Chromosomal and sub-chromosomal abnormalities in infertility

M. R. Zamanian MD, PhD

Department of Genetics

Reproductive Biomedicine Research Center

ROYAN Institute

November 2021

Categories of Genetic Defects

• Chromosome aberrations

• DNA copy number variants (microdeletions and duplications)

• Single-gene disorders

• Complex conditions

• Epigenetic disorders

Zorrilla and Yatsenko. Curr Genet Med Rep. (2013)

Overview

• Infertility: a highly complex disorder of the reproductive system.

• 1 out of 6 people worldwide suffer from infertility.

• Evaluation of both members of the couple is mandatory.

• Diagnostic timeline of infertile couples:– Biochemical and instrumental analyses: diagnosis in 65% of cases

– 35% undiagnosed cases: genetic tests are performed

• Approximately 15% of genetic disorders are associated with infertility.

Cariati et al. J Transl Med (2019)

Relevancy in Genetic Screening

Implications for the:

• Reproductive health of the patient

• General health of the patient and his children

• Prognostic value (sperm retrieval)

• Help in personalizing hormone therapy in near future

Cariati et al. J Transl Med (2019)

Routine diagnostic work up: genetic diagnosis in 20% of cases

Chromosomes

• In contrast to other cell organelles, the size of chromosomes shows a remarkablevariation depending upon the stages of cell division.

• Interphase: chromosome are longest & thinnest

• Prophase: there is a progressive decrease in their length accompanied with anincrease in thickness

• Anaphase: chromosomes are smallest.

• Metaphase: Chromosomes are the most easily observed and studied duringmetaphase when they are very thick, quite short and well spread in the cell.

Chromosome measurements are generally taken during

mitotic metaphase.

Tissue samples for postnatal chromosomal analysis

• Peripheral blood

• Fibroblasts from skin biopsy

• Epithelial cells from buccal smear (in rare cases for Barr body identification or FISH)

• Bone marrow

• Solid tumor

• Autopsy material

• Product of conception

Basic cytogenetic examinations

• Interphase cells:

– Barr body (sex chromatin)

• Metaphase cells: staining of chromosomes

– Solid staining

– G-banding

– R-banding

– C-banding

– Q-banding

Heterochromatin vs Euchromatin

KaryotypeThe representation of entire metaphase chromosomes in a cell, arranged in order of size and other characteristics (centromere position and banding pattern).

ISCN (From 1995)International System for Human Cytogenetic Nomenclature

group A (1-3)

group B (4-5)

group C (6-12, X)

group D (13-15)

group E (16-18)

group F (19-20)

group G (21,22,Y)

Types of Chromosomal Abnormalities

• Numerical– Aneuploidy (monosomy, trisomy, tetrasomy)– Polyploidy (triploidy, tetraploidy)

• Structural– Translocations– Inversions– Insertions– Deletions– Rings– Isochromosomes– ESAC (marker chr.)

Numerical

• Aneuploidy

– Autosomal trisomy: 47 (Down Syndrome, …)

– Sex chromosomes: 45, 47, 48, 49 (Klinefelter Syndrome)

• Polyploidy: Whole chromosomal set

– Triploidy: 69

– Tetraploidy: 92

Aneuploidy

• Almost all seen in oocytes and early embryos: trisomies and monosomies

• Most lethal: up to 60% in products of early onset miscarriages

• Rarely in fetus or live born

• Exceptions: sex chromosomes and Down Syndrome

• Some aneuploidies are age related

Meiotic Nondisjunction

Structural Chr. Abnormalities

Terminal

• Cri du chat: 5p15

• Wolf-Hirschhorn: 4p36

Interstitial

• Williams: 7q11.2 microdeletion (FISH)

• Retinoblastoma: 13q14

• Prader-Willi: 15q11.2

• Angelman: 15q11.2

• DiGeorge: 22q11.2

Deletions

Translocation

The most common chromosomal rearrangement

Reciprocal Translocation

Robertsonian Translocation

Chromosomal Translocations: Clinical Relevance

Men with Oligospermia

Classified into two types:

– Paracentric:

Does not include the centromere

– Pericentric:

Transpasses the centromere

Inversions

Crossing over occurs:

– Normal gametes

– Balanced gametes (inverted)

– Acentric

– Dicentric

Paracentric inversion

Cross over occurs:

– Normal gametes

– Balanced gametes (inverted)

– Unbalanced gametes (Duplication or deletion)

Pericentric inversion

Reciprocal translocation

45,XX,der(13;14)(q10;q10)

Robertsonian translocation

46,XY,t(6;9)(q24;p23)

Inversion

46,XX,inv(9)(p13q13)

Duplication

Deletion

46,X,dup(X)(p11.2p22.1)

46,XY,del(18)(p11.2)

Sex chromosomes

Abnormalities are more tolerated

• Extra Y: few genes mainly for sex determination

• Extra X: excess X is inactivated

Monosomy X: Turner syndrome

– Majority die during embryonic development

– Only small proportion survive to birth

– Short stature

– Infertile

ESAC

Extra Structurally Abnormal Chromosome

• Abnormal chromosome in addition to 46

• Small and difficult to identify

• Sometimes called marker chromosomes

• Difficult to work out effect on person

• May be benign or cause serious mental disability

Chromosome Abnormalities

Mixoploidy

Mosaicism• Two or more cell lines, differing in genetic constitution in

an individual or a tissue, derived from a single zygote

• Usually from non-disjunction in an early embryonic mitotic division with the persistence of more than one cell line.

• Cell line with 45 chromosomes would probably not survive

• Embryo shows approximately 33% mosaicism for trisomic chromosome.

Chromosomal Aberrations in Female

• The three main classes of abnormalities:– Monosomy (45,X)

– Trisomy

– Polyploidy

• Events increase with maternal age.

Oktay et al. J Pediatr Adolesc Gynecol. 2016

• Women with a normal karyotype produce a variable percentage of oocytes with chromosomal abnormalities due to errors occurring by:

– Crossing-over

– Meiotic nondisjunction

Chromosomal Aberrations in Female

Chromosomal disorders:

Significantly impact fertility and the miscarriage risk

Karyotype analysis is always advisable

The most clinically important structural disorders in infertile females are:

– Translocations (Reciprocal or Robertsonian) responsible for blocks of meiosis

– Structural alterations of the X chromosome

– X-autosome translocations: rare, variable phenotypes including amenorrhea, ↓ sex hormones,…

Morin et al. 2017. Fert. Steril.

Chromosomal Aberrations in Female

Balanced rearrangements:

• Do not create health problems for their carriers because they cause neither loss nor duplication of genetic information.

• They can give rise to gametes in which the genetic information is unbalanced and can thus become a cause of infertility or multiple miscarriage.

Morin et al. 2017. Fert. Steril.

X Chromosome and female infertility

• In the normal ovary, the primordial germ cells carry two X chromosomes, one of which is initially inactivated similarly to any other somatic cell.

• The second X chromosome is reactivated prior to meiosis as the presence of two transcriptionally active X chromosomes is essential for oogenesis.

Arnold et al. Phil Trans R Soc B 2016

X Chromosome abnormalities and female infertility

• Monosomy X (Turner syndrome)

• Cytogenetically visible deletions/duplications

• Balanced and unbalanced X-autosome rearrangements

Associated with an accelerated loss of primordial oocytes during female fetal development, resulting in streak gonads at birth.

Yatsenko and Rajkovic. Biology of Reproduction, 2019

They account for near to 10% of cases of POI

X Chromosome Derangements

• Structural/numerical abnormalities of the X chromosome: the largestsubgroup with primary ovarian insufficiency (POI)

• Although one X chromosome is sufficient to allow ovarian differentiation, oocytes need two active X chromosomes.

• Reproductive disorders in women with Turner’s syndrome (45,X) arise from the lack of all or part of the X chromosome.

Devos et al. 2010; Kuo et al., 2004; Kim et al., 2014;

X Chromosome Derangements

Genetic mechanisms contributing to infertility:

• Reduced gene dosage

• Non-specific chromosome effect impairing meiosis

• Decreasing the pool of primordial follicles

• Increasing atresia due to apoptosis

• Failure in follicle maturation

Devos et al. 2010; Kuo et al., 2004; Kim et al., 2014

Turner syndrome (Monosomy X or 45,XO)

• 1:2000 births.

• Due to nondisjunction in meiosis.

• Skeletal abnormalities, congenital heart defects, short stature, webbed neck, low hairline, flat chest, and gonadal dysgenesis with signs of amenorrhea or ovarian failure.

• Females mosaic for Turner syndrome (45,X/46,XX) present with a milder form, often noted due to infertility.

Simpson and Rajkovic. Am J Med Genet (1999).

47,XXX syndrome or Trisomy X

• One of the most common causes of premature ovarian insufficiency (POI)

• 1 in 1000 female births.

• Mostly present as normal, some suffer from POI, malformations of the genitourinary tract, learning disability, emotional/behavioral difficulties.

• Due to chromosome nondisjunction

Tartaglia et al. Orphanet J Rare Dis. (2010)

Premature Ovarian Failure

Chromosomal (X)

• Rearrangements

• Deletions

• Turner’s syndrome

• Ovarian leukodystrophy

• Ataxia telangiectasia

• Progressive external ophthalmoplegia

• BPES

(blepharophimosis, ptosis, and epicanthus inversus syndrome)

Single Genes:

Autosomal• STAG3• FOXL2• FSHR• LHCGR • GDF9 • ….

X linked:• FMR1 • BMP15

Syndromic Disease Isolated Disease

Genetics of Male Infertility

• 7% of male population are infertile

• 2000 genes involved in spermatogenesis

• Unknown etiology: 40%

• Genetic factors: 15%

• Azoospermia: highest frequency of known genetic factors (25%)

Etiologic Categories

1. Spermatogenic quantitative defects (75% of cases)

2. Ductal obstruction or dysfunction

3. Hypothalamic axis dysfunction

4. Spermatogenic qualitative defects

.

Adopted from: Wosnitzer, Transl Androl Urol. 2014;3(1):17-26.

Chromosomal Karyotyping in Male Infertility

First genetic test in patients with quantitative spermatogenic impairement

Incidence of chromosomal abnormalities in male infertility:

– Overall 5.8% (4.2% sex chr, 1.5% autosomal)

– 15% of NOA cases

– 4% of oligozoospermic patients

Indicated in:Men with <10 mil/mL sperm count

And a family history of:RPL, mental retardation, congenital malformation, infertility of unknown cause

.

Klinefelter Syndrome (47, XXY)

• General population: 1 in 600

• Azoospermia: 1 in 7

• 90% azoospermic, 10% severe oligozoospermic

• Syndrome manifestations are due to:

– Hypoandrogenism

– Dosage of X linked genes

– Epigenetic alterations

Klinefelter Syndrome (47, XXY)

• 2-25% sperm aneuploidy: increased probability of chromosomal abnormality in their offspring

• Children born to Kline men are usually healthy (only one 47,XXY reported)

Due to significant increase in sex chromosome and autosomal abnormalities in

embryos of Klinefelter men, PGD or PND may be considered

Jacob Syndrome (47, XYY)

• 1 in 1000 male newborns

• Most cases of JS are not inherited.

• Mainly due to nondisjunction at meiosis II

• Common features:

– Infertility in adulthood

– Behavioral and cognitive disorders

– Facial dysmorphia, micropenis, non-palpable testes, and decreased total testosterone

45,X0/46,XY mosaicism (mixed gonadal dysgenesis)

• Prevalence: 1 in 15,000 newborns

• Often caused by the loss of the Y chromosome through nondisjunction in some somatic cells after normal fertilization.

• Can repress the SRY genes, resulting in abnormal genitals (incomplete sexual differentiation) and testosterone levels

• Variable phenotypes due to differential distribution of two cell lines

• Can also cause conditions such as azoospermia, oligospermia, sperm DNA fragmentation, and increased gonadotropins

• Sexual ambiguity in 60% of cases

46, XX Male Syndrome (de la Chapelle)

• 1 in 20,000 children

• Definite azoospermia

• 80-90% due to translocation of SRY onto X chromosome

• TESE is not indicated: due to lack of AZF regions & focal sperm production

Y Chromosome Structural Abnormalities

• X-autosome translocations impair pairing during meiotic recombination, disrupting gametogenesis, and resulting in spermatogenic failure

• Isodicentrics, truncated, ring, inversions

Important: AZF regions affected?

• Severity of phenotype: rate of mosaicism, AZF deletion

• TESE can be performed based on the type of AZF deletion

• ↑ 45,X mosaic cells: TESE poor prognosis

Chromosomal Structural Abnormalities

Autosomes

• Robertsonian translocations, inversions, reciprocal translocations

• Robertsonian translocation: reduce the volume of testicles and testosterone,impairing spermatogenesis, and resulting in azoospermia or oligospermia

• 10 folds more frequent in oligozoospermic men (4-8%): Genetic counselingshould be offered

• PGD in patients seeking IVF/ICSI is necessary:

(a) The most frequent non-familial reciprocal translocation t(11;22)(q23;q11)

(b) Robertsonian translocation der(13;14)(q10;q10)

(c) partial karyotype of a patient with Klinefelter syndrome (47,XXY)

(d) partial karyotype of a patient with 47,XYY syndrome

Yatsenko and Rajkovic. Biology of Reproduction, 2019

References

• Yatsenko S.A. and Rajkovic A., Genetics of human female infertility, Biology of Reproduction, 2019, 0(0), 1–18.

• Pylyp, et al. Chromosomal Abnormalities in Patients with Infertility. Cytology and Genetics, 2015, Vol. 49, No. 3, pp. 173–177.

• Cariati et al. The evolving role of genetic tests in reproductive medicine. J Transl Med (2019) 17:267

• EAU Guidelines. Edn. presented at the EAU Annual Congress Copenhagen 2018. ISBN 978-94-92671-01-1.

• Baratt et al. The diagnosis of male infertility: an analysis of the evidence to support the development of global WHO guidance challenges and future research opportunities. Human Reproduction Update, 2017; 23 (6): 660–680.

• ASRM practice committee report. Diagnostic evaluation of the infertile male: a committee opinion. Fertil Steril. 2015; 103(3):e18-25.

• Krausz C., Riera-Escamilla A., Genetics of male infertility. Nat Rev Urol. 2018; 15(6):369-384.

• Wosnitzer MS. Genetic evaluation of male infertility. Transl Androl Urol. 2014;3(1):17-26.

• Morin SJ, Eccles J, Iturriaga A, Zimmerman RS. Translocations, inversions and other chromosome rearrangements. Fertil Steril. 2017;107:19–26.

• Committee on Ethics, American College of Obstetricians and Gynecologists, Committee on Genetics, American College of Obstetricians and Gynecologists. ACOG Committee Opinion No. 410: ethical issues in genetic testing. Obstet Gynecol. 2008; 111:1495–502.

• Oktay K, Bedoschi G, Berkowitz K, Bronson R, Kashani B, McGovern P, et al. Fertility preservation in women with turner syndrome: a comprehensive review and practical guidelines. J Pediatr Adolesc Gynecol. 2016;29:409–16.

• Arnold AP, Reue K, Eghbali M, Vilain E, Chen X, Ghahramani N, Itoh Y, Li J, Link JC, Ngun T,Williams-Burris SM. The importance of having two X chromosomes. Phil Trans R Soc B 2016; 371:20150113.

• Michelle Zorrilla and Alexander N Yatsenko. The Genetics of Infertility: Current Status of the Field. Curr Genet Med Rep. 2013 December 1; 1(4)

• Tartaglia NR, Howell S, Sutherland A, Wilson R, Wilson L. A review of trisomy X (47,XXX). Orphanet J Rare Dis. 2010; 5:8.

• Simpson JL, Rajkovic A. Ovarian differentiation and gonadal failure. Am J Med Genet. Dec 29; 1999 89(4):186–200.