1. *
Julia Loginova
The International Centre of
Reproductive Medicine (MCRM)
Russian Federation
The 5th Congress of the Asia Pacific Initiative on Reproduction
Brisbane, Australia, 4 - 6 April 2014.
2. 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
3. Birth of children with unbalanced
karyotype 1-17%
Stillbirth / infant death 5-8%
Miscarriage 20-30%
Unbalance karyotype
Revealed by amniocentesis 14-28%
(second trimester)
Effect of the parental balance chromosomal
rearrangement for offspring:
4. balanced chromosomal rearrangement
Chromosomal 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
5. 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
6. 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 chromosome
FISH markers: locus-specific probes,
subtelomeric/centromeric probes
patients:
45,XX,der(15;22)(q10;10)
46,XY
15ceno, 15qtelo ,
22q11o
15p11.2o, 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
9. REC segregation pattern according to the carrier’s sex
carrier ♂
embryos n= 225
carrier ♀
embryos n = 220
alternate
(n/b)
adjacent-1
ploidy
abnormalities
adjacent-2
recombinants
mosaic chaotic3:1 4:0
0
5
10
15
20
25
%
ns ns
6.1% 7.7%
10. 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
*% ♂
♀
n=103 n=140
n=79 n=141
ns
*
11. 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
recombinants
%
ns
8.9%
ploidy
abnormalities
12. PGD
cycles
n
Embryos (n)/
abnormal
embryos (n)
Chromosomes
n
Abnormal
chromosomes
n
control <35 38 219/50 1533 56
Rec <35 46 300/104 2100 135
Rob <35 29 207/76 1449 84
Inv <35 7 39/20 273 26
control 35-39 82 363/178 2541 220
Rec 35-39 17 116/38 812 48
Rob 35-39 12 76/38 532 57
Inv 35-39 3 11/6 77 7
control ≥40 166 466/303 3262 417
Rec 40+ 2 8/4 77 4
Rob 40+ 3 9/6 63 9
Inv 40+ 3 14/8 98 7
3 groups according maternal age:
35, 35-39, ≥40
Interchromosomal effect (ICE) analysis
inclusion criteria
control groups:
normal karyotype - 46,XX;46,XY
diploid blastomeres
PGD of 7-12 (or more) chromosomes
35 – PGD without medical indication
(patients’ desire)
studied groups:
carriers of: reciprocal and
Robertsonian 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 rearrangement
in control groups – any 7 chromosomes
13. ICE
control
reciprocal
Robertsonian
pericentric inversion
♂♀ ♂♀ ♂♀
<35 35-39 ≥40
<35 35-39 ≥40
*
*
0
5
10
15
20
25
3.7
6.4 5.8
9.5 8.7
5.9
10.7
9.1
12.8
5.2
20.6
7.1
according to sex of the
carrier, maternal age
and the rearrangement
type
according the
rearrangement type and
maternal age
0
5
10
15
20
25
30
♂♀ ♂ ♀ ♂♀ ♂ ♀ ♂ ♀ ♂♀
%
%
ns
ns
ns
ns
ns
14. Outcome
PGD cycles - 125
with 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 – 3
Ongoing 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
15. 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
16. 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:
17. 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.
18. IVF units:
Russian Latvia
Federation
St.Petersburg: Riga:
MCRM, Jusu Arsti
Privatklinika
Mother and Child
Moscow:
Altra-Vita,
Art-IVF
FertiMed
Ufa:
LLS Medical
technologies center
PGD unit:
Russian
Federation
S.Petersburg
MCRM
Editor's Notes
The incidence of balanced chromosomal rearrangement in the general population is just 0.2%, but it increases dramatically among patients of IVF clinics. The highest % has been detected among the couples with recurrent miscarriage.
Most balanced rearrangement carriers are phenotypically normal because they do not have gain or loss of genetic material. These patients can be detected when they attempt to reproduce. The parental balanced chromosomal rearrangements are associated with:
- birth of children with genetic abnormalities or stillbirth
- miscarriage
Chromosomal segregation during gametogenesis in these carriers may lead to production of gametes with gains and losses of genetic material. Theoretically 50% of the gametes are balanced, but empiric data give us large variability. The reasons of this variability can be:
Type of rearrangement
Involved chromosomes
Breakpoints
Carrier's gender
Genetic background
IVF – Environment
Understanding the segregation mechanisms of the chromosomes involved and not involved in the rearrangements is important to estimate the risks of pregnancy loss, birth defects in the offspring and prediction the PGD cycles outcome. This defined the OBJECTIVES of the study we performed. Analysis of chromosomal segregation patterns and estimation an inter-chromosomal effect (ICE) in embryos obtained from male and female carriers of different types of chromosomal rearrangement.
In most cases biopsies were performed on day 3 and 95% embryo transfers were in fresh cycles. PGD was done by FISH. Our standard procedure involves 3-7 steps of re-hybridization with 3 - 8 markers for rearrangement chromosomes and PGS for 7 or more chromosomes.
We analyzed 907 cleavage-stage embryos derived from 125 PGD cycles performed between the years 2010-2013 in 6 IVF centers. Most cycles were done for male and female carriers of reciprocal translocations and female carriers of Robertsonian translocation. Other groups of carriers were treated at the IVF-centers less frequently, probably because the frequency of other rearrangements is lower in the population and reproductive risk is lower for inversion carriers and male carriers of Robertsonian translocations
This slide shows the segregation patterns of the chromosomes involved and not involved in rearrangements according to rearrangement type and carrier’s gender.
The number of balanced and euploid embryos was significantly lower in both female and male carriers of the reciprocal translocations than in others groups of carriers. The female carriers of Robertsonian translocations have twice more balanced and euploid embryos than female carriers of reciprocal translocations. And other groups have almost equal number of balanced and euploid embryos – nearly 40%.
Red and purple segments of the charts represent embryos aneuploid for chromosomes not involved in the translocation - balanced and unbalanced on parental rearrangement. Male carriers of a pericentric inversion have significantly more embryos with aneuploidy for chromosomes not involved in arrangement than male carriers of reciprocal translocations. The statistically significant difference was not found for other groups.
We compared segregation pattern of embryos from reciprocal translocations carriers according to the carrier’s gender. The alternate segregation was 6% higher in male carriers than in female - 24% versus 18%, but it’s not statistically significant. Moreover, we detected higher percentage of segregation with recombination in the case of female carriers, but difference was not significant again.
463 embryos from carriers of reciprocal translocations were analyzed to investigate whether involvement of acrocentric chromosomes combined with carrier gender is related to meiotic segregation pattern. The highest incidence – 27% of normal or balanced embryos was detected in male carriers with translocations that involve acrocentric chromosomes. The embryos from female carriers with translocations involving acrocentric chromosome showed significantly lower incidence of adjacent-1 and significantly higher rate of 3:1 segregation than female carriers without involvement in translocation of acrocentric chromosome. The embryos from male carriers had the same tendency – in the case of the presence of acrocentric chromosome rate of adjacent-1 was lower and 3:1 segregation was higher, but without significant difference
330 embryos from Robertsonian translocations carriers were analyzed to investigate the segregation patterns according carrier’s sex. The alternate segregation was 9% higher in male carriers – 50% versus 41% in female carriers, but the difference was not statistically significant, However that allowed us to say that male carriers have lower reproductive risk than female
For the study of inter-chromosomal effect (ICE) control and studied groups were formed according to maternal age.
Inclusion criteria for control groups were - normal karyotype for booth partners, the youngest control group - with maternal age lower than 35 years old, included patients who used PGD without medical indication.
Only diploid blastomeres were estimated in control and studied groups, mosaic and chaotic blastomeres, and blastomeres with aneuploidy for more than 3 chromosomes were excluded.
ICE estimation was done on 7 chromosomes, and in studied groups – the 7 chromosomes not involved in the rearrangement were analyzed.
The inter-chromosomal effect (ICE) analysis was done according to maternal age and the rearrangement type and then according to maternal age, rearrangement type in combination with the gender of a carrier.
The ICE was detected only in groups with the maternal age lower than 35 regardless of a carrier gender. This provides evidence that female age has a stronger effect on the level of aneuploidy than ICE. I have to say that studied groups were not large and we are planning to continue data collection for this study.
Here is the outcome of the work done. We have the information about 116 PGD cycles. Clinical pregnancy rate per Embryo Transfer was – 37%, 21 healthy children were born and 3 pregnancies are progressing. We had 2 miscarriages, in one case the abortus karyotyping was done, it was 47,XX,+D. Chromosomes from group D not involved in the mother’s translocation and unfortunately couldn’t be find by method we’ve used.
