Complex chromosomal rearrangements (CCRs) are structural abnormalities involving at least three chromosomal breaks with exchange of segment . The CCRs range from simple 3-way rearrangement between three chromosomes to highly complex genetic rearrangement involving four or five chromosomes with multiple breakpoints, insertions, or inversions [2,3]. Although CCR carriers are usually phenotypically normal, they have an increased risk of developing significant reproductive failures by making meiotic abnormalities in males and chromosomal imbalances in female gametes . The production of embryos from unbalanced gametes typically results in implantation failure, recurrent miscarriage, or the birth of a baby with congenital abnormalities [4,5].
Once CCR identified,
Next-generation sequencing (NGS) is a newer technology that is increasingly being used for PGT . NGS has been shown to result in superior pregnancy outcomes compared with the use of fluorescence in situ hybridization, and array comparative genomic hybridization . NGS has a high sensitivity and specificity for detecting the segmental and whole chromosome . The accurate detection of NGS for imbalanced segmental and whole chromosome aneuploidies might improve the IVF success rate and increase the chances of having a healthy live birth for translocation or CCR carriers .
Herein, we reported the case of an unexpected CCR found based on the embryonic profiles obtained after NGS/PGT-SR and demonstrated that IVF-derived embryo NGS analysis could help identify chromosomal structural abnormalities in patients carrying CCRs.
A 26-year-old female visited our clinic under the problem of primary infertility for 2 years. She had polycystic ovaries with regular menstruation, and karyotype was 46, XX normal. Her husband was 28 years old and found to have severe oligoasthenoteratozoospermia on semen analysis. Serum level of gonadotropins and testosterone were found to be normal. Cytogenetic analysis revealed balanced reciprocal translocation, 46,XY,t(3;11)(p26;p14).
The couple chose to undergo PGT-SR to increase the possibility of pregnancy per embryo transfer and reduce the risk of miscarriage and birth of anomalous baby with unbalanced chromosome. The PGT-SR process was explained to the patients and their queries were answered. The genetic risk, the success rates, the risk of false negative PGT and the importance of prenatal confirmation of fetal chromosomes in case of pregnancy were specifically discussed with the couple. The couple signed an informed consent form for PGT-SR testing.
Two cycles of controlled ovarian stimulation were performed using recombinant follicle-stimulating hormone (Gonal F™; Merck KGaA, Darmstadt, Germany, or Follitrope™; LG Life Sciences Ltd., Seoul, Korea) and human menopausal gonadotropin (IVF-M HP Multidose™; LG Life Sciences, Ltd.), followed by a gonadotropin-releasing hormone antagonist protocol. The ovulation and final oocyte maturation were triggered with human chorionic gonadotropin (Ovidrel; Merck, Kenilworth, NJ, USA) administration 36 hours before oocyte retrieval. A total of 90 oocytes (1st cycle – 47 oocytes, 2nd cycle – 43 oocytes) have been retrieved and intracytoplasmic sperm injection was used in order to prevent DNA contamination. Trophectoderm (TE) biopsy was performed on embryos of blastocyst stage. Morphological evaluation was performed according to the criteria set by Gardner and Schoolcraft grading scheme . After biopsy, the embryos were vitrified and stored.
In house PGT-SR was performed. A commercial VeriSeq PGS kit (Illumina, Inc., San Diego, CA, USA) was used for NGS-based aneuploidy screening. DNA obtained from TE biopsies cell was amplified, then diluted, fragmented, adaptor ligated, and subjected to PCR-based SurePlex Amplification System (Illumina Inc.). Whole Genome Amplification samples were used for library preparation and sequenced using Illumina MiSeq system in our center. Subsequent comprehensive chromosome screening was tested using Illumina BlueFuse Multi software. Embryo classification was performed with reference to the Preimplantation Genetic Diagnosis International Society guidelines and recommendations for embryo prioritization .
Karyotyping was performed by GTG (G-bands after Trypsin using Giemsa; 550 bands)-banding technique with metaphase chromosomes staining of lymphocytes in peripheral blood. Chromosome aberrations were classified with reference to the International System for Human Cytogenomic Nomenclature (ISCN2020).
In the first IVF cycle, PGT-SR analysis was performed on nine biopsied blastocysts, from which only one embryo had unbalanced translocation between chromosome 3 and 11. In addition, another segmental aneuploidy on chromosome 21q21.3-qter was found. Interestingly, this segmental aneuploidy occurred repeatedly in five embryos at the same break point. Terminal segmental aberrations involving chromosomes 3, 11 and 21 (Embryo 1, 3, 4, 6, 7, and 9); and three (Embryo 2, 5 , and 8) had a chaotic aneuploidy showing more unbalance of than two chromosomes. All embryos had abnormal chromosome.
Based on the first NGS results, we suspected that this couple may have CCR instead of balanced reciprocal translocation. We recommended to repeat parental karyotype analysis. As a result, the karyotype was 46,XY,t(3;11;21)(p26;p14;q21) (Fig. 1): the segment on chromosome 3 distal to 3p26 has been translocated to chromosome 11 at band 11p14, the segment on chromosome 11 distal to 11p14 to chromosome 21 at 21q21, and the segment of chromosome 21 distal to 21q21 to chromosome 3 at 3p26 (Fig. 2).
In the second IVF cycle, PGT-SR analysis was performed on 10 biopsied blastocysts. Only one embryo was euploid (Embryo 1); nine embryos had unbalanced chromosome involving distal regions of chromosomes 3, 11, and 21 (Embryo 2-10).
Segregation analysis showed that among a total of 19 embryos only one embryo (5.2%) was 3:3 alternate segregation resulting in a normal or balanced chromosome. Thirteen unbalanced embryos (68.4%) were 3:3 adjacent segregations, of which twelve (63.2%) was adjacent-1 and one (5.2%) was adjacent-2 segregation. Two unbalanced embryos (10.5%) were 4:2 segregations and none was found with 5:1 or 6:0 segregation. The probable mode of meiotic segregation could not be ascertained for three embryos (10.5%) due to complex mosaicism or aneuploidy (Table 1).
Based on the chromosome screening outcome of embryo from the second IVF cycle, Embryo No. 1 with euploid (normal or balanced) was selected for transfer. For frozen-thawed embryo transfer cycle, the endometrium was prepared using 6 mg of estradiol valerate (Progynova®; Bayer Schering pharma AG, Berlin-wedding, Germany) daily starting from the third day of the menstrual cycle for 15 days, then combined with 50 mg of progesterone subcutaneous injection (Prolutex® 25 mg; IBSA Institut Biochimique SA, Lugano, Switzerland) daily. Embryo was transferred back to the uterus after 5 days of progesterone injection. Serum level of β-human chorionic gonadotropin on day 14 after the transfer was 180.3 mIU/mL. Transvaginal ultrasound examination was performed at 5 and 7 gestational weeks to confirm the pregnancy. Single gestational sac and single fetus with fetal heartbeat were found. To confirm the PGT-SR result, amniocentesis was performed at 17 week of gestation. Karyotype of the fetus was of normal chromosomal microarray test using amniotic fluid also showed normal finding, arr(1-23)x2. The patient delivered a healthy girl weighing 3.05 kg via cesarean section at 37th week of pregnancy.
In this case report, we described an incidental finding of unbalanced CCR embryos during NGS analysis of PGT-SR performed in couple with balanced translocation. This study demonstrates that careful NGS analysis of the preimplantation embryos can identify parental chromosomal structural abnormalities such as balanced reciprocal translocation or CCR.
As chromosomal structural rearrangement carriers exhibit alternations in meiosis, hexavalent segregation during the meiosis can produce germ cells with normal and balanced karyotypes, and/or cells with unbalanced translocated karyotype, according to 3:3 (alternate or adjacent), 4:2, 5:1 or 6:0 chromosomal segregation . Theoretically, of the over 64 different possible segregation modes resulting from a three way CCR only two would produce a normal/balanced gamete . Interestingly, all unbalanced chromosome 21 was found in 4:2 segregation mode in this study. This is in agreement with the published data suggesting that the involvement of acrocentric chromosomes in a hexavalent formation favors segregation into the 4:2 mode [15,16].
Recently, NGS based PGT-SR came into use widely. NGS is a comprehensive, high-throughput genetic technique used to screen all 24 chromosomes of multiple samples in a single sequencing run . NGS provides more accurate detection of mosaicism, segmental aneuploidy [18,19], and unbalanced rearrangement [20,21]. NGS-based PGT-SR is a valuable alternative for chromosomal microarray (i.e., single nucleotide polymorphism and comparative genomic hybridization—arrays)-based testing. Increased sensitivity and specificity of NGS technology has enabled the identification of additional errors such as segmental abnormalities, which in turn have resulted in better pregnancy outcomes in couples with chromosomal structural rearrangement [11,22].
Treating patients with chromosomal structural rearrangements is technologically complex and limited by the patients’ genetic characteristics, resulting in a low percentage of normal or balanced embryos . Unfortunately, in this type of CCR, only 9% of biopsied embryos will be normal or balanced . As a result, these cases require more attention.
Most CCR carriers do not know their condition, until genetic analysis of either abortus or affected baby or parental karyotyping is performed. Therefore, selective karyotyping, adequately adopted prior to IVF, may have additional clinical and financial advantages, especially in patients with a history of recurrent miscarriages or severe male infertility . First of all, the parental karyotyping may provide the useful information for genetic cause of infertility due to chromosomal abnormalities leading to gametogenesis failure . This information can be vital and help clinicians have patients counselling and the best treatment options. Second, if structural rearrangements such as balanced translocations or CCR are identified, PGT-SR can be marked to avoid the transfer of embryos with unbalanced translocation [7,26].
In conclusion, we reported a case of retrospective CCR carrier identification via TE biopsy analysis. Suspicion about translocation or CCRs is recommended in cases of the repeated presence of chromosomal breakage associated with segmental aneuploidy within the same chromosomal regions during NGS reading. Given that karyotyping is not routinely performed in the treatment and diagnosis of infertility, the PGT-SR with NGS will facilitate the detection of previously undiagnosed balanced translocation or CCR carriers in IVF patients.
The authors declare that they do not have any conflicts of interest.
Conception and design: ISK. Acquisition of data: MJK, EAP, YSH, SOP, SHP, YBL, TKY. Analysis and interpretation of data: MJK, SOP, SHP. Drafting the article: EJY. Critical revision of the article: EJY, ISK. Final approval of the version to be published: all authors.
Results of preimplantation genetic testing for structural rearrangement