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Next Generation Sequencing (NGS) in chromosome translocation 46, XX, t (9; X) (q22; q28) - a case report

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This paper reports the case of a patient who sought assisted reproductive technology (ART) treatment and was referred to pre-implantation genetic diagnosis (PGD) on account of a chromosomal translocation presented with secondary infertility. The patient underwent a highly complex ART treatment and had 14 metaphase II oocytes collected on the day of follicular aspiration. The embryos were taken to extended culture and five were biopsied and vitrified. The embryo genetic report showed aneuploidy in four of the blastocysts, while the other resulted in 46, XX. In conclusion, chromosome translocations involving the X chromosome might result in the deregulation of gene expression and defective ovarian formation. Therefore, the genes present in the X chromosome are believed to be essential in normal ovarian function.
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Received September 18, 2017
Accepted April 15, 2018
Case report
Next Generation Sequencing (NGS) in chromosome translocation
46, XX, t (9; X) (q22; q28) - a case report
Monise Santos1, Ivan Henrique Yoshida1, Caroline Zulim1, Michelli Suemi Tanada1, Emerson Barchi Cordts1,2,
Caio Parente Barbosa1,2
1Instituto Ideia Fértil de Saúde Reprodutiva
2Faculdade de Medicina do ABC
ABSTRACT
This paper reports the case of a patient who sought
assisted reproductive technology (ART) treatment and
was referred to pre-implantation genetic diagnosis (PGD)
on account of a chromosomal translocation presented
with secondary infertility. The patient underwent a highly
complex ART treatment and had 14 metaphase II oocytes
collected on the day of follicular aspiration. The embryos
were taken to extended culture and ve were biopsied and
vitried. The embryo genetic report showed aneuploidy in
four of the blastocysts, while the other resulted in 46, XX.
In conclusion, chromosome translocations involving the X
chromosome might result in the deregulation of gene ex-
pression and defective ovarian formation. Therefore, the
genes present in the X chromosome are believed to be
essential in normal ovarian function.
Keywords: Next generation sequencing, chromosome
translocation, assisted human reproduction, pre-implanta-
tion genetic diagnosis
JBRA Assisted Reproduction 2018;00(0):000-000
doi: 10.5935/1518-0557.20180034
INTRODUCTION
For decades embryo viability has been assessed based
on embryo morphology (Ebner et al., 2003), although
many cycles do not result in gestation. Thus, it is believed
that morphologically normal embryos might be aneuploid.
In fact, some studies have shown that 50% of the formed
blastocysts suer from aneuploidy (Alfarawati et al., 2011;
Goldman et al., 2016). However, the advancements in
reproductive medicine have made it possible to evaluate
embryo viability through chromosomal and gene mutation
analysis.
In 1990, pre-implantation genetic diagnosis (PGD) was
performed for the rst time in a couple with genetic dis-
ease to rule out the possibility of transmitting the condition
to their ospring (Harper, 2009). The technique is indicat-
ed to couples with known genetic or chromosomal alter-
ations, with the purpose of preventing the transmission of
the alterations to their ospring. Embryo biopsies may be
performed in the zygote, cleavage or blastocyst stages. At
present, most of the biopsies are performed in the blasto-
cyst stage, since studies found that the rate of mosaicism
in the cleavage stage is 50%, whereas in the blastocyst
stage it is 3-5% (Munné et al., 1994; Brezina et al., 2013).
Chromosomal abnormalities include complete or par-
tial deletions, the presence of an additional chromosome
or translocations involving or not the sex chromosomes
(Goswami & Conway, 2005). Chromosomal translocations
occur because of a rearrangement between chromosomes
and are 18% more likely to form euploid embryos. Cyto-
genetic abnormalities related to the X chromosome usually
lead to early ovarian failure. A region of the long arm of the
X chromosome known as "critical region Xq" - ranging from
Xq13 to Xq28 - has been associated with the formation
of the female gonad and ovarian function maintenance;
therefore, the genes present in this chromosome are be-
lieved to be essential for normal ovarian function (Simpson
& Rajkovic, 1999).
The most modern molecular technology used today is
Next Generation Sequencing (NGS). The technique may
be used to rule out chromosomal and genetic alterations,
genetically analyze an embryo's twenty-four chromosomes
with accuracy levels greater than 90%, identify losses and
gains of genetic material, and examine aneuploidies of
whole chromosomes and chromosomal trisomies. Howev-
er, extremely small chromosomal changes, depending on
the site of the chromosomal alteration, might not be de-
tected by NGS.
CASE REPORT
This is the case of a 29-year-old patient who had her
menarche at the age of 12, with regular cycles lasting for
four days, no history of gynecological surgery, and normal
hormonal tests. Her partner was 28 years old and had nor-
mal hormonal tests and spermograms. The couple sought
ART treatment at a fertility center (RHA) and was referred
to PGD on account of chromosome translocation, 46, XX, t
(9; X) (q22; q28).
Induction was performed with Pergoveris 150UI/75UI
(rFSH/rLH), 0.25mg Cetrotide and Choriomon 5000IU.
Fourteen metaphase II oocytes were harvested in follicular
puncture and fertilized using the intracytoplasmic sperm
injection technique (ICSI); semen analysis showed 3.9M,
96% progressive spermatozoa. The fertilization rate was
78.57% (11/14) and all embryos were taken to the blasto-
cyst stage on extended culture.
Five blastocysts were biopsied and subsequently vit-
ried. The cells were removed from the trophectoderm,
stored in appropriate solutions, and sent to the laboratory
for embryo genetic analysis. NGS was used to analyze the
24 chromosomes.
The embryo genetic report revealed four aneuploid blas-
tocysts (80%) and one euploid blastocyst (20%) (Table 1).
Table 1. Embryo genetic report results
Nº Blastocyst Embryo Genetic Analysis
1 47,XY,+9
2 45,XX,-13
3 45,XX,-2
4 45,XX,dup(2q),-9
546,XX
2
Case report
JBRA Assist. Reprod. | v.00 | nº0 | / 2018
Endometrial preparation was performed with Primogyna
8mg/day and Utrogestan 600mg/day for later embryo trans-
fer with a Sydney catheter. Ten days after embryo transfer,
the BHCG test read 1,710 mIU/mL.
DISCUSSION
Patients with known chromosome or gene anomalies
and a track record of unsuccessful transfers undergoing ART
treatment may resort to screening and genetic diagnostic
tests to have embryos free of the assessed conditions trans-
ferred. In the present case report, the cycle of a patient
with chromosomal translocation yielded a fertilization rate
of 78.57% (11/14); embryo biopsies and molecular genetic
tests with NGS were performed, leading to the transfer of a
euploid embryo (20%).
Liss et al. (2015) studied 42 couples with reciprocal trans-
location and 35 with Robertsonian translocation through the
FISH technique. The authors assessed fertilization rates, the
number of biopsied embryos, and normal embryos. They ob-
served that the group with reciprocal translocation had a fer-
tilization rate of 70.8% versus 66.8% of the individuals with
Robertsonian translocation. The overall fertilization rate was
68.9%. Five hundred and forty-eight embryos were biopsied
and 101 euploid embryos were found, yielding a euploidy
rate of 19.4%. A study carried out by our group examined
nine patients with translocations identied with CGH-array
and/or NGS tests and found fertilization and euploidy rates
of 84% and 38%, respectively, similarly to Liss et al. (2015).
Zhang et al. (2016) investigated 21 couples with history of
translocation and miscarriages oered RHA cycles with PGD.
Molecular genetic analysis was performed in 98 embryos,
and a euploidy rate of 30.6% was found.
Chromosomal translocations alter the spatial organiza-
tion of the "critical region Xq". PGD is of great importance
in cases of genetic alterations to prevent the transmission
of genetic disorders to the ospring. The age of the patient
enabled the harvesting of a good number of oocytes, thus
increasing the chance of nding a euploid blastocyst. In this
cycle, in addition to having a healthy embryo transferred, the
patient became pregnant.
CONFLICTS OF INTEREST
The authors have no conict of interest to declare.
Corresponding Author:
Monise Santos
Instituto Ideia Fértil
Faculdade de Medicina do ABC
Santo André/SP - Brasil
Email: monise.santos@ideiafertil.com.br
monisesantos_@hotmail.com
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Ovarian failure can result from several different genetic mechanisms-X chromosomal abnormalities, autosomal recessive genes causing various types of XX gonadal dysgenesis, and autosomal dominant genes. The number and precise location of loci on the X are still under investigation, but it is clear that, in aggregate, these genes are responsible for ovarian maintenance, given that monosomy X shows germ cells that undergo accelerated atresia. Despite recent hypotheses, at present there is no evidence for a gene directing primary ovarian differentiation; this process may be constitutive. Phenotypic/karyotypic correlation and limited molecular confirmation have long shown that proximal Xp and proximal Xq contain regions of the most importance to ovarian maintenance. Terminal deletions at Xp11 result in 50% primary amenorrhea and 50% premature ovarian failure or fertility. Deletions at Xq13 usually produce primary amenorrhea. Terminal deletions nearer the telomeres on either Xp of Xq bring about premature ovarian failure more often than complete ovarian failure. The X-linked zinc finger gene (ZFX) and diaphanous 2 Drosophila homologue (DIAPH2) are the only candidate genes for ovarian maintenance that map to the X chromosome. Additional, as yet unidentified, genes along the X chromosome must be involved. The search for these genes in humans is hampered by the lack of candidate genes that map to the X chromosome, the scarcity of patients with fortuitous autosomal translocations, and small pedigrees, which hinder mapping of the loci. In addition, difficulties with human germ cell research also make it challenging to dissect genes important to ovarian development. Autosomal genes also are involved in ovarian differentiation and gonadal failure. Follicle-stimulating hormone receptor and ataxia telangiectasia are examples of autosomal genes known to cause human ovarian failure. Transgenic mouse models point to many other candidate autosomal genes, and sequencing of the human homologues in affected women should lead to the discovery of new genes responsible for human ovarian failure. Identification, functional analysis, and mapping of novel genes specifically expressed in the ovary of mice and women eventually should lead to fruitful dissection of essential genes in mammalian ovarian development and maintenance.