Chromosomal abnormalities inembryos from couples with a previous aneuploid miscarriage Nasser Al-Asmar, M.Sc.,a,bVanessa Peinado, M.Sc.,aMaría Vera, M.Sc.,aJose Remohí, M.D., Ph.D.,d Ant
Trang 1Chromosomal abnormalities in
embryos from couples with
a previous aneuploid miscarriage
Nasser Al-Asmar, M.Sc.,a,bVanessa Peinado, M.Sc.,aMaría Vera, M.Sc.,aJose Remohí, M.D., Ph.D.,d
Antonio Pellicer, M.D., Ph.D.,dCarlos Simon, M.D., Ph.D.,c,dTerry Hassold, Ph.D.,eand Carmen Rubio, Ph.D.a,b
a
Preimplantation Genetic Diagnosis Unit, Iviomics, Paterna, Spain;bPreimplantation Genetic Diagnosis Unit, Instituto
Valenciano de Infertilitad, IVI-Valencia, Valencia, Spain; c Scienti fic Director, Iviomics, Paterna, Spain; d Medical
Biosciences and Center for Reproductive Biology, Washington State University, Pullman, Washington
Objective: To compare the incidence of chromosomal abnormalities in preimplantation embryos from couples undergoing preimplan-tation genetic screening (PGS) after previous aneuploid miscarriage after either natural conception (NC) or assisted reproductive tech-nology (ART) versus fertile couples who underwent PGS for sex-linked diseases as a control group
Design: Retrospective study
Setting: IVF clinic
Patient(s): Patients with previous aneuploid conception undergoing PGS
Intervention(s): Embryo biopsy,fluorescence in situ hybridization
Main Outcome Measure(s): Embryo aneuploidy rates and pregnancy and implantation rates in couples with a previous aneuploidy for autosomes or sex chromosomes
Result(s): The overall rates of chromosomal abnormalities in groups with previous autosomal aneuploidy were significantly higher compared with the control group (67.8% for those whose previous aneuploidy arose after NC and 65.8% for those previously arising after ART, vs 34.0%) No significant differences were observed in those with previous sex chromosome abnormalities compared with control subjects Within couples with previous aneuploidies after NC, no difference existed in the incidence of chromosomal abnormalities compared with the ART groups Clinical outcomes were better (trend) in patients with previous autosomal aneuploidy after NC Conclusion(s): In preimplantation embryos, the incidence of chromosomal abnormalities due to a previous aneuploid miscarriage after either NC or ART is significantly higher than in the control group Furthermore, this incidence is higher when the previous aneuploidy was for autosomes; PGS is recommended in these couples (Fertil SterilÒ2012;98:145–50 Ó2012 by American Society for Reproductive Medicine.)
Key Words: Preimplantation genetic screening,fluorescence in situ hybridization, chromosomal abnormalities, natural conceptions, assisted reproductive technology
No fewer than 10% of clinically
recognized human pregnancies
end in spontaneous abortion
A large proportion, if not a majority, of
these involve chromosomally abnormal
conceptuses In fact, estimated rates of
chromosomal abnormality in
spontane-ous abortions arising from natural
con-ceptions (NC) vary from 47.9% to 83.0%
(1–5) The most common chromosome
abnormality in humans is aneuploidy, i.e., a missing (monosomy) or extra (trisomy) chromosome Aneuploidy is the main genetic cause of miscarriages (6, 7) A few autosomal trisomies (13,
18, and 21) and sex chromosome aneuploidies (45,X; 47,XXY; 47,XXX;
or 47,XYY) are compatible with life, but they typically result in serious congenital malformations and/or
cognitive or behavioral abnormalities; indeed, aneuploidy is the most frequent known cause of mental retardation and congenital birth defects in humans (6–8) As a group, sex chromosome aneuploidies are the leading type of chromosome abnormality in newborns (6, 8) Compared with sex chromosomes, autosomal aneuploidies produce more adverse phenotypic effects and are less compatible with an ongoing pregnancy(9)
The increasing use of assisted re-productive technology (ART) has gen-erated concern about possible increases in chromosome abnormalities
in ART-initiated pregnancies, but the data are equivocal Some studies found
Received December 22, 2011; revised March 15, 2012; accepted March 17, 2012; published online April
21, 2012.
N.A.-A has nothing to disclose V.P has nothing to disclose M.V has nothing to disclose J.R has
noth-ing to disclose A.P has nothnoth-ing to disclose C.S has nothnoth-ing to disclose T.H has nothnoth-ing to
dis-close C.R has nothing to disdis-close.
Reprint requests: Nasser Al-Asmar, M.Sc., Iviomics, Preimplantation Genetic Diagnosis Unit, Parc
Cientí fic Universitat de Valencia, Catedratico Agustín Escardino Street n 9, Biotec Building 2,
lab 2.10, 46980, Paterna, Spain (E-mail: nasser@iviomics.com ).
Fertility and Sterility® Vol 98, No 1, July 2012 0015-0282/$36.00
Copyright ©2012 American Society for Reproductive Medicine, Published by Elsevier Inc.
doi: 10.1016/j.fertnstert.2012.03.035
Trang 2no statistical difference in the total frequency of
chromo-somal abnormalities after a pregnancy arising by NC or
ART [specifically, in vitro fertilization (IVF) or
intracytoplas-mic sperm injection (ICSI)](4, 5, 10, 11) However, the type of
chromosomal abnormality may differ: Bettio et al (2008)
described a twofold increase in polyploidy after ART
compared with NC, whereas Martínez et al (2010) described
an increase in the incidence of monosomy X and a decrease
in polyploidies in miscarriages after ICSI(4, 5) Furthermore,
within ART itself, the total aneuploidy rate between ICSI
and IVF is not significantly different (4, 10–12), but sex
chromosome aneuploidy is more frequent in ICSI-related
pregnancies than in pregnancies associated with
conven-tional IVF(10–12)
Additionally, studies have reported that the risk of fetal
aneuploidy increases in couples with previous spontaneous
abortions or aneuploid conceptions due to both autosomes
and sex chromosomes (13–17) Women who have had
a previous trisomic pregnancy, particularly those<35 years
old, appear to be at increased risk for subsequent trisomic
pregnancies(17) The relative risk of trisomy 21 subsequent
to trisomy 21 is greater for women <35 years old at the
previous pregnancy, as is the risk of the same trisomy and
of a different trisomy subsequent to trisomy 13 or 18 The
relative risk of a different trisomy subsequent to trisomy 21
is similar for women <35 and women R35 years old at
their previous pregnancy(17) No differences were observed
whether the previous trisomy was viable or not(13)
The main aim of the present study was to compare the
in-cidence of chromosomal abnormalities in preimplantation
embryos from couples undergoing preimplantation genetic
screening (PGS) due to a previous aneuploid miscarriage
aris-ing through NC or ART Additionally, the frequency of sperm
aneuploidy and diploidy was analyzed in sperm samples from
a subset of these couples to investigate the possibility of a
pa-ternal origin of the chromosomal abnormalities observed in
the miscarriages
MATERIALS AND METHODS
Patients
This was a retrospective study carried out from July 2001 to
April 2011, in which 70 PGS cycles were performed in 56
cou-ples with a previous aneuploid conception from NC or ART
Female age was %37 years in all study groups The study
was reviewed and approved by the Institutional Review Board
(IRB) of the Instituto Valenciano de Infertilidad To assess the
incidence of chromosomal abnormalities on preimplantation
embryos, four groups of patients were considered (Fig 1):
Group 1: 28 PGS cycles in patients with previous
autoso-mal aneuploidy following NC
Group 2: 22 PGS cycles in patients with previous
autoso-mal aneuploidy following ART
Group 3: 12 PGS cycles in patients with previous sex
chromosomal aneuploidy following NC
Group 4: 8 PGS cycles in patients with previous sex
chro-mosomal aneuploidy following ART
Control group: 33 PGS cycles in patients with sex-linked diseases
For statistical comparisons, a control group of 28 fertile couples who underwent PGS for sex-linked diseases (n¼ 33 cy-cles) was included in the study In the control group, all male partners were normozoospermic and female age was %37 years All patients and control subjects had normal karyotypes
Ovarian Stimulation and Embryo Culture
After ovarian stimulation, oocyte retrieval was carried out via transvaginal aspiration of ovaries under ultrasound guidance Fertilization was assessed 17–20 hours after ICSI (day 1) and embryo cleavage 24 hours thereafter (day 2) At this time, em-bryos were grown in IVF medium (CCM medium, 1:1; Vitro-life) and subsequently cocultured in CCM medium with
a monolayer of heterologous EEC (previously screened for HIV, HBV, HCV, and syphilis) from day 2 until day 5, when embryo transfer was performed(18)
Embryo Biopsy and Fixation
Embryos were placed on a droplet containing Ca2þ- and Mg2þ -free medium (G-PGD; Vitrolife), and Tyrode solution (Vitrolife)
or laser technology (Octax) was used to perforate the zona pellu-cida Only embryos withR5 nucleated blastomeres and %25% fragmentation degree were biopsied, and one or two blastomeres were removed depending on the cell number on day 3 (one blastomere was biopsied in embryos with 5–7 blastomeres, two blastomeres in embryos withR8 blastomeres) Individual blastomeres were fixed in glass slides (Superfrost; Cole-Palmer) under an inverted microscope using a slightly modified Tarkowski protocol without hypotonic pretreatment(19)
FISH Protocol for PGS
Our current protocol includes analysis of chromosomes 13,
15, 16, 17, 18, 21, 22, X, and Y in two consecutive fluores-cence in situ hybridization (FISH) rounds In thefirst hybrid-ization, chromosomes 13, 16, 18, 21, and 22 were analyzed using Multivysion PB panel probe (Vysis) In a second hybrid-ization, chromosomes 15, 17, X, and Y were analyzed with Multivysion 4 Color Custom panel probe (Vysis) Nuclei with nonconclusive signals (overlapping,fiber, or split sig-nals) or with absence of signals for any of the tested chromo-somes were reanalyzed using subtelomeric probes In our protocol, ambiguous/uncertain/indeterminate FISH signals are resolved by means of reanalysis using subtelomeric probes (20) Cycles performed before 2004 did not include analysis of chromosomes 15 and 17 Detection washings and signal scor-ing were carried out followscor-ing manufacturer's instructions FISH analysis was performed using an Olympus AX70 epi-fluorescence microscope equipped with a triple-band pass fil-ter for 406-diamidino-2-phenylindole/Texas red/fluorescein isothiocyanate (FITC), and single-band passfilters for FITC, Texas red, and aqua blue
FISH on Sperm
Sperm samples were prepared for FISH to analyze chromo-somes 13, 18, 21, X, and Y (Vysis) as previously described
VOL 98 NO 1 / JULY 2012
Trang 3(21), and spermatozoa with disomy and diploidy for these
chromosomes were scored as abnormal Sperm samples
were classified as abnormal when the number of spermatozoa
with abnormalities for at least one chromosome was signi
fi-cantly higher than that observed in a control group of ten
nor-mozoospermic donors(22) To decrease the subjectivity of the
observations, the following criteria were used: 1) Overlapping
spermatozoa or sperm heads not well defined were not
eval-uated; 2) in cases of disomy or diploidy, all signals had the
same intensity and were separated from each other by a
dis-tance longer than the size of one signal; and 3) nullisomies
were not directly scored and were conservatively considered
as equivalent to the incidence of disomies(23)
Statistical Analysis
Fisher exact test with Yates correction was used to compare
the percentage of abnormal embryos and chromosomal
aneu-ploidy among study groups and the control group We also
compared the clinical outcome binary variables by the same
test Mann-WhitneyU test was used to compare the different
study groups for number of previous miscarriages,
implanta-tion rate, and sperm concentraimplanta-tion Chi-square test was used
to compare sperm FISH results among study groups and
con-trol group, and Bonferroni correction was applied for multiple
comparisons
RESULTS
No statistical differences were detected in mean female age
among the four study groups Differences were detected in
mean number of previous miscarriages; specifically, group
3, with previous sex chromosome aneuploidies following
NC, had the highest incidence of previous miscarriage (com-pared with group 1: 1.8 1.6 vs 0.7 1.0; P¼.021)
We observed statistically significant differences in the in-cidence of chromosomal abnormalities in groups undergoing PGS (Table 1) The overall rates of chromosomal abnormali-ties in groups with previous autosomal aneuploidy (groups
1 and 2) were significantly increased compared with the con-trol group (67.8% and 65.8%, respectively, vs 34.0%; P<.001) Further, these rates were higher in the two groups with previous autosomal aneuploidy compared with the two groups with previous sex chromosome aneuploidy However,
no differences were detected in the incidence of chromosomal abnormalities between couples with aneuploidies arising from NC compared with those from ART When analyzing the incidence of aneuploidy in couples with previous preg-nancies with autosomal aneuploidy (groups 1 and 2), group
1 had significantly more frequent aneuploidy for sex chromo-somes compared with the control group (19.4% vs 7.5%; P¼.007); in contrast, group 2 had a significant increase for all autosomes tested compared with the control group (P<.05) For patients with previous sex chromosome aneu-ploidies (groups 3 and 4), chromosome 13 was the only auto-some with an increased incidence of aneuploidy (19.6% vs 5.2%; P¼.037) The percentage of aneuploid embryos suf-fered a slight increase when chromosomes 15 and 17 were added in the panel We checked for these differences in all study groups and the control group, and the increase was ho-mogeneously distributed in all of them
We found similar rates of haploid embryos among groups: 1.78% in NC, 0.59% in ART, and 1.50% in the control group For polyploidy, the rates were 1.33%, 0%, and 2.0% in
NC, ART, and control groups, respectively
FIGURE 1
Flow diagram of patients in the study.
Al-Asmar PGS with previous aneuploid miscarriages Fertil Steril 2012.
Trang 4Interestingly, clinical results indicated a trend toward
a better outcome in patients with previous autosomal
aneu-ploidy in NC, but these differences did not reach statistical
significance (Table 2) Notably, in both groups of patients
with previous aneuploidy in NC, there were no miscarriages
after the PGS cycle
We also performed FISH analysis on spermatozoa in 16
couples (Table 3) Abnormal results were obtained for only
one patient, from group 4, who had a significantly increased
rate of sex chromosome disomies compared with the control
group (0.59% vs 0.20%;P<.0005) There were no statistical
differences for all other autosomes tested The total diploidy
rate for all chromosomes was similar between patients and
control subjects (0.07% vs 0.10%) Sperm concentration in
the sample with the abnormal FISH result was 12.0 106
sperm/mL, and mean concentration in the remaining samples
with normal FISH results was 48.0 106 sperm/mL Mean
sperm concentration was 44.7 31.4 106in group 1; 48.3
42.4 106in group 2; 55.1 32.8 106in group 3, and
30.1 35.9 106in group 4; these differences were not
sta-tistically significant
DISCUSSION
Our data from the past 10 years confirmed a significantly
higher rate of abnormal embryos in patients with previous
aneuploidy compared with control subjects, independently from the origin of the previous pregnancy (NC or ART) Fur-thermore, the incidence was increased when previous aneu-ploidies were in autosomes Aneuploidy for chromosomes
16 and 22 were more common in patients with previous auto-somal aneuploidy in NC; an increase in aneuploidy for all chromosomes was detected in previous aneuploid pregnan-cies derived through ART The rate of sex chromosome aneu-ploidies was higher in all study groups compared with the control group, but it was statistically significant only for those with previous autosomal aneuploidy in NC
Earlier studies(13–17)suggest a risk of recurrence of both autosomal and sex chromosome fetal aneuploidy in couples with previous spontaneous abortions or aneuploid conceptions Our results confirm these findings for all study groups when the previous aneuploidy was in autosomes However, after a previous sex chromosome aneuploidy, we found a significant increase in aneuploidy recurrence only for chromosome 13 in previous pregnancy by NC
The FISH study on sperm samples found only one patient with abnormalities, specifically a higher rate of sex chromo-some disomy compared with the control group However, this result was not surprising, because the majority of autosomal trisomies arise from errors in maternal meiosis, which typi-cally occur during metaphase I, as in Down syndrome For
TABLE 1
Description of the incidence of chromosomal abnormalities in preimplantation genetic screening cycles.
a Statistical differences between group 1 and group 3: P¼ 021 (analysis of variance with Bonferroni multiple test post hoc comparison).
b Statistical comparisons of each group vs control group: P %.05 (Fisher exact test).
Al-Asmar PGS with previous aneuploid miscarriages Fertil Steril 2012.
TABLE 2
Clinical outcome after preimplantation genetic screening in the study and control groups.
Note: PR ¼ pregnancy rate; IR ¼ implantation rate; Ong ¼ ongoing.
Al-Asmar PGS with previous aneuploid miscarriages Fertil Steril 2012.
VOL 98 NO 1 / JULY 2012
Trang 5sex chromosome aneuploidies, some are as likely to be
pater-nal as materpater-nal in origin, such as 47,XXY (Klinefelter
syn-drome; 50%) In contrast, nondisjunction of the paternal
sex chromosomes is predominant in 45,X (Turner syndrome;
74%) (7, 8, 24) Interestingly, an increase of aneuploid
spermatozoa has been reported in fathers of children with
Down syndrome and couples with spontaneous abortions or
children with sex chromosomal abnormalities such as
Turner or Klinefelter syndromes(25–31)
PGS for the selected panel of nine chromosomes in the
present study resulted in similar outcomes in all groups To
date, the only way to screen aneuploid embryos is through
PGS, a method that can detect most of the numeric
chromo-somal abnormalities described in miscarriages by using
ex-tended panels for a selected number of chromosomes (32)
Lathi et al.(32)found statistical differences between the
lim-ited (5-probe) and extended (9-, 10-, and 12-probe) panels,
but not among the extended panels, and suggested that
FISH for chromosomes 13, 15, 16, 18, 21, 22, X, and Y should
identify80% of the most common chromosomal anomalies
in samples of spontaneous abortions In 2004, Munne et al
(13)examined whether the rate of aneuploidy among women
having PGS is increased by a previous aneuploid conception
Women having PGS because of previous aneuploidy were
compared with two control groups: women having PGS for
diagnosis of X-linked disorders and women having PGS
be-cause of repeated IVF failure A higher rate of aneuploidy
was reported for embryos from young patients having IVF
be-cause of a previous trisomic conception than for embryos of
the control groups The authors concluded that a history of
a trisomic conception is associated with an increased risk of
another aneuploid conception(13)
The present study is, as far as we know, thefirst to
sepa-rately analyze previous aneuploid conceptions involving
au-tosomes and those involving sex chromosomes, as well as
differentiating between ART and NC pregnancies for better
counseling of couples attending an IVF center We
acknowl-edge that these are preliminary results owing to the number of
patients included in the study, but the difficulty in recruiting
patients with these characteristics should be taken into
account
In conclusion, in preimplantation embryos, the incidence
of chromosomal abnormalities associated with a previous an-euploid miscarriage derived through NC or ART is signifi-cantly higher than in individuals without a previous aneuploid conception Additionally, this incidence is higher when the previous aneuploidy was in autosomes The recur-rence of aneuploidies can be avoided using PGS for a selected panel of chromosomes or in the future with CGH arrays for all
24 chromosomes
Acknowledgments: The authors thank the clinicians, em-bryologists, and technicians of the Instituto Valenciano de In-fertilidad clinics for their cooperation in the development of this study Nasser Al-Asmar expresses special thanks to all
of his colleagues in the PGD lab at Iviomics in charge of the FISH analysis on embryos and sperm The authors are very grateful to Drs Marcos Meseguer and Nicolas Garrido for sta-tistical support
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