Genetic Issues and Male Infertility, Azoospermia

Dr Sujoy Dasgupta delivered a talk on "Genetic Issues in Male Infertility" in a webinar organised by the North East India Genomic Consortium on 25 June, 2024. Number of experts in the field of Medical Genetics participated in it.

1. Genetic Issues in Male Infertility Dr Sujoy Dasgupta MBBS (Gold Medalist, Hons ) MS (OBGY- Gold Medalist) DNB (New Delhi) MRCOG (London) Advanced ART Course for Clinicians (NUHS, Singapore) M Sc, Sexual and Reproductive Medicine (South Wales, UK) Clinical Director, Genome Fertility Centre, Kolkata Managing Committee Member, BOGS, 2023-24 Executive Committee Member, ISAR Bengal, 2022-24 Clinical Examiner, MRCOG Part 3 Examination Winner, Prof Geoffrey Chamberlain Award, RCOG World Congress, London, 2019 Delivered, Dr Kamini Rao Oration, AICOG, 2024 7 0741+/ +9

2. Thank you NE India Genomic Consortium

3. Disclaimer Written consent from all the patients Understanding genetics?

4. Do we understand- “Male Infertility?”

5. Men’s fertility potential depends on female factors Assessment of tests and treatments for the male is challenging due to inconsistent endpoints and the observation that many of these endpoints are dependent upon and measured from the female partner. Ideally, the endpoint for fertility trials should be "live birth or cumulative live birth (WHO, 2021)

6. We cannot treat We bypass

7. Reference ranges

8. Limitations of WHO Guideline 5 percentile and time-to-pregnancy (TTP) concept Not true reference values but recommends acceptable levels. Day to day variation Functional ability of the sperms?

9. Severe Male Factor is NOT ONLY a fertility problem Diabetes Cardiovascular diseases Lymphoma, extragonadal germ cell tumours , peritoneal cancers Repeated hospitalization Increased mortality Testicular Cancer Choy and Eisenberg, 2020; Bungum et al., 2018; Eisenberg et al., 2013; Jungwirth et al., 2018; Hotaling and Walsh, 2009 Self-Testicular Examination Atrophic Testes H/O undescended testicles Testicular microcalcification (post-mumps or others)

10. ICSI bypasses natural “protection” The spermatozoa of infertile men show an increased rate of aneuploidy , structural chromosomal abnormalities, and DNA damage Carrying the risk of passing genetic abnormalities to the next generation (AUA, 2018)

11. Genetic issues in Male Infertility

12. Genetic issues in Male Infertility

13. Chromosomal abnormalities Numerical- Trisomy Structural- Inversion. Translocation etc The frequency of chromosomal abnormalities increases in Severe testicular deficiency spermatozoa count < 5 mil/ml NOA Incidence in infertile men- 5.8% (Johnson, 1998) Sex chromosome abnormalities- 4.2% Autosomal abnormalities 1.5%.

14. Sex Chromosome abnormalities The most common - the Klinefelter’s syndrome (KS) 47,XXY or 46,XY/47,XXY mosaicism KS mosaic can have variable extent of germ cell production inside the testicles Sperms carrying abnormalities in sex chromosomes (24,XY sperms) and autosomes ( disomy for chromosomes 13, 18 and 21) Needs PGT-A

15. Klinefelter Syndrome

16. Klinefelter’s with “normal” phenotype 37 yr FSH 35.42, LH 10.13, testo 93, E2 14.45 Undiagnosed Diabetes Prev FNAC - Lt side- Sertoli Only Syndrome TESE – Rt side- No sperms, Lt side- Motile Sperms

17. What next? Straightaway donor sperm IUI Testicular FNAC

18. Problems with indiscriminate FNAC B/L testes- 6 cc each FNAC- B/L maturation arrest FSH 37.2, LH 24.4, Testo 245.53, E2 37, ratio <10 Not keen for IVF-ICSI

19. Non-targeted investigations ? Delayed puberty Testo 100.86. FSH 28.33. LH 13.65. E2 27.83 Testosterone injection started at puberty - sec sex charac , voice, genital size improved MRI pitutary microadenoma GH, TSH, Cortisol, PRL, - all normal Karyo - 47,XXY Pituitary Incidentaloma

20. Other sex chromosome abnormalities LH 30.10, FSH 43.70, E2 38.48, Testo 432 Karyo - 46,XX

21. 39 yr FSH 25.4, LH 12.6, Estradiol 14, Testo 61. 46,XX

22. 46,XX SRY+ sex reversal

23. Autosome abnormalities Mostly structural anomalies Increased associated risk of aneuploidy or unbalanced chromosomal complements in the fetus Needs PGT-SR or prenatal invasive testing

24. Translocation of autosomes 45, XY rob (14, 21), (q10, q10) Azoospermia Robertsonian Translocation 46,XY;t(2:22)(q37;q11.21) Severe OAT Reciprocal Translocation

25. Alternative- Prenatal testing 46,XY22ps+ Oligospermia → Azoospermia YCM normal Spermes obtained by TESA Amniocentesis Normal K aryo & CMA Live born by 34/40

26. Other Translocations 46, XY,t (15:17) (q10;q10) Azoospermia FNAC- B/L SCO YCM normal 46,XY;t(2:22)(q37;q11.21) Azospermia TESE- no sperms available YCM normal

27. Other chromosomal aberrations 46,X,del(Y)(q11.23) 46,X,del(Y)(q11.2) 46,X,+mar

28. Familial Azoospermia 46,XY, dup(9)(q11-q12) Duplication of long arm of chromosome 9- partial trisomy FNAC B/L Late maturation arrest Family History of Azoospermia in Own brother 2 maternal uncles 2 Cousin brothers (of same maternal aunt)

29. Congenital anomaly Transverse testicular ectopia  (TTE), or crossed testicular ectopia (CTE) 46,X,Yqh-

30. Genetic abnormality ≠ Advanced interventions 46,XYqh- Severe OAT 46,XY,16qh+ Azoospermia

31. Keep Geneticist on board 46,XY,15ps+ 46,X,Y,q+ 46,X,inv(Y)(p11.q11) 46,X,inv(Y)(p11.2q11.2)

32. Other genetic disorders associated with infertility Prader -Willy Syndrome Bardet-Biedl Syndrome Noonan’s Syndrome Myotonic dystrophy Autosomal dominant polycystic kidney disease 5 α- reductase deficiency

33. Genetic issues in Male Infertility

34. Sperm chromosome abnormalities FISH- more accurate risk estimation of affected offspring Limited role clinically Only specific indication- Macrocephalia ( Themset et al., 2009)

35. Sperm Aneuploidy FISH Panel at LPL Abnormal sperms

36. Genetic issues in Male Infertility

37. X-linked disorders Androgen insensitivity syndrome Kallmann syndrome

38. Partial Androgen Insensitivity Syndrome X-linked inheritance Very rare in absence of genital abnormalities Male infertility can be the sole symptoms Rarely seen in fertile men

39. Kallmann syndrome Mutation of KAL-1 (ANOS-1) gene located on the X chromosome X-linked recessive inheritance Autosomal (dominant and recessive) inheritance has also been reported This gene encodes for a protein anosmin-1 which promoted migration of GnRH neurons and olfactory neurons to the hypothalamus from the olfactory placodes ( Legouis et al., 1991) Treatment- GnRh or gonadotrophin replacement

40. 32, office worker H/O delayed puberty Was on TRT (17-23 yr age) Gynaecomastia surgery, 22 yr LH 0.06, FSH 0.02, Testo 0.63, PRL 1.18, TSH 2.48 Low libido, ED Anosmia , MRI- B/L olfactory bulb absent Genetic tests awaited

41. Y chromosome microdeletion Most common genetic abnormality in infertile men (after KS)

42. Y chromosome microdeletion Never found in normozoospermic men Highest frequency in azoospermic men (8-12%), followed by oligozoospermic (3-7%) men. Extremely rare with a sperm concentration > 5 million/ mL (~0.7%). AZFc deletions are most common (65-70%), followed by AZFb and AZFb+c or AZFa+b+c (25-30%). AZFa region deletions are rare (5%). AZFa - Sertoli cell only syndrome AZFb - maturation arrest AZFc - variable phenotype

43. Y chromosome microdeletion AZF-a, AZF-b, AZF-c, AZF-d AZF a/b Poor prognosis TESA should NOT be attempted AZF c/d Good prognosis Some may have sperms in semen

44. Surgical Sperm Retrieval ( SSR ) in Azoospermia (OA>NOA)

45. Predictors of sperm retrieval? FSH Testicular Size LH , Testosterone BMI AMH - semen, serum Inhibin B- semen, serum Age Ultrasound parameters No reliable positive prognostic factors guarantee sperm recovery for patients with NOA The ONLY negative prognostic factor is the presence of AZFa and AZFb microdeletions .

46. Genetic counseling in YCM (AZF-c) Father to son transmission (can be larger mutation) Depends on environmental factor. Often spermatozoa from men with complete AZFc deletion are nullisomic for sex chromosomes → risk of 45,X0 Turner’s syndrome May develop ambiguous genitalia In practice, infants are often phenotypically normal Reduced implantation rate Spontaneous miscarriage of embryos bearing a 45,X0

47. Clinical features related with infertility male : atrophy, fibrose or congenital absence of vas deferens female : reduced fertility, thick dehydrated mucus in the cervix About 98% of males affected with CF are infertile Congenital Absence of Vas Deferens ( CAVD) 1-2% male infertility, 6% obstructive azoospermia Mutations (>1300) in CFTR gene (Cystic Fibrosis Transmembrane Conductance Regulator) Cystic fibrosis mutation

48. Congenital bilateral absence of vas deferens ( CBAVD ) CLINICAL DIAGNOSIS Semen- Volume <1.5 ml, pH <7.0, fructose negative TRUS Renal ultrasound Cystic fibrosis mutation (CFTR) testing (EUA, 2018; ASRM< 2020) Partner testing Indian prevalence- 1:10,000- 1:40,000 ( Kapoor et al., 2006; Prasad et al., 2010)

49. Testing for CFTR Not practical to test for all known mutations A very low prevalence in a particular population Routine testing is usually restricted to the most common mutations in a particular community through the analysis of a mutation panel

50. CBAVD, TRUS, CFTR mutation TRUS- B/L agenesis of seminal vesicles Male partner- CFTR carrier Fem ale partner- CFTR carrier

51. CBAVD is NOT uncommon CFTR negative CFTR carrier Wife- normal CFTR refused CFTR carrier Wife- normal CFTR negative

52. Unilateral vas absent Usually fertile CFTR testing if no renal agenesis

53. Sperm DNA Fragmentation Advanced paternal age Inadequate diet Drug abuse Tobacco use Environmental factors such as pesticide exposure or air pollution Varicocele Systemic diseases Genital inflammation

54. DNA Fragmentation Index (DFI): ICSI treatment is more likely to result in pregnancy than IUI and IVF if DFI value is above 30% Number of TUNEL negative morphologically normal sperm X 100 Total number of sperm cells evaluated Four statistical types of fertility potency : Less than or equal to 15% DFI outstanding to sound sperm DNA credibility Between 15 to 25% DFI best to good sperm DNA credibility Between 25 to 50% DFI good to weak sperm DNA credibility Greater than or equal to 50% DFI exceptionally poor sperm DNA credibility

55. Clinical Management of SDF Frequent ejaculation Antioxidant therapy Treatment of subclinical infection Varicocele repair TESA ICSI

56. Sperm DNA Fragmentation (SDF) Infertile men with: Repeated IUI or IVF failure Recurrent spontaneous miscarriages (ESHRE, 2018) Previous low fertilization, cleavage or blastulation rate Varicocele with normozoospermia Advanced male age (>40 y) Significance of SDF Live birth after IUI/ IVF/ ICSI- ? Oocytes can repair the damaged DNA Lack of standardization Lack of definitive treatment DNA fragmentation Should not be routine (ASRM, 2020; ESHRE, 2023)

57. Genetic testing- Summary Sperm concentration <10 million/ml Azoospermia Testicular atrophy Elevated FSH Karyotyping Y chromosome Microdeletion (YCM) CFTR testing in CBAVD

58. Other indications RPL Family H/O congenital malformations or mental retardation

59. In presence of genetic defect PGT-SR (previously- PGD) Prenatal invasive testing (EUA, 2018; ASRM, 2020)

60. Semen analysis Mild problem Severe problem Lifestyle changes Antioxidants History Physical Exam Repeat semen ASAP Hormonal evaluation Low FSH, LH Pituitary imaging hCG / FSH supplementation High FSH Karyotype YCM ICSI TESA for azoospermia Donor sperms Repeat semen after 3 months Normal hormones Cannot afford ICSI No sperms in TESA S/O obstruction Idiopathic Obstructive Azoo TRUS CFTR test for CBAVD Pituitary failure Testicular failure

61. Treatment burden for MALE infertility falls on FEMALE Thank you