oxydatif et diminution de la capacité antioxydante • Carnitine levels 50% lower than normal (Fencki 2008) Les niveaux de carnitine 50% plus bas que la normale • Insulin resistance • Abnormal lipid profile • High homocysteine levels

38. Polycystic Ovarian Syndrome (PCOS) Metformin and L-carnitine are effective in women with polycystic ovary syndrome. La metformine et la L-carnitine sont efficaces chez les femmes atteintes du syndrome des ovaires polykystiques.FSH, L-carnitine and hMG are effective for ovulation induction in women With clomiphene-resistant polycystic ovarian syndrome (PCOS).

39. 6. Genomics and DNA Stability

40. Ovarian aging Vieillissementovarien • As women age, a higher and higher percentage of their eggs are abnormal • À mesure que les femmes vieillissent, un pourcentage de plus en plus élevé de leurs œufs sont anormaux • Increase in pregnancy failure • Augmentation de l'échec de la grossesse • meiotic errors resulting in chromosomally abnormal conceptions. • Les erreurs méiotiques entraînant des conceptions chromosomiques anormales.

43. Influence of Nutritive Agents on Epigenetics And Genomic Stability Influence des agents nutritifs sur l'épigénétique et la stabilité génomique Folic acid (B9), B vitamins, vit D, iodine, choline, carnitines, etc. useful in prevention of birth defects (cleftpalate, spina bifida, anencephaly) How?..... Folic acid - synthetic form of B9 All B vitamins help the body convert food (carbohydrates) into fuel (glucose), which is used to produceenergy. Other actions? Cytoskeletaland DNA stability

44. Conclusion Protégerl'ovocyte Maintain/Restore DNA/Methylation Pattern

45. THANK YOU FOR YOUR ATTENTION MERCI DE VOTRE ATTENTION

46. Supports oocyte quality and female fertility to increase the chances of conception

47. Conclusions • L-Carnitine, acetyl-L-carnitine and antioxidant supplementation can improve metabolism and hormonal profile. • La L-Carnitine, l'acétyl-L-carnitine et la supplémentation en antioxydants peuvent améliorer le métabolisme et le profil hormonal. • Improvements could be due to effects via mitochondrial improvement in energy and reduction in ROS • Des améliorations pourraient être dues à des effets via l'amélioration de l'énergie mitochondriale et la réduction des ROS • Studies show improvements in female fertility and IVF reproductive outcome • Des études montrent des améliorations de la fertilité féminine et des résultats de la fécondation in vitro

48. In a single human cell under normal physiology 50.000 -100.000 DNA lesions occur each day (Friedberg EC 2006b). Table 2 gives an overview over the endogenous sources, frequencies and types of DNA lesions. Table 2. Numbers of endogenous DNA lesions arising and repaired in mammalian cells in 24 hours (adapted from. (Friedberg EC 2006b)). Endogenous source Lesions pr day Single strand breaks 50,000 Hydrolysis Depurination 18,000 Depyrimidination 600 Cytosine deamination 500 5-mC deamination 50 Oxidation 8oxoG 1,000 -2,000 Ring saturated pyrimidines 2,000 Lipid peroxidation products 1,000 Nonenzymatic methylation by S-adenolsylmethionine 7-mG 6,000 3-mA 1,200 1-mA, 3mC 200 Nonenzymatic methylation by nitrosated polyamines and peptides 6-mG 20 - 100 The most quantitatively important damage forms are single strand breaks and depurination by hydrolysis.

49. Table 3. DNA damaging agents, lesions, repair pathways and prototypical human repair enzymes (adapted from (Dalhus et al. 2009)) . Damaging agent Prototypical lesions Major repair mechanism Prototypical human repair enzymes Alkylating agents6-mG DR Tranferases: Agt 1-mA DR Oxidoreductases: Abh2 3-mA, 3mG, 7-mA, 7-mG BER Glycosylases: Aag HydrolysisAbasic sites BER Endonucleases: Ape1 Deamination forming uracil BER Glycosylases: Ung Deamination forming hypoxanthine NIR Endonucleases: EndoV ROS 8-oxoG. faPyA/G, TG, 5-ohC, BER Gylcosylases: Ogg1, Nth1 DHU, DH DHU,DHT, 5-ohC NIR Endonucleases: Ape1 Replication errors(a) Base mismatches MMR Mismatch proteins: (b) Insertion/deletion loops MutS/ UV radiation Bulky adducts NER Xpa –Xpf + others CPDs, 6-4PDs DR Photolyases mismatch repair pathway (MMR), base excision repair (BER), nucleotide excision repair (NER)

50. Talk Title: Female infertility: Role of Anti-Oxidants Female infertility can result from a number of factors. Some of these important risk factors are related to metabolic dysfunction as a result of age and/or inadequate nutrition. Research is showing that strategies can be developed to ameliorate female fertility. These strategies focus on for example, improving oocyte quality and IVF success rates in polycystic ovary syndrome (PCOS) and endometriosis patients, after supplementation with specific metabolic compounds such as L-carnitine and acetyl-L-carnitine and antioxidants such as vitamin C. The talk will discuss the role of oocyte quality in female fertility and especially in IVF and poor responders. The various risk factors such as the hormonal, metabolic and psychological will be discussed. The underlying cellular and especially the biochemical processes associated with fertility and their relationship to mitochondria are becoming important. Role of mitochondria in metabolic dysfunction and factors that may improve their function, especially carnitines will be highlighted. Research studies, animal and human studies showing role of the metabolic factors and ovulation in various experimental and clinical studies will be presented.