1. Gonadal function and Fertility issues in Thalassaemia 3rd Pan-European Conference on Haemoglobinopathies & Rare Anaemias Limassol, 24 – 26 October 2012 Nicos Skordis, MD Makarios Hospital, Nicosia

6. The Thalassaemia map ? Original sin

9. AnteroVentral PeriVentricular nucleus

12. Leptin acts on hypothalamic neurons expressing its receptor (Ob-R). Neurons containing POMC and NPY mRNA also express Ob-R mRNA, and both POMC and NPY mRNA are regulated by leptin. Changes in the synthesis and/or release of these or other hypothalamic peptides could then act at GnRH neurons to affect GnRH release.

13. Leptin levels in patients with Thalassaemia Del Giudice BrJ Haematology 1999 Vlachopapadopoulou E. Ann Clin Ped Un Ath 2004

15. Sexual Complications in Thalassaemia 1.Delayed puberty : Lack of pubertal development ( girls by 13 yr, boys 14 yr ) 2.Arrested puberty : Lack of pubertal progression for longer than 12 months with reduced growth velocity 3.Primary Hypogonadism : Total absence of pubertal development by the age of 16 yrs 4.Secondary Hypogonadism : Gonadal failure after puberty has been completed

16. De Sanctis, 2002 PER 2007 Toumba M, Skordis N. PER 2007

17. Endocrine events and development of secondary sexual characteristics Endocrine event Sexual maturation Increased GnRH increased LH/FSH Increased steroid production Oestrogen secretion Breast development Testosterone secretion Penile and scrotal development FSH secretionTesticular enlargement /follicular development Adrenal androgen secretionPubic and axillary hair Sex Steroid and GH secretionGrowth acceleration Oestrogen withdrawalMenarche (anovulatory) Induction of LH surgeOvulatory menstrual cycles ~3-4 years

20. Adapted from Modell B, Mathews R. Birth Defects Orig Artic Ser. 1976;12:13. 86420 Fe % d.w. Minimum Maximum Skeletal muscle Testes Kidney Heart Adrenal Salivary gland Thyroid Pancreas Liver Parathyroid Distribution of body iron at post-mortem in TM in pre-chelation TM = thalassaemia major; d.w. = dry weight.

21. Normalization of total body iron load with very intensive combined chelation reverses cardiac and endocrine complications of Thalassaemia major Farmaki K. Br J Haematol. 2010 1. 39 patients with abnormal glucose metabolism, 44% normalized. 2. 18 on thyroxin for hypothyroidism, 10 were able to discontinue, and 4 reduced their thyroxin dose. 7/14 hypogonadal males on testosterone therapy, stopped treatment - one male became father 19 females, who were hypogonadal on DFO monotherapy, six were able to conceive.

22. Acute effects of blood transfusion on pituitary gonadal axis and sperm parameters in adolescents and young men with Thalassaemia major: a pilot study Soliman A. Fertility Sterility, 2012 Total sperm count increased significantly from 57.8 to 166 million/mL, and rapid progressive sperm motility increased from 20.6 to 79.7 % Blood transfusion is associated with significant acute enhancement ?? of sperm parameters and increased concentrations of serum T, LH, FSH, and IGF-1

24. Chalcolithic Fertility Goddess Cyprus 3000-2500 BCE Limestone Goddess of Fertility Dionysus

25. Fertility in Cypriot Thalassaemic women Skordis N, Ped Endocr Rev 2004 Endocrine statusNormal Menses Primary Αmenorrhea Secondary Αmenorrhea Number 72 11 17 Total Number 100 35 % of women older than 20 years Children 161

26. 266 53 40 3 Outcome of 362 pregnancies in 316 women Singleton340 Twin20 Triple2

27. Update on fertility in Thalassaemia major 359 successful pregnancies - Skordis N. PER 2004 Absence of any deleterious effect on the course of Thalassaemia Safety of the pregnancy Spontaneous fertility can occur in well chelated and transfused patients Ovarian function is well preserved in women suffering primary or secondary amenorrhea as they become able to conceive following a closely monitored stimulation therapy

28. Over the last 15 years, 22 women with TM have completed 29 pregnancies The major pre-pregnancy issues, medications, and pregnancy care are reviewed Experience suggests that, with proper care and guidance, pregnancies among women with Thalassaemia major are practical and can have successful outcomes. Fertility and Pregnancy in Thalassemia Major SUSAN M. TUCK, JAN 2006

29. Current perspectives of fertility and pregnancy in Thalassaemia. Bajoria R, Chatterjee R Hemoglobin. 2009 Pregnancies following ART in 11 TM women Pregnancy is feasible and safe in women with TM with normal resting cardiac performance and optimized iron overload in specialist centers under a multi disciplinary team

30. Pregnancy and beta-thalassemia: an Italian multicenter experience. Origa R, Haematologica. 2010 46 women with TM (58 pregnancies) 11 women with TI (17 pregnancies ) Pregnancy was safe in most women with TM or TI However, women with TI who had never previously been transfused or who had received only minimal transfusion therapy were at risk of severe alloimmune anemia if blood transfusions were required during pregnancy

31. Cardiac monitoring during pregnancy in 44 pregnancies in 35 women with TM from 1997 to 2009 Echocardiograms were performed before pregnancy, at first, second, third trimester and after labour Kypris L et al. 2011 No significant cardiac complications were encountered Pregnancy can be safe for cardiac function in women with TM provided they have started early on proper treatment and have a normal resting cardiac performance

32. 1.Pregnancy is safe 2.No severe obstetric complications 3.No deleterious effects of pregnancy on the course of Thalassaemia 4.Women with Primary or Secondary Amenorrhea preserve their Ovarian function and therefore Induction of Ovulation can be easily achieved 5.? The incidence of preterm labour and growth restriction

34. Issues Medical history Emotional dilemmas Legal issues Ethical issue s

37. Evaluation of Eligibility 1.Cardiac function: ECG, Echo 2.Liver function tests, Ultrasound 3.Vessels: Clotting factors, Doppler 4.Endocrine: Hypothalamic – Pituitary – Gonadal axis Thyroid, Calcium - OGTT 5.Viral infections: HBV, HCV, HIV 6.Iron status 7.Genetic counseling – Partner’s carrier status and fertility

38. Management during pregnancy 1. Maintenance of Hb level at 10 gr/dl 2.Frequent low volume blood transfusions 3.Serial Ultrasound to monitor fetal growth 4.Regular cardiac monitoring 5.Assessment of endocrine function, OGTT Multidisciplinary approach by all specialist involved in the medical care of Thalassaemic women

39. stated simply 1. The desire of Thalassaemic women to achieve motherhood should be recognized, respected and approached with special caution and sensitivity by all physicians involved in their medical care 2. Ambitions of this sort pose numerous medico legal and ethical issues that need to be addressed prudently if the patients' quality of life is to be optimized 3. Should balance improvement in the quality of life of the woman and the safety of her life

41. Induction of ovulation Primary Amenorrhea Secondary Amenorrhea Oligomenorrhea Normal Menstrual function who fail to conceive Planned pregnancy where both partners are Thalassaemics

42. two significant risks Powerful drugs - growth of two or more follicles, with risk of twin or triplet pregnancy Ovarian Hyperstimulation Syndrome – enlarged ovaries - fluid retention with bloating, breathlessness and nausea. Although uncommon, serious cases require hospitalization and would be particularly complicated to manage in a patient already unwell with Thalassaemia.

44. Ovarian Reserve Testing (the capacity of the ovary to provide eggs that are capable of fertilization resulting in a healthy and successful pregnancy) is based on indirect measures to evaluate the size of the residual ovarian follicle pool

45. Antral follicle count (AFC ) reflects the pool of primordial follicles, those follicles that decline with age

46. Reproductive aging is directly related to the decline in the pool of these follicles

47. AMH : Earliest marker of change with age and has very little intercycle and intracycle variability AMH prevents recruitment of non-dominant follicles

52. Paternity in Cypriot Thalassaemics Skordis N, Ped Endocr Rev 2004 Endocrine Status Normal Hypogonadism Number 54 4 Total number 58 20% of all men > 20 years Children 96

53. Abnormal sperm concentrations, quality, motility and morphology in males with TM DeSanctis in Fert Ster;50:969,1988 Induction of spermatogenesis with hCG and hMG

54. Endocrine complications Cryopreservation - preserved by freezing - of ejaculated semen, which can be used for insemination or ICSI whenever pregnancy is desired

58. Oxidative stress and Infertility Induced by Reactive Oxygen Species – ROS Normal levels of ROS are required for sperm physiology but excessive levels can negatively affect sperm quality Contributing factor in 30 – 80 % of all cases The generation of ROS can be exacerbated by environmental, infectious and life style factors Oxidative stress can be measured

60. F Hypothesis suggesting one of the mechanisms by which DNA damage in human spermatozoa can increase the mutational load subsequently carried by the embryo Aitken R J et al. Reproduction 2011;141:139-150 © 2011 Society for Reproduction and Fertility

63. INTERVENTIONS: assessment of the degree of spermatozoa undergoing apoptosis by terminal deoxynucleotidyl transferase mediated assay (TUNEL) and measurement of the degree of those with compromised structural integrity as measured by the sperm chromatin structure assay (SCSA) using flow cytometry. MAIN OUTCOME MEASURES: the degree of spermatozoal DNA damage by TUNEL and SCSA and the correlation between these measures andsperm motility, concentration, morphology and serum FSH, LH, sex hormone binding globulin, free and total testosterone, ferritin, zinc and vitamin E using regression analysis and Student t-test. RESULTS: comparative analysis showed that beta-thalassaemia patients had significantly more sperm DNA damage (mean TUNEL=18.5%, SCSA=0.28) than controls (mean TUNEL=11.4%, mean SCSA=0.18) (p<0.001). Among thalassaemia patients there was a negative correlation between itchromatin structure damage and sperm concentration (r2=0.3, p<0.006). There was a significant negative correlation between serum ferritin levels and abnormal sperm morphology (r2=0.2, p<0.05). CONCLUSIONS: compared to controls there was a higher degree of DNA damage in spermatozoa of beta-thalassaemia patients. Thalassaemic patients with low sperm concentrations were more likely to have a higher degree of defective chromatin packaging. The negative association between ferritin levels and abnormal sperm morphology suggests a possible detrimental effect on spermatogenesis by the iron chelator desferrioxamine, which is used to reduce iron overload. Thalassaemic patients especially those being considered for assisted conception procedures should be counselled accordingly 1. Significantly more sperm DNA damage (mean TUNEL=18.5%, SCSA=0.28) than controls (mean TUNEL=11.4%, mean SCSA=0.18) 2.Negative correlation between itchromatin structure damage and sperm concentration (r2=0.3, p<0.006). 3.There was a significant negative correlation between serum ferritin levels and abnormal sperm morphology - Possible detrimental effect on spermatogenesis by the iron chelator DFX Spermatozoal DNA damage in patients with β- thalassaemia syndromes. - 28 patients with TM and TI De Sanctis V Pediatr Endocrinol Rev. 2008

64. 1. TM patients (median ferritin: 2251 μg/l) had more sperm DNA damage 2. The sperm DNA damage by SCSA and TUNEL were positively correlated 3. The age of onset of chelation and sperm DNA damage were positively associated with both SCSA and TUNEL 4. No other biochemical or clinical data were associated with sperm DNA damage. Sperm DNA damage in potentially fertile homozygous β- thalassaemia patients with iron overload Doreen Perera Results

65. 1. The increase in sperm DNA damage and the negative correlation between sperm motility and DNA damage suggest that iron overload predisposes sperm to oxidative injury. 2. This finding has important implications in ART – ICSI where there is increased risk of transmitting defective DNA to the offspring Sperm DNA damage in potentially fertile homozygous β- thalassaemia patients with iron overload Doreen Perera Conclusions

66. Therapeutic management of Oxidative Stress

68. Semen quality North America Europe Sperm concentartion 10 6 /mL

71. Mother, don’t kill my sperm !

72. Vitamin D

73. Hormone (Vitamin ) D and Reproduction Steroidogenesis in healthy women Higher clinical pregnancy rates in IVF Endometriosis Polycystic Ovary Syndrome Semen Quality Androgen status - Testosterone levels

74. 1. Thalassaemic patients with low sperm concentrations were more likely to have a higher degree of defective chromatin packaging. 2. Possible detrimental effect on spermatogenesis by the iron chelator desferrioxamine 3. Thalassaemic patients especially those being considered for assisted conception procedures should be counselled accordingly Spermatozoal DNA damage in patients with β- thalassaemia syndromes. - 28 patients with TM and TI De Sanctis V Pediatr Endocrinol Rev. 2008

76. Use of laptop computers connected to internet through Wi-Fi decreases human sperm motility and increases sperm DNA fragmentation Conrado Avenda Fertility and Sterility, 2012

81. epilogue