* CHROMOSOME SEGREGATION AND INTERCHROMOSOMAL EFFECT IN

HUMAN EMBRYOS FROM CARRIERS OF CHROMOSOMAL REARRANGEMENT.

MULTICENTRAL STUDY

Julia LoginovaThe International Centre of Reproductive Medicine (MCRM)Russian Federation

The 5th Congress of the Asia Pacific Initiative on ReproductionBrisbane, Australia, 4 - 6 April 2014.

general population (newborns ) - 0.2 %

infertile couples – 0.6-1.3 %couples with repeated implantation failure (RIF) – 3.2 %

couples with recurrent miscarriage (RM) - 4.7- 9.2 %

infertile males requiring ICSI (severe male factor) - 2-3.2%

The incidence of balanced chromosomal rearrangement

Birth of children with unbalanced karyotype 1-17%

Stillbirth / infant death 5-8%

Miscarriage 20-30%

Unbalance karyotypeRevealed by amniocentesis 14-28%(second trimester)

Effect of the parental balance chromosomal

rearrangement for offspring:

balanced chromosomal rearrangementChromosomal segregation during gametogenesis may lead to production of gametes with gains and losses of genetic material -

theoretically - 50%, empiric date – large variability 0? - 100? %

Rate of balanced and unbalanced gametes depends on: • Type of rearrangement• Involved chromosomes • Breakpoints • Carrier's sex• Genetic background • IVF - Environment

OBJECTIVES: analysis of chromosomal segregation patterns and estimation an inter-chromosomal effect (ICE) in embryos obtained from male and female rearrangement carriers (Robertsonian translocations, reciprocal translocations, pericentric inversions).

understanding the segregation mechanisms of the chromosomes involved and not involved in the

rearrangements GENETIC COUNSELING: estimation the risks of pregnancy loss and birth defects in the offspring

IVF:prediction the PGD cycles outcome

IVF / ICSI

FISH

Biopsy: Day 3 (95% cases);Day 5 (5% cases)

ET fresh – 90%, frozen 10%

genetic counseling

Testing of the specific FISH probes on the metaphase chromosomes from the couples (carrier and his/her partner)

Methods:

Day 5Day 4

FISH: 3 - 7 re-hybridization steps (depending on the fixed nuclei quality)3 - 8 markers for rearrangement chromosomes +PGS for 7-12 chromosomes not involved in rearrangement (1-3 markers for every tested chromosomeFISH markers: locus-specific probes, subtelomeric/centromeric probes

patients:45,XX,der(15;22)(q10;10)46,XY

15ceno, 15qtelo ,

22q11o15p11.2o, 15ceno, 14q11.2o+o , 22q11o

MultyPB, CEPXo+o,CEPYo+o

18q21o+o, 16qtelo , 21q22 o

18q21o+o, 22q11o , 21q22 o

FISH steps: 1st 2nd 3rd 4th

31

3431

136 7

13

Reciprocal (REC)

Robertsonian (ROB)

pericentric inversion (INV)

other

PGD cycles (n=125) Embryos (n=907)

Other rearrangements:46,XY,t(1;8)(q32.1;q24,1),der(15)t(Y;15)(q12;q10)46,XY,der(15)t(Y;15)(q12;p11.2)45,X[25]/46,X,r(X)[5]46,X,r(Y)[30]/45,X[20]

233

249242

8827 47

1 20

♀

♂

♀ ♂

♀

♂

♂♀

♂

♂

♂

♂♀

♀

♀

♀

- balansed, euploid

- balansed, aneuploid

- unbalanced, euploid

- unbalansed, aneuploid

- other unbalans

carriers ♀

REC (n=451)

carriers ♂

ROB (n=330)

INV (n= 72)

14%

6+22=28%

14+26=40%

31+20=51%

Segregation patterns according rearrangement type and carrier’s sex

14% 4%

44%

32%

7%17%

6%

45%

22%

10%

28%

14%

24%

22%

12%

40%

12%11%

26%

10%

37%

22%

15%

19%

7%

36%

31%

20%

13%

28%

37%

17%

40%

36%

**

*

4+32=36%

14+22=36%

22+19=41%

REC segregation pattern according to the carrier’s sex

carrier ♂ embryos n= 225carrier ♀ embryos n = 220

alternate (n/b)

adja

cent

-1 pl

oidy

ab

norm

aliti

esadja

cent

-2

reco

mbi

nan

ts

mosaic

chaotic3:1 4:0

0

5

10

15

20

25

%ns ns

6.1% 7.7%

REC segregation pattern according to chromosome types (acrocentric chromosome involvement) and to the carrier’s sex embryos n= 463

n/b adj1 adj2 3:1 4:0 recomb ploidy mos chaotic abnorm.

0

5

10

15

20

25

30 *% ♂♀

with

acr

o

with

out a

cro

n=103 n=140

n=79 n=141

ns*

9.5%

mosaic

chaotic

ROB segregation pattern according to sex of the carrier

carrier ♂ embryos n= 91

carrier ♀ embryos n = 240

0

10

20

30

40

50

60

alternate (n/b)

adjacent

reco

mbi

nant

s

%ns

8.9%

ploi

dy

abno

rmal

ities

PGD cycles

n

Embryos (n)/abnormal embryos (n)

Chromosomes n

Abnormal chromosomes n

control <35 38 219/50 1533 56Rec <35 46 300/104 2100 135Rob <35 29 207/76 1449 84Inv <35 7 39/20 273 26

control 35-39 82 363/178 2541 220Rec 35-39 17 116/38 812 48Rob 35-39 12 76/38 532 57Inv 35-39 3 11/6 77 7

control ≥40 166 466/303 3262 417Rec 40+ 2 8/4 77 4Rob 40+ 3 9/6 63 9Inv 40+ 3 14/8 98 7

3 groups according maternal age: 35, 35-39, ≥40

Interchromosomal effect (ICE) analysis inclusion criteriacontrol groups:normal karyotype - 46,XX;46,XY diploid blastomeresPGD of 7-12 (or more) chromosomes 35 – PGD without medical indication (patients’ desire)

studied groups:carriers of: reciprocal andRobertsonian translocation,pericentric inversion

exclusion criteria: mosaic, chaotic, blastomeres with aneuploidy for 3 chromosomes

ICE estimation – on 7 chromosomes in studied groups – 7 chromosomes not involved in the rearrangementin control groups – any 7 chromosomes

ICE

controlreciprocalRobertsonianpericentric inversion

♂♀ ♂♀ ♂♀

<35 35-39 ≥40

<35 35-39 ≥40

*

*

0

5

10

15

20

25

3.76.4 5.8

9.5 8.75.9

10.79.1

12.8

5.2

20.6

7.1

according to sex of thecarrier, maternal age and the rearrangement type

according the rearrangement type and maternal age

0

5

10

15

20

25

30

3.76.0 6.8 6.2 5.7

10.8

5.78.7

6.3 5.58.3

12.410.2

7.1

12.8

8.6

4.8

28.6

14.3 14.3

5.2

♂♀ ♂ ♀ ♂♀ ♂ ♀ ♂ ♀ ♂♀

%

%

ns

nsns

ns

ns

Outcome

PGD cycles - 125with euploid (transferable) embryos – 77% (96 cases)

available data - 116 PGD cycles With fresh ЕТ – 56% (65 cases)

Clinical pregnancy rate x ET – 37% (24 cases)

Delivery: Singletons – 13 Twins – 3Ongoing pregnancies 12 weeks – 3

ectopic pregnancy - 3

Miscarriages – 2 (9%): 1 – (9-10 weeks) 47,XX,+D – mother 46,XX,t(4;18)2 - not karyotyped

Conclusion:

Carriers of different chromosomal rearrangements have different reproductive genetic risk:

- the carriers of reciprocal translocations regardless of their gender have a higher risk of aneuploid embryos than other carriers.

- the male carriers of Robertsonian translocations have lower risk of aneuploid embryos, than female. The possible explanation is specific chromosome segregation in meiosis and/or selection during subsequent spermatogenesis stages

There is no significant influence of gender on the rearranged chromosomes segregation pattern for reciprocal and Robertsonian translocations carriers

For reciprocal translocations the modes of segregation pattern depend on the involvement of acrocentric chromosomes

Conclusion:

Conclusion:

ICE regardless of gender is more evident if the maternal age is less than 35. This suggests that the embryonic aneuploidy in older women depends more on the woman’s age than the rearrangement itself

In male carriers the embryonic aneuploidy rate caused by ICE depends on the rearrangement type

The strongest ICE was detected in both gender carriers of Robertsonian translocations and pericentric inversions.

IVF units:

Russian LatviaFederationSt.Petersburg: Riga: MCRM, Jusu ArstiPrivatklinika Mother and Child

Moscow: Altra-Vita, Art-IVFFertiMed

Ufa: LLS Medical technologies center

PGD unit:

Russian Federation

S.Petersburg

MCRM