Infertility if quite common. Understanding genetic basis of infertility can help in making the right decision on what diseases can be transmitted to the offspring and which IVF technique would be most helpful in overcoming infertility.

1. Genetic Basis of Male & Female Infertility
Work up of infertile couple
Prof. Ashok Rattan,
MD, MAMS,
Common Wealth Fellow, INSA DFG Fellow, SEARO Temporary Advisor,
WHO Lab Director (CAREC/PAHO)
Advisor: Pathkind Labs Knowledge Forum, R & D and Quality.

4. Where should we start from ?

5. Definition of Infertility
Failure to conceive after one year of regular unprotected intercourse
Observation:
• Couple having regular, unprotected
intercourse
• 50 to 60 % will conceive in 3 months
• 70% in 6
• 85% in 1 yr
• 90% in 2 yrs
Goal of evaluation:
• To identify the cause of infertility
• To prescribe adequate therapy
• Provide accurate information to
the couple
• Correct the misinformation
gained from friends, family and
Google
• Provide emotional support
during evaluation and treatment

6. When to initiate evaluation
A. Initiate evaluation after 12 months of unprotected & frequent intercourse
in women < 35 yrs of age without risk factors of infertility
B. Initiate evaluation after 6 months of unprotected & frequent intercourse
in women between 35 and 40 years of age
C. Initiate evaluation upon presentation despite less than 6 months of
unprotected & frequent intercourse in
1. Women over 40 years
2. Women with amenorrhoea / oligomenorragia
3. Women with history of chemotherapy, radiation therapy, advanced endometrosis
4. Women with known or suspected uterine / tubal disease
5. Women whose male partner has
1. a history of groin or testicular surgery,
2. Adult mumps
3. Impotence
4. Subfertility with other partner
5. Chemotherapy, radiation

7. Evaluation strategy
• Couple must be considered as a
single unit as each partner
contributes a share to the
infertility potential of the couple
• Many couple have more than
one contributory cause which
would be identified early in the
evaluation
infertility causes
female male both

8. Copyrights apply

9. Elements of Evaluations
1. Medical History of both female and male partners
2. Focused Physical Examination
3. Semen Analysis
4. Detection of Ovulation
5. Ovarian reserve (if indicated)
6. Patency of fallopian tubes
7. Uterine Evaluation
8. Genetic Evaluation
9. Preconception counselling
10. PGS or PGD
11. IVF

10. FC # : Flow Chart

11. 2. Focused Physical Examination of couple

12. 3. Semen Analysis
Nomenclature
Classification Definition
Normospermia Normal values
Oligozoospermia Sperm count < 20 x 10 6/ml
Astherozoospermia < 50% sperms with forward
progression or
< 25% rapid linear progression
Teratozoospermia < 30% sperm with normal
morphology
Oligoastherozoospermia Attributes of all three
Azoospermia No sperm in ejaculate*
Aspermia No ejaculate
Normal Value
Element Value
Volume 1.5 ml [1.4 to 1.7 ml]
pH 7.2 – 8
Sperm concentration > 20 x 10 6/ml
Total sperm count > 40 x 10 6/ml
Motility within 60 min > 50% sperms with forward
progression or
> 25% rapid linear progression
Morphology > 4 % sperm with normal
morphology
Vitality > 75% live
WBC < 1 x 10 6/ml

14. Tests & Procedures for Male Fertility
• Acrosome Stimulation test
• Anti-sperm Antibody test
• Cervical mucus interaction
• Hamster egg penetration test
• Hormone analysis
• Krueger or Strict Criteria Morphology
• Hypo-osmatic solution assay
• Post coital test
• Retrograde semen analysis
• Semen culture
• Sperm Chromatin Integrity Test
• Sperm longevity

15. Sperm DNA fragmentation test
• Abnormal genetic material within the sperm which leads to
• Male infertility
• IVF failure
• Miscarriage
• Studies shoe that
• High sperm DNA fragmentation affects blastocyst development
• Higher DNA fragmentation levels, higher chances of failed IVF & miscarriage
• Sperm DNA fragmentation is higher in subfertile men with abnormal sperm
parameters
• Advantages of sperm DNA fragmentation test
• Provides reliable analysis of sperm DNA integrity
• Provides information helpful in clinical diagnosis, management & treatment of male
fertility
• Provides prognostic value in assessing outcome of assisted conception treatment

16. Sperm DNA fragmentation test
causes
• Infection
• Elevated testicular temperature
• Recreational drugs
• Smoking
• Alcohol
• Stress
• Diet
• Environmental & occupational pollutants
• Advanced chronological age
• varicocoele
Indications for the tests
• Unexplained infertility
• Arrested embryo development
• Poor blastocyst development
• Multiple failed IVF/ ICSI treatments
• Recurrent miscarriages
• Advanced chronological age
• Varicocoele
• Poor semen parameters
• Exposure to harmful substances

17. AO Test
[Acridine Orange]
• Metachromatic shift in
fluorescence of AO when bound
to ssDNA  Red
• Ds DNA  green
• Rapid, simple & inexpensive
• Inter-laboratory variations
• Lack of reproducibility
• Subjective

18. AB staining
[Aniline blue]
• Increased affinity of AB dye to
loose chromatin of sperm
nucleus
• DNA fragmentation  stain
• Rapid, simple, inexpensive
• Inter laboratory variations
• Lack of reproducibility
• Subjective

19. CMA3 staining
[Chromomycin A3]
• CMA3 competitively binds to DNA
• Protamine deficient sperms 
bright yellow
• Normal sperms  yellowish green
• Indirectly visualizing protamine
deficient DNA
• Yields reliable results
• Inter observer variability
• subjective

20. TB staining
[Toulidine blue]
• Increased affinity of TB to sperm
DNA phosphate residues
• Normal sperms  blue
• DNA fragmentation  violet
• Rapid, simple, inexpensive
• Inter observer variability

21. Tunel
[Terminal deoxynucleotidyl transferase dUTP nick end labelling
• Quantifies enzymatic
incorporation of dUTP into DNA
breaks
• Uses optical microscopy,
fluorescent microscopy and flow
cytometry sorting histogram
showing % of SDF
• Sensitive, reliable, reproducible
• Can be performed on few
sperms

22. SCSA
[Sperm chromatin structure assay]
• Measures susceptibility of sperm
DNA to denaturation. Cytometric
version of AO test
• Uses flow cytometry
• Reliable estimate of % of DNA
damaged sperms

23. SCD or Halo test
[Sperm chromatin dispersion]
• Assess dispersion of DNA
fragments after denaturation
• Uses optical or fluorescent
microscopy
• Simple test
• Interobserver variability
• Subjective

24. SCGE or COMET
• Electrophoretic assessment of
DNA fragments of lysed DNA
• Uses fluorescent microscopy
• Can be done in very low sperm
count
• Sensitive & reproducible

25. 4. Detection of Ovulation
More often in ovulatory cycles
• Menstrual history
• Menstrual cycle length:
• 21 – 35 days
• Moliminal symptoms:
• Breast tenderness
• Mood changes
Causes of Menstrual irregularity
• PCOS
• Testosterone
• Follicular phase 17 – OHP
• Thyroid dysfunction
• TSH
• Hyperprolactinemia
• Prolactin
• Hypothalmic dysfunction
• Primary Ovarian insufficiency

26. Copyrights apply

27. Follicular Stimulating Hormone (FSH)
levels to be considered along with other hormones
• Associated with development of eggs in women & sperm in men
• Produced by Pituitary gland,
• controlled by hypothalamus vis Gonadotropin releasing hormone
• Can be measured in blood or urine
• FSH stimulates growth & maturation of eggs (follicles) during follicular phase of the
menstrual cycle; FSH levels 2.8 to 11.3 mIU/ml
• FSH initiates production of estradiol by the follicle
• Near end of follicular phase surge of FSG & LH  ovulation; FSH level 5.8 to 21 mIU/ml
• FSH facilitates ovary to respond to LH, FSH level during Luteal Phase 1.2 to 9 mIU/ml
• Inhibin, estradiol & progesterone help modulate FSH release by pituitary
• At menopause ovarian function wane, FSH & LH levels rise; FSH level 22 to 153 mIU/ml
• In men FSH stimulates testes to produce mature sperm
• FSH levels in men are relatively constant after puberty; 0.9 to 11.8 mIU/ml

28. Luteinizing Hormone (LH)
• LH is produced by the pituitary gland, controlled by hypothalamus
• It is released intermittently throughout the day
• LH is associated with reproduction & stimulation of release of egg from ovary
(ovulation) in females and testosterone production in men
• Measured in blood or urine (24 hours sample is more representative)
• LH stimulates ovulation & production of estradiol & progesterone; LH 17 to 77 mIU/ml
• During Luteal phase, ruptured egg follicle become corpus luteum & produce
progesterone, FSH & LH levels decrease while progesterone & estradiol levels increase,
then decrease if egg is not fertilized Start of menstruation
• At menopause as ovarian functions wane, LH levels rise; 11.3 to 40 mIU/ml
• In men, LH stimulates Leydig cells in testicles to produce testosterone
• LH levels are relatively constant after puberty in men. 1.0 to 8.65 mIU/ml
• A high testosterone level provides negative feedback to pituitary & LH decreases

29. Estrogen
• A group of steroid hormones responsible for
• Development & function of reproductive organs
• Formation of secondary sex characteristics in women
• Regulate menstrual cycle (along with Progesterone)
• Involved in growth of breasts & uterus
• Help maintain a healthy pregnancy
• Play a role in bone metabolism
• Three components
• Estrone (E1)
• Estradiol (E2)
• Estriol (E3)
• Measured in blood or urine

30. • During menstruation Estradiol (E2) & other hormones rise & then fall in a
specific sequence
• During pregnancy estriol (E3) is the primary estrogen. It is produced by
placenta, starts to rise in 8th week & continues throughout
• Sharp rise in E3 occurs 4 wks prior to onset of labour
• E1 also rises during pregnancy, increasing to 10x between 24 & 40 wks
• After delivery, E1 falls & E3 again becomes essentially undetectable
• During menopause Estrone (E1) is the primary estrogen
• E2 conc. Significantly decrease as ovarian function declines.
• Reference Range:
• Adult male : ND to 56 pg/ml
• Adult Female: Follicular phase ND to 160 pg/ml
• day 2 & 3 ND to 84
• periovulatory 34 to 400
• luteal 27 to 246
• menopausal ND to 30

31. Progesterone
• A steroid hormone, main role to prepare woman’s body for pregnancy
• While estrogen causes endometrium to grow, LH surge leads to ovulation, a
corpus luteum forms on ovary produces progesterone
• Progesterone Stops endometrial growth & readies uterus for possible
implantation of a fertilized egg
• If no fertilization, corpus luteum degenerates, progesterone levels drop &
menstrual bleeding begins
• If fertilized egg is implanted in uterus, corpus luteum continues to produce
progesterone
• Egg forms a trophoblast that produces human chorionic gonadotropin (hCG)
• Soon Placenta replaces corpus luteum as main source of progesterone
• In males progesterone is responsible for development of sperm.

32. Prolectin
• Is a hormone produced by anterior pituitary gland
• It is measured in blood
• Primary role to promote breast milk production (lactation)
• Elevated in women during pregnancy & just after childbirth
• Dopamine & estrogen control production & release of prolactin
• Following childbirth, prolactin helps initiate & maintain milk supply
• If mother doesnot breast feed, prolactin level soon drops
• Sucking by infant plays an important role in release of prolactin
• Prolactinoma, a benign pituitary tumour leads to elevated levels
• Prolactinoma can lead to infertility and irregularities in menstruation

33. Testosterone
• Is the main sex hormone in men
• Responsible for male physical characteristics
• Produced by Leydig cells in male testicles & adrenal glands of both sex
• High level during puberty & adult male
• Regulate sex drive, Maintains muscle mass
• Present in women too, small quantity also produced by ovaries
• Converted to estradiol I females
• Controlled by LH, testosterone acts as a negative feedback for LH
• Diurnal variation, peaking early morning, lowest in evening
• Levels increase after exercise & decrease with age
• Two third circulates bound to sex hormone binding globulin (SHBG), slightly less
than one third bound to albumin. Only 4% is free testosterone, which is active

34. Progestrone
Estradiol
LH
FSH

35. Basal Hormonal Levels in Males
Clinical Condition FSH LH Testosterone Prolactin
Normal spermatogenesis N N N N
Hypogonodatropic
hypogonatropism
L L L N
Abnormal spermatogenesis H/N N N N
Complete testicular failure H H N/L N
Prolactin secretory Pituitary
tumor
N/L N/L L H

36. 5. Ovarian Reserve
OOCTES
• All females have a fixed number
of gametes
• Max number is 7 million oocytes
at 7 months of gestation
• By birth it is down to 1 million
• By Puberty there are only
300,000 oocytes
• By menopause  Zero

37. 5. Ovarian Reserve (if indicated)
1. Tests
• Early follicular (day 3) serum FSH & Estradiol
• FSH level at menopause > 20 mIU/ ml
• FSH > 10 to 11 mIU & Estradiol > 80 pg /ml associated with decrease in egg quality
and fertility
• Single elevated cycle day 3 FSH values suggest poor prognosis
2. Clomiphene citrate challenge test (CCCT)
3. Serum inhibin : no longer recommended
4. Anti Mullerian Hormone (AMH)
Produced by granulosa cells in antral follicle
Reflect size of remaining follicle pool,
correlates well with AFC, but not predictive

38. Anti-mullerian Hormone
(AMH)
• AMH is a glycoprotein produced by reproductive tissues, including testicles in males & ovaries
in females.
• It is measured in blood
• In Baby Boy: AMH inhibits development of female reproductive organs while promoting
development of other male reproductive organs.
• In Boys, level of AMH remains high until puberty, when it begins to taper off.
• In girls, low levels are produced and remain low till puberty
• AMH levels increase after puberty and then steadily decline, undetectable after menopause
• AMH has balancing effect on monthly cyclic actions of FSH & LH during process of egg
maturation & ovulation
• AMH levels are useful in determining remaining egg maturation potential & likihood of
conceiving
• AMH plays a vital part in sexual differentiation in the foetus
• Elevated AMH levels are associated with polycyctic ovary syndrome (PCOS)

39. Model of AMH action in the Ovary

40. Anti-mullerian Hormone
(AMH)

42. 6. Patency of Fallopian Tubes
Causes of damage
• STD: Gonococcus, Chlamydia
• TB
• Peritonitis: ruptured appendix
• Prior pelvic surgery
• Endometriosis
Evaluation
• Hysterosalpingogram (HSG)
• Laparoscopy
• Sonohysterogram
• Tests for STD, TB

43. Sites of infection in FGTB
90 – 100 % 
50 – 80 % 
20 – 30 % 
5 – 15 % 
1 – 2 % 

45. CD8 T-cells
• Suppress Mycobacteria tuberculosis growth
• Kill infected cells
• Directly lyse intracellular Mycobacteria
• TB specific CD8 T cells that produce IFN-Ύ have been
• More frequently detected in those with active TB disease vs latent infection
• Associated with recent exposure
• Detectable in active TB subjects with HIV co-infection & young children

47. 7. Uterine Evaluation
• Endomatrial cavity assessment
• HSG
• SonoHSG
• Hysteroscopy
• 3D Ultrasound or MRI for Mullerian
anomalies
• Endometrial biopsy
• Markers of implantation

48. 8. Genetic causes of Infertility
Male
• Klinefelter syndrome
• Translocations
• Y chromosome microdeletion
• X chromosome defects
• CFTR gene mutation
• Trisomy 13, 18, 21
• Epigenetics
Female
• Turner syndrome
• Translocations
• Hypogonadotropic Hypogonadism
• Premature Ovarian Insufficiency
• Trisomy 13, 18, 21
• Genetics of Endometriosis
• Genetics of Polycystic Ovarian Syndrome
• Epigenetics

49. Laboratory Techniques for investigations of
Genetic Disorders
• 1. Chromosomal analysis
• 2. Fluorescent in situ hybridization (FISH)
• 3. PCR & Sequencing
• 4. PCR & Microarray
• 5. Next Generation Sequencing (NGS)

50. Genetic Basis of Male & Female Infertility
• Infertility affects 50 million couples world wide (WHO) and prevalence is
rising
• 25 to 30% of couples with Idiopathic infertility are likely to have a genetic
etiology for their condition
• Application of Next Generation Sequencing has added to our knowledge
• Genetic causes may be due to
• 1. Cytogenetic abnormalities
• 2. Gene defects
• 3. Epigenetic aberrations

51. Human Genome
• Gene is the basic unit of inheritance
• Genes are organised on chromosomes
• In humans there are 23 pairs of chromosomes [46 chromosomes]
• 22 chromosomes are somatic and one pair sex chromosome
• Human Genome consists of 6 million base pair
• 20,000 genes encode proteins (exone, 1.5%)
• Rest are either regulatory (8%) or ? Junk (interons)
• Each chromosome consists of two chromatids
• Each chromatid has a centromere which divided the chromosome into two
arms
• If centromere is in the centre = Metacentric
• Towards one end = Submetacentric: small p arm and long q arm
• Near terminal end = Acrocentric

54. Mitosis Meiosis

57. Laboratory Techniques for investigations of
Genetic Disorders
• 1. Chromosomal analysis
• 2. Fluorescent in situ hybridization (FISH)
• 3. PCR & Sequencing
• 4. PCR & Microarray
• 5. Next Generation Sequencing (NGS)

58. Chromosomal Examination: Karyotyping
• A. Numerical Changes:
• Normal : Euploidy: 46, XX or 46, XY
• Aneuploidy :
• Somatic Trisomy 21: 47, XX, +21 [Down Syndrome]
• Trisomy 18: 47, XY, +18 [ Edward Syndrome]
• Trisomy 13 : 47, XX, +13 [Patou Syndrome]
• Sex Chromosome 45, X [ Turner Syndrome]
• 47, XXY [Klinefelter Syndrome]
• B. Structural changes:
• Balance
• Reciprocal
• Deletion: Macro, Micro

59. Preparation of Metaphase Chromosomes

60. Karyotyping

63. Balanced chromosomal abnormalities Robertsonian translocation
Robertsonian translocation commonly Involves chromosome 13,14,15,21 or 22, but have 50 to
70% of healthy live birth if closely monitored.
Patients with unbalanced translocation have 5 to 10% chance of pregnancy that may have
abnormalities, should get antenatal diagnosis

65. Turner Syndrome
45, x or 45,x / 46,xx
• Monosomy X but may have mosaicism of x chromosome
• Short stature
• Dysmorphic features are common & include
• Posteriorly rotated ears
• Webbing of the neck
• Shield like chest
• Cubitus valgus
• Short fourth & fifth metacarpals
• Hypoplastic nails
• Lymphedema
• Pigmented nevi
• Congenital heart defects
• Streak gonads with ovarian failure & puberty delay

67. Klinefelter Syndrome
47,XXY
• Most common sex chromosome
abnormality causing primary
hypogonadism
• 47, XXY karyotype results from
nondisjunction of sex
chromosomes
• Most cases are detected
postnatally
• Diagnosed during evaluation for
infertility or gynecomastia

68. Klinefelter Syndrome
• Infertile man with
• Elevated serum FSH
• Elevated LH
• Sperm count < 10 million / ml
• Increased risk for
• psychiatric disorders
• Autism
• Social problems
• Karyotyping 47, XXY

69. XXY Klinefelter syndrome Mosaic Yq del Inv (Y)
Males
Females
Turner Syndrome and variants del(Xp) del(Xq) i(Xq)
KARYOTYPE PATTERNS IN INFERTILITY

70. Laboratory Techniques for investigations of
Genetic Disorders
• 1. Chromosomal analysis
• 2. Fluorescent in situ hybridization (FISH)
• 3. PCR & Sequencing
• 4. PCR & Microarray
• 5. Next Generation Sequencing (NGS)

71. Fluorescent In Situ Hybridization (FISH)

75. Laboratory Techniques for investigations of
Genetic Disorders
• 1. Chromosomal analysis
• 2. Fluorescent in situ hybridization (FISH)
• 3. PCR & Sequencing
• 4. PCR & Microarray
• 5. Next Generation Sequencing (NGS)

77. Laboratory Techniques for investigations of
Genetic Disorders
• 1. Chromosomal analysis
• 2. Fluorescent in situ hybridization (FISH)
• 3. PCR & Sequencing
• 4. PCR & Microarray
• 5. Next Generation Sequencing (NGS)

78. Microarray

79. Conventional Karyotyping Comparative Genomic Hybridisation /
Microarray
Involves cell culturing No cell culture required
High Failure rate High Success rate
Detects only large chromosomal re-arrangements,
(resolution 5-10Mb)
Detects sub-microscopic chromosomal re-arrangements
(resolution depending on array)
Sample collection more tedious Easy sample collection
Cannot rule out maternal contamination Maternal cell contamination testing done on all DNA
samples
Can pick up balanced translocations and inversions Limitation – cannot pick up balanced translocations (not
cause of abortion)
Comparison

81. Y Chromosome microdeletions
• Increasing being recognized as genetic
causes of azoospermia & severe
oligozoospermia
• Upto 20% infertile men with sperm conc.
< 5 million / ml have microdeletion in
long arm of Y chromosome, maps to Yq11
• Named azoopermia factor (AZF)
• Has three regions: AZF a, AZF b & AZF c
• Deletion of AZFa & AZFb  azoospermia
• Testicular biopsy will show germinal cell
maturation arrest or Sertoli cell only
• Deletion of AZFc causes infertility but has
variable phenotype ranging from
oligozoospermia to azoospermia
• Test if
• Sperm conc. < 5 million/ml

85. Cystic fibrosis transmembrane conductance regulator
CFTR gene
• Present with obstructive azoospermia with or without manifestations
of cystic fibrosis
• Normal testicular volume
• No vas deference on palpation of external genitalia
• Normal FSH, LH & testosterone conc.
• May give a family history of cystic fibrosis
• Both partners should be tested for CFTR gene mutation

86. Karyotypic abnormalities:
46,XXY
45,X/46,XY mosaicism
Structural abnormalities inv Y
Sperm FISH with X & Y probes (low count)
CFTR mutations for Cystic Fibrosis
Y chromosome 20 Microdeletions
MALE FACTOR INFERTILITY

87. Laboratory Techniques for investigations of
Genetic Disorders
• 1. Chromosomal analysis
• 2. Fluorescent in situ hybridization (FISH)
• 3. PCR & Sequencing
• 4. PCR & Microarray
• 5. Next Generation Sequencing (NGS)

88. •

89. ABI 3700

90. •
Next Generation Sequencing

91. Strategies for Next Generation Sequencing
1. Whole Genome Sequencing (WGS)
100+ Gb sequencing
2. Whole exome sequencing (WES)
Capture all (200,00) exons & sequence them
Less sequencing & analysis (38 Mbs)
3. Target sequencing
Capture regions of interest & sequence them
4. Transcriptone sequencing (RNAseq)
What is being actively transcribed in the cell of interest
20,000 protein coding genes

92. •
Cost of Sequencing is falling every year

93. Next Generation Sequencing (NGS)

97. Consultation with a qualified Genetic Counsellor
Preimplantation Genetic Diagnosis
PGS / PGD / PGT
In Vitro Fertilization (IVF)
Intra Uterine Insemination (IUI),
IVF
Intracytoplasmic Sperm Injection (ICSI)
Donor Egg

98. When to order Genetic tests in infertile couple
• 1. Physical examination shows complete bilateral absence of vas deference
(CBAVD)
• mutation of CFTR gene located on chromosome 7
• 2. Sperm analysis shows Oligozoospermia (< 5 x 106 sperms /ml)
• Karyotyping & FISH
• Suspected Klinefelter Syndrome (XXY)
• Yq chromosome microdeletion (Y)
• 3. Infertility in female
• Karyotyping & FISH
• Turner syndrome (X)
• POF syndrome (FMR 1 gene)

99. Thanks you for your attention