Nino Guy Cassuto ART Unit Drouot Paris

Influence du matériel génétique du spermatozoïde sur la qualité du conceptus

Journée AMP Hôpital des Diaconesses Paris 18 Janvier 2014

SPERM PARAMETERS

Male fertility can't be investigated just by the sperm parameters (WHO not a Norm, but Threshold 5 p.15M; 40%; 4%)

Spermatozoa from infertile men had high percentage with DNA damage Barroso et al. 2000; 2007

Today it’s necessary to explore the functional capacity of the spermatozoa and its DNA integrity Paternal DNA packaging in spermatozoa: more than the sum of its parts? DNA, histones, protamines and epigenetics. Miller D,Reproduction 2010.

Chromosomal abnormalities and spermatogenesis

Abnormalities of structure

Robertsonnian Translocations

t(13;14) Translocations

t(13;13)

Microdeletion AZF

Abnormalities of number

Klinefelter syndrome X XY

Double XX Avec SRY

Y

AZFa

AZFb AZFc

AZFa

Rares; Phénotype testiculaire sévère: SPZ O

AZFb

Rares; Blocage en méiose SPZ O

AZFc

Les plus fréquentes: Azoospermie ou OATS sévère

Transmission au garçon

Micro délétion de L’Y

P

Q

Bras court

Bras long

FISH sur spermatozoïdes Fluorescent In Situ Hybridization

• Permet de repérer les anomalies chromosomiques par un système AC- Fluorophore

• Évaluer le profil de ségrégation et la qualité du génome

• Comprendre certaine anomalies du sperme

Anomalies chromosomiques et conséquences

On peut observer: – Non activation de l’ovocyte – Blocage précoce de l’embryon – Blocage avant blastocyste – Non implantation – Avortement précoce (<11s) ou tardif – Naissance avec une ségrégation normale ou

équilibrée

SPERMATOGENESIS

Quality control of the Chromatin: Epigenetic information Chromosomes: Genetic information APOPTOSIS AND R O S DNA Fragmentation Chromatin Decondensation Abnormal maturity of DNA + PROTAMINES

0 100

100

Fragmented DNA (Blue fluorescence)

Nat

ive

DNA

Stai

nabi

lity

(Gre

en fl

uore

scen

ce)

TUNEL: Terminal Uridine Nick End Labeling

Fragmentation DNA • Radiotherapie RX, Gamma,UV • Chimiotherapie

• Anomalies de la spermatogénèse cassures des ponts entre

les 2 brins ADN. • Apoptose, Infection, Temperature, Tabac, Pesticides,

Phtalates, Pollution, ROS, OH, Age, AC anti SPZ

Décondensation de la Chromatine du SPZ • Marqueur de la mauvaise qualité de la chromatine • Technique au bleu d’Aniline N < 25%

Compaction du spermatozoïde

• Un mauvais remplacement des histones par les

protamines , • Un ratio P1/ P2 déséquilibré, • Un environnement inadéquat, un stress oxydatif • Altération de l’activité mitochondriale affectant la

conservation des transcrits

– ARN non conservés correctement – Possible déséquilibre de methylation et d’acétylation

Genomic imprinting

The imprinting is a chemical process through methylation and histone acetylation, without altering the DNA sequence Methylation and Acetylation are associated with the activation or repression of the transcription genes

Morphology is yet the only tool to

evaluate sperm quality prior to ICSI (x400)

Head - Cytoplasmic droplet - Tail

We cannot see what is happening inside the sperm !!!!

We can only see how it looks and how it moves…..

NOT TO SEE TO SEE

Correlation between normalcy of the sperm;

fertilization and early embryo development

Which sperm defects affect this development ??? Strict morphology criteria

Our first study

Score = Head x2 + Vacuole x3 + Base x1 = 6 Vanderzwalmen P. RBMO 2008 Balaban B. RBMO 2011 Knez K. Rep Bio Endoc 2011 and RBMO 2012 Setti AS. J A Rep Gen 2012 Tanaka A. F S 2012 El Khattabi L. F S 2013 Greco E. F S 2013

a b c

g

d e f

h i

Chelli MH. J A Rep Gen 2013

Boitrelle F. H R 2011 Perdrix A. H R 2011 Franco JG.Jr Int J Andr 2012 Leandros L. RBMO 2013

During spermatogenesis chromatin is packaged tightly protecting the

Histones are widely replaced by Protamines Tail Histones methylation activating and repressing genes transcription

Carrell DT HR U 2007

Perturbations result in sperm epigenetic abnormalities This sperm impact the outcome, diseases, genetics disorder and male factor infertility

One link

Repeated ICSI failures Score 0 as an abnormal chromatin compaction With more Histones, Immature sperm I Low score 0 rate ICS I II Intermediate score 0 rate and young oocytes ICSI III High score 0 rate IMSI IV Aging oocytes IMSI

Antinori M. RBM 2008 Gonzalez Ortega C. Gin Obst Mex 2010 Setti AS. RBMO 2012 Klement AH. F S 2013 Delaroche L. E J Ob Gyn Rep Bio 2013

Consequences A good spermatozoon with normal DNA prior injection is a crucial step and helps to optimize the outcome! What are the consequences of DNA defect spermatozoon injection on the outcome?

Prospective follow up cohort study 2005 to 2010 1028 Infants studied 578 From ICSI (56%) 450 From IMSI (44%)

NormalBirth defectsLost for follow up

996 96.89%

11 1.09%

32 3.11%

Birth defects after natural conception are estimated at 2.40%

0

100

200

300

400

500

600

Child

ren

(n)

ICSI IMSI

Major Malformations

22 3.80%

6 1.33%

556

444

Fisher test p-value = 0.014 Odds ratio (OR) = 0.35

(IC 95%) from 0.14 to 0.87

Healthy Child

Low birth defects by deselecting abnormal spermatozoa before ICSI

6

2

16

4

0

2

4

6

8

10

12

14

16

Girls Boys

ICSIIMSI

p value=0.009

ICSI IMSI

Girls 6 2

Boys 16 4

Birth defects according to the gender

Mainly affecting the urogenital system Girls Boys

Conclusions 1 High magnification permits to deselect the worst

spermatozoon. 2 Highlights the impact of the DNA spermatozoa on

the outcome. 3 ICSI risk to select abnormal spermatozoa and to increase by 3 the risk of major malformations