RNDr. I. Černáková, PhD.

Introduction in medical genetics 6

Slovenská zdravotnícka univerzita, Bratislava, 27.3.2017

(In)fertility

Inferility frequency

1 : 6 couples

or

15% of couples

Link between genetics and infertility disorders

• 1940s – gynecomastia, aspermatogenesis and increased secretion of FSH (Harry Klinefelter) – Klinefelter syndrome

• 1956 – determination of karyotype in KS patients: 47,XXY

• 1938 – the first description of Turner syndrome by Henry Turner

• 1964 – karyotype 45,X determination in patient with Turner syndrome

3 000 – 5 000 genes directly or indirectly influencing human fertility:

- genes expressed just in germ cells

- genes expressed just or also in gonads

- genes expressed in ontogenesis

Laboratory tests available:

- Genetic screening:

- basic panethnic: CF, SMA, hemoglobinopathies

- expanded population specific –Middle Eastern, Askenazi Jewish, Mediterranean

- expanded panethnic - few hunderds of autosomal recessive and X-linked diseases

Link between genetics and infertility disorders

- Genetic diagnosis of monogene disease in the family

- Genetic examination for some infertility-related mutations

(FSHR, Fragile X, FMR I, AR gene)

- Whole genome testing of infertility

(Atypical ovarian response, premature ovarian insufficiency, male factor infertility, disorders of sexual development, recurrent pregnancy loss) polycystic ovarian syndrome,

- Preimplantation genetic diagnosis /screening of chromosomal anomalies

(prevention of genetic diseases)

- Prenatal genetic diagnosis

(managment of delivery, treatment and care about child with genetic disease)

Clinical genetic counseling

Why clinical genetics in IVF treatment of infertility?

Infertile and sterile patients are a source of genetic pathologies - occurence of genetic pathology in infertile patients - occurence of particular genetic disease in family - transmission of genetic disease and infertility to the child by IVF treatment

Laboratory test of: - most offen occuring genetic pathologies in infertility - genetic diseases potentially passing to the next generation (karyotype + genetic-based infertility conditions + genetic screening – CF, SMA, hemoglobinopathies...)

Genetic counseling of: - results of genetic tests - risk of transmission of genetic disease to the next generation - explanation of reproductive option of examined couple - clinical genetic examination and laboratory genetic test of relatives at risk of diagnosed disease

Chromosomal abnormalities Preimplantation

period

Implantation I. trimester

II. trimester

III. trimester Term

Inherited birth defects

Early miscarriage 50 to 60 % when gross structural abn. is present

Late miscarriage 12 % 15% 0,5 % 4 – 5 %

50% loss before implantation at 5 to 6 days postconception

Spontanneous miscarriages - 15% of clinically recognized pregnancies before 12th weeks ´gestation

The frequency of chromosomal abnormalities after birth

General population 1 : 200 0,5 %

Infertile couples (spontaneous miscarriages, stillbirths)

1 : 48 2 %

Sterile couples 1 : 10 10 %

Prenatal/Postnatal

genetic diagnostics

Preimplantation

genetic diagnostics

and PGS + Mitoscore

Genet. dg PB Genet. dg of sperm

ET of good embryos

8 cell stage

Infertility treatment

- Endometrial receptivity assay

Zygota ET

REPRODUCTION

OF COUPLE

GENETIC EXAMINATION OF INFERTILE COUPLE

Embryo biopsy –

Day 3 or Day 5

Genetic examination of couple:

- Karyotype - Genetic tests of infertility

- Cystic fibrosis - Carrier genetic test

- Trombophilia mutations - Pharmacogenetics in infertility treatment

- Y-chromosome microdeletion

Genet. dg of spont. miscarriage

The reasons of male infertility

Frequency of infertility world-wide 15 – 20%

Gynecological reasons 40 %

Andrological reasons 40 %

Idiopathic infertility 20 %

WHO GUIDELINES AND

DEVELOPMENT OF SPERM QUANTITY

0

10

20

30

40

50

60

70

80

90

1951 1964 1987 2023

2010

15 mil/ml

The reasons of male infertility

Endogenneous factors

Disorders of sperm production

Disorders of seminal ductus

Genetic factors (translocations, numerical chromosomal abnormalities, Y-deletions, CFTR

gene mutations and mutations in other genes)

Other (anatomical, hormonal, age of man ...)

Hominid ancestry

Exogenneous factors

Industrial exhalation

Chemicals – herbicids, pesticids, insecticids

X-ray, microwave, mobil cells

Industry – heavy metals

Life style factors – smoking, alcohol, drugs, obesity, sauna/thermal baths

Inflammation diseases, radio-/chemotherapy

Human male germ cells

Characterization:

Poor quality exhibiting frequent morphological defects

Impaired motility

Poor chromatine compaction and condensation

High incidence of DNA fragmentation

Impaired capacity for oocyte recognition and fusion

Major source of of disease-causing mutations in humans

Infertility Dominant single gene disease

Childhood cancer

Birth defects

Immunological disorders

Sperm analysis - basic

(WHO 2010)

Volume > 1,5 ml

Count > 15 mil/ml

Motility > 40 %

Progressive motility > 32 %

Morphology > 4% of normal sperm

pH ≥ 7,2

Liquification up to 30 min

Leucocytes < 1 mil/ml Leu

SPERM DNA DAMAGE

Sperm cell disorders

Paternal imprinting disorders

Chromosomal aneupoidy

Defects of morphology

Defects of vitality (mitochondria insufficiency)

Residual cytoplasma

Protamines insufficiency

VitalSperm Vitality examination – Progressive vitality < 40%

Living immotile sperm Dead sperm - necrozoospermia

Pathological finding

of epididymis

Structural defects of tail

+

Defects/lower count of mitochondria

Urological, sperm donation DNA fragmentation

Antioxidants + ICSI

VitalSperm

LeucoTest

Examination

of leucocytes in ejaculate

Small round cells

> 1 mil/ml

Epitelial cells Leu > 1 mil/ml

Urological examination

Why is important to do correct semen analysis?

To know correct diagnosis of male infertility

To suggest and realize additional genetic tests

To propose to the couple their real opportunities to have baby

To choose the appropriate method of ART

(IVF/ICSI, PICSI, MACS or TESE)

or to decise for a sperm donation

ProtamineSperm

Examination of presence of protamines in sperm nucleus

– Morphology < 4%

– Idiopatic infertility

Protamine test When the strands are not packed well –

long DNA strands succeptible to damage

Sperm DNA fragmentation

Low probability of fertilization of oocytes

Infertility

Counselling of couple, IVF / ICSI treatment

Oxidative stress

Production of DNA adducts

DNA endonukleases cleavage

Imprinting disorders

DNA damage and

mutations

Assisted reproduction

Thermal stress in scrotum

DNA Reparation normal

Normal

embryogenesis

Abnormal development of

embryos

Unsuccessful implantation

Spontanneous miscarriages

Single gene disease in

children

Children tumor diseases abnormal

Infection

Disorders of spermatogenetis:

- residual of cytoplasma

- abortive apoptosis

- abnormal protamination

Disorders of DNA replication

increased by age

Xenobiotics:

- life style

- medications and oncotherapy

- professional environment

FragmentSperm

Direct examination of DNA damage

-ssDNA a dsDNA –

by TUNEL method

(fluorescence microscopy)

INDEPENDENT PROGNOSTIC

FACTOR for measurement

of sperm quality relating to the

MALE INFERTILIY

Sperm DNA fragmentation

Sperm DNA fragmentation

Normal sperm

Up to 15 % - normal level

> 15 % - IUI

> 20 % - IVF/ICSI

> 30 % - small probability of

spontaneous pregnancy

Men with OAT + Men with normal SPG

idiopatic infertility (normal SPG)

repeated unsuccessful IVF cycles: non-fertilized oocytes

no cleveage of embryos

embryo fragmentation

no clinical pregnancy

spontanneous miscarriages

men older than 40 years

exposition of toxins/medications

smoking

infections, inflammation

Sperm donation – testing of donors

INDICATIONS – Sperm DNA fragmentation

AneuSperm

FISH examination of sperm karyotype – chromosomal

aneuploidy

Examination of aneuploidy level

in sperm sample

- chromosomes 13, 18, 21, X a Y

OAT gravis

Normálny SPG

Men with

sperm aneuploidy

Infertile male patients with sperm

of pregnancy is miscarried by couples

with normal aneuploidy rate in sperm 60%

Embryos: aneuploidy in embryos

triploidy in embryos

Pregnancy: decreased pregnancy rate of the treatment of infertility

increased frequency of spontaneous miscarriages

Births: increased risk of aneuploidy in baby

Identification of men with low reproductive success

Personalized genetic counseling of infertile couple in relation to IVF treatment

Preimplantation genetic screening of aneuploidy – goals:

- to increase the pregnancy rate

- to decrease the risk of spontaneous miscarriage

- for a higher chance to have healthy baby

Sperm aneuploidy effects

Impact of sperm aneuploidy on pregnancy rate/ET

in IVF treatment and the incidence of spontaneous miscarriages

Men with OAT + Men with normal SPG

Repeated unsuccessful IVF cycles: non-fertilized oocytes

no cleavage of embryos

embryo fragmentation

no clinical pregnancy

Repeated spontanneous miscarriages with normal karyotype of couple

Chemicals /Medication exposition /Chemotherapy/Radiotherapy

Preimplantation genetic diagnostics

Donation programme

The indications - AneuSperm

Why to do the comprehensive sperm analysis?

• To know the correct diagnosis of male infertility

• To suggest the additional tests for correct diagnosis (karyotype, CF mutation screening, Y-chromosome deletions, FSH gene and FSH receptor gene polymorphism for hormonal treatment ...)

• To explain possible causes of infertility and the risk of transmission of infertility, recurrent miscarriages and genetic diseases to their children

• To guide the treatment of male infertility: - antioxidants - hormonal treatment by low dose-long term FSH according to the genotype - to recommend the appropriate methods of IVF: ICSI, TESE for non-obstructive azoospermia, MACS / PICSI preimplantation genetic screening for aneuploidy, for CF ....)

Why clinical genetics in IVF treatment of infertility?

Infertile and sterile patients is s source of genetic pathologies - occurence of particular genetic disease in family - occurence of genetic pathology in infertile patient - transmission of genetic disease and infertility to the child by IVF treatment

Laboratory test of: - most offen occuring genetic pathologies in infertility - genetic diseases potentially passing to the next generation (karyotype + genetic-based infertility conditions + genetic screening – CF, SMA, hemoglobinopathies...)

Genetic counseling of: - results of genetic tests - risk of passing of genetic disease to the next generation - explanation of reproductive option of examined couple - clinical genetic examination and laboratory genetic test of relatives at risk of

diagnosed disease

CLINICAL GENETICIST - Genealogy, physical examination,

genetic counseling

- Karyotype

- Cystic fibrosis

- Trombophilic mutations

- Y-chromosome microdeletion

- examination of genetic diseases occuring in some population

(thalassemias, familial mediterrean fewer, Jewish specific disorders,

or in particular patients/family (monogene diseases)

Foresta C, Ferlin A, Gianaroli L, Dallapiccola B:

Guidelines for the appropriate use of genetic tests in infertile couples.

Eur J Hum Genet, 2002, 10, 5, 303 – 312.

Preconception clinical genetic service of infertile couples

Chromosomal abnormalities and human reproduction

Disturbance of gametogenesis

- male infertility: oligoasthenoteratozoospermia

azoospermia

- female infertility: disturbance of menstrual cycle

premature ovarian insufficience

Unbalanced chromosome abnormalities in gametes

- reproduction failures : spontaneous miscarriages

stillbirths

birth defects

neonatal death

Medical indications of cytogenetic examination of infertile couple

1. Occurence of chromosomal abnormality in family

2. Congenital abnormality in development and/or mental disabbility in family

3. Infertility (minimal 2 spont. miscarriages, stillbirths, repeated implantation failures in IVF cycles)

4. Idiopathic infertility (more than 1 year or 6 months when woman is older than 35 yrs.)

5. Andrologic infertility (OAT, azoospermia)

6. Disorders of menstrual cycle (amenorhea, oligomenorhea)

7. Anatomical defects of male/female genitalia

8. Professional and medical exposition

9. Couples before PGS of aneuploidy

Cystic fibrosis

Indication of examination of CFTR gene mutation

Male infertility - oligozoospermia ( 1< 106/ml )

- azoospermia

Men with congenital bilateral absence of vas deference (CBAVD)

Familial occurence of cystic fibrosis - based on genealogy

Indications of examination - Trombophilic mutations

Leiden mutation (Factor V gene) Protrombin gene mutation (Factor II)

- Deep venal trombosis in personal and/or familial history

- Repeated spontaneous miscarriages

- Repeated unsuccessful IVF cycles

- Reproductive history:

stillbirth, premature delivery, abruption of placenta, preeclampsia, IUGR

Y-chromosome microdeletions

Men with severe oligozoospermia or azoospermia and Y-chromosome microdeletion:

- 100% of probability of microdeletion

and infertility transmission to the sons

- Decreased fertilization rate of oocytes

- Less quality of embryos

Indication of examination:

- Oligozoospermia (1<106/ml) – 5% of men

- Azoospermia – 10% of men

Genetic counseling of result of test and explanation

of reproductive option and IVF treatment (TESE/MESA)

The cause of spontaneous miscarriages

Genetic laboratory tests

- couples with repeated spontaneous miscarriages

Test Who? Material

Karyotype Both partners Peripheral blood

Annexin Both partners

Peripheral bloos / buccal cells

Trombophilia mutations FV, FII

Woman Peripheral bloos / buccal cells

Sperm DNA fragmentation + ROS

Men Sperm

Sperm chromosome aneuploidy

Men Sperm

+ Clinical genetic counseling

Annexin 5

ANXA5 gene – chromosome 4q27 4 polymorphisms in promotor of the gene: 19G>A (rs112782763) +1A>C (rs28717001) 27T>C (rs28651243) 76G>A (rs113588187)

M2 haplotype M1 haplotype

N (WT) – standard alllele M1 haplyotype – non-pathologic, no complications in pregnancy, no increased risk of spontaneous miscarriage M2 haplotyp – defect of placental vasculature, venal tromboembolism, 4x increased risk of spontaneous miscarriage, IUGR and intrauterine death of fetus, gestational hytertension, preeklampsia M2/M2 haplotype of fetus – severe form of IUGR

Annexin 5

Activity of ANXA5 gene promoter variants in luciferase reporter gene assays.

Nadia Bogdanova et al. Hum. Mol. Genet. 2007;16:573-578

© The Author 2007. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oxfordjournals.org

Genotypes: N/N N/M1 M1/M1 N/M2 alebo M1/M2 M2/M2 Incidence: of IVF patients has M2 haplotype of persons has M2/M2

The fetus has an increased risk to inherit minimal one M2 maternal or paternal

Men has no risk himself, nor woman when is not pregnant!

44%

6%

• Idiopathic infertility – 37% • Repeated miscarriages • OAT - 40% • PCOS – 35% • Premature ovarian insufficiency

Annexin 5

The treatment – low molecular weight of heparine NO ASPIRIN!

from embryo transfer – to end of puerperium

Which material to examine? • Peripheral blood • Buccal cells

How? • Genetic test • NO IMMUNOLOGIC !

Chorionic villi biopsy

Amniocentesis

molecular-genetic diagnostics

Invasive prenatal diagnostics

PGD / PGS Preimplantation genetic diagnostics - analysis of genetic disorders in early human embryos before their embryotransfer in uterus as a prevention of single gene disorders

• Introduced in routine: 1990 – adrenoleukodystrophy

Preimplantation genetic diagnostics – prevention of genetic disorder

transmission to the next generation

- single gene disorders - chromosomal translocations

Preimplantation genetic screening af aneploidy – prevention of transfer of

chromosomally abnormal embryos of „poor responder“ couples

- older women (35+)

- repeated unsuccessful impalntation of embryos

- repeated spontaneous miscarrages

- male infertility

PGD medical indications

Preimplantation genetic diagnosis

- Diagnostics of single gene disorders

- Diagnostics of translocations

- Diagnostics of late-onset genetic diseases or cancer diseases occuring

in adulthood

- HLA typization of embryos

- Mitochondrial diseases

PGD - steps

Haplotype analysis of family

– PGD set-up

IVF/ICSI

Hormonal stimulation

Oocyte pick-up

Fertilization

Embryo biopsy – Day 3,

Day 5 or blastocentesis

Genetic analysis

IVF – Embryo transfer

Hormonal stimulation of ovaries

Oocytes pick-up

Embryo culture

Vyšetrovaný materiál

1st polar body 2nd polar body

Trophectoderm of blastocyst – Day 5 Blastomere - Day 3

Embryo biopsy

PCR – PGD steps

A. Biopted of cell (cells) is transfered

in lysis buffer solution in eppendorf

tube

B. Its genome is released from nucleus

of cell.

C. Whole genome amplification (WGA)

– amplification of all genome of cell

D. Fragmentation analysis of examined

gene using capillary electrophoresis

– analysis of genotype of embryo

Haplotype analysis

Allelic drop-out risk

Analysis of 5 fully informative DNA

markers linked to the mutation

Haplotype analysis of the family

Marker –IVS10CA

proband - sick

mother

father

healthy daughter

IVS10CA Alela 1 Alela 2

Proband - sick 318 330

Mother 320 330

Father 318 320

Healthy daughter 320 320

Analysed

12 DNA markers

before, inside and

after CFTR gene

Transfer of healthy embryo

PGS medical indications

Preimplantation genetic screening of aneuploidy

FISH: 8 chromosomes

aCGH / NGS: all chromosomes

- Women older than 35 - 37 years

- Repeated unsuccessfull IVF cycles

- Repeated spontaneous miscarriages

- Trisomic fetus in previous pregnancy (Down syndrome)

- Male infertility (OAT gravis, TESE/MESA)

Chromosomal abnormalities in oocytes and maternal age

0,001,002,003,004,005,006,007,008,009,0010,0011,0012,0013,0014,0015,00

16 X 212215 131814 9 2 4 7 8 2010 1217 6 5 11 3 19 Y

Chromozóm

Pe

rce

nto

%

Frequency of chromosomal abnormalities

in spontaneously miscarriages – Ist trimester

Frequency of aneuploiy in preimplanattion period – abnormalities detected by CGH method

Munné et al. , 2010

PGD/PGS schematic representation Day 0 Oocyte pick-up

Day 1 Fertilisation

Day 3 Biopsy of blastomere

Day 3 + Day 4 results, report FISH, aCGH, PCR-PGD

Day 5 Embryo transfer Biopsy of vitrification of biopted embryos trophoectoderm aCGH, NGS ET in next menstrual cycle

arrayCGH

arrayCGH

Examples of aCGH results

-10 -22

-5p parc. +19

blastomera

6denní embryo

Spontaneous miscarriages I. Aneuploidy

Natural conception: 15 %

ART: 23 – 37 %

PGS: 12 - 15 %

2 x comparing to ICSI

Spontaneous miscarriages II. Translocations

Spontaneous miscarriages:

Natural conception 87 %

PGD 18 %

5 x

Take-home-baby rate:

Natural conception: 11,5 %

PGD: 81,4 %

7 x

Trisomy child delivery

Natural conception:

2,6 % trisomy 13, 18 a 21 (chorionic villi examinations)

PGD cycle:

0,6 % trisomy 13, 18 a 21

independent on type of hormonal stimulation

4 x

Mitochondria during embryogenesis

Mitoskóre Implantation rate %

< 18 81%

18-24 50%

24-50 65%

> 50 18%

> 160 none

Importance of PGD / PGS

Diagnosis - diagnosis of chromosomal anomaly/single gene disease according to the patient´s medical indication

Prevention - decreased risk of spontaneous miscarriage in IVF couples to 15%

PREVENTION OF DELIVERY OF CHILD WITH GENETIC DISORDER !

Treatment - higher effectivity of infertility treatment in poor prognosis patients

Prognosis - prognosis & change of infertility treatment in next IVF cycle

Finance - next examinations & IVF treatment

Psychology - psychic status of infertile couple

Single gene disorders

• Fenotyp effects are caused by the mutation in one gene

• Mendelian type of inheritance

• 7 000 single gene disorders

WHO: Prevalence 10 : 1000

20% of cases of child mortality

in developed countries

40% of medical interventions

in children hospitals

(Kanada – Scriver, 1995)

Carrier screening of single gene diseases

single gene diseases AR a X-linked inheritance

General population – affected 1 : 100

Carriers

Cystic fibrosis 1 : 25 4 %

Spinal muscular atrophy 1 : 50 2 %

Αlpha-/Beta-hemoglobinopathy 1 : 48 2 %

- analysis of 549 genes, 600 single gene diseases using NGS

- high risk of transmission of disease with recessive inheritance

(AD or X-linked) to the next generation

2.1 pathogenic mutation/person

7.1 % of tested persons don´t have pathogenic mutation

8% couples carry the mutation in the same gene

( so called „genetic incompatibility“)

Carrier screening of single gene diseases

Preimplantation genetic diagnostics

1 : 100 1 : 100 000

Risk of the delivery of the child affected by single gene disorder

For whom? Before natural conception

Before assisted conception

Oocytebanking and spermbanking

Carrier screening of single gene diseases

Single gene disorders can´t be cured, but they can be prevented

Primary prevention of occurence of single gene disorders in family

- Preimplantation genetic diagnostics

- Carrier screening of single gene disorders

Genetics and IVF clinic a multidiscipline dialogue in assisted reproduction

Clinical geneticist

- physical examination

- genealogy

- pre-test counseling

- indication of genet. lab. tests

- post-test counseling

IVF clinic

Genetic lab

- Molecular cytogenetics

Genetic lab

- DNA lab

Genetic lab

- Cytogenetics

Urology

Gynecology

Genetic lab

- PGD lab

Endocrinology

Bring genetics in your practice

TREATMENT

GUIDE treatment, such as gonadotropin dosage and prescription of blood thinners

MANAGE treatment risks, such as ovarian hyperstimulation syndrome

DECISION-MAKING

INFORM decisions about reproductive options, plan and treatment protocol

RECOMMEND preimplantation genetic diagnosis/screening (PGD/PGS)

GUIDANCE

PREDICT and prepare patients for possible outcomes of infertility treatment

EXPLAIN possible causes of infertility or recurrent miscarriage

Thank you for your attention

Genetic lab ReproGen

Bratislava, Slovakia

Tel: 0948 230 661

www.reprogen.sk