DOI: https://doi.org/10.21203/rs.3.rs-2859924/v1
Background:Two chromosomes undergo a break and exchange with each other, then the formation of two new derived chromosomes without a mitotic break is a reciprocal translocation. Reciprocal translocations are relatively common structural aberrations that can occur on any chromosome. Reciprocal translocations are called balanced translocations when there is only a change in position and no visible gain or loss of chromosome segments. Chromosomal translocations are rearrangements based on chromosomal breaks resulting in abnormal chromosome structure and are an important cause of habitual abortion and fetal malformations[1].
Method: The karyotypes of patients were analyzed by collecting peripheral blood for lymphocyte culture, chromosome harvesting, filming, and G dominant band staining.
Result: The chromosomal results of the couples were 46, XY,t(13;16)(q34;q12.1) and 46, X,t(X;3)(p22.1;q26.2). They are both chromosomally balanced translocation carriers, with translocations occurring on chromosomes 13, 16, X, and 3. Their chromosome breakpoints are q34, q12.1, p22.1, and q26.2.
Conclusion: It is clinically rare for couples to be both chromosomal translocation carriers simultaneously. Abnormal chromosome structure is one of the leading causes of primary infertility in males or females[2]. Preimplantation genetic testing (PGT) allows genetic testing of gametes or embryos of chromosomal translocation carriers and selection of healthy or translocated embryos for transfer to solve the fertility problems faced by chromosomal translocation carriers.
Research Subjects
The couple married for five years had everyday sexual life, but the wife was not pregnant. Examination: The husband was 30 years old, 177cm in height, 73kg in weight, with a bachelor's degree, a bank employee, endocrine test results were in the normal range; varicocele on the left side had been treated surgically; semen examination was performed three times, suggesting 13% sperm motility (a+b>50%) and 10% sperm malformation rate (reference value <4%). Wife is 28 years old, height 164cm, weight 53kg, bachelor's degree, teacher, irregular menstruation, endocrine measurement: testosterone 110.00 (0.1-0.75ng/ml).
Ultrasound examination: 14 7-8 mm diameter follicles in the unilateral ovary, wheel-shaped arrangement; no dominant follicle and no signs of ovulation were seen on continuous monitoring—clinical diagnosis: polycystic ovary syndrome.
Apparatus and reagents
Thermostatic incubator (Thermo); Ultra-clean bench (Suzhou Antai Air Technology Co., Ltd.); BX51 microscope (OLYMPUS); Peripheral blood lymphocyte culture medium, colchicine and trypsin (Guangzhou Dahui Biotechnology Co., Ltd.); Giemsa staining solution (Zhejiang Hangzhou Baorong Technology Co., Ltd.); Chromosome karyotype analysis system (Leica GSL- 120 high-throughput automated scanning chromosome analysis system).
Method
Specimen collection: about 3 ml of venous blood was collected under aseptic conditions and placed in a vacuum blood collection tube containing lithium heparin anticoagulant.
Cell culture: aspirate about 2 ml of peripheral blood with a 5 ml syringe under aseptic conditions, then inoculate 30 to 35 drops in peripheral blood lymphocyte medium, 2 bottles of each specimen, and incubate in a constant temperature incubator at 37℃ for 68h-72h. Production of harvest: add 114 μl of colchicine (20 μg/ml) to each bottle of medium and place in a constant temperature incubator for 40min, after which the cells have then centrifuged the cells at 2000 rpm for 10 min, discard the supernatant, pour 6 ml of 0.075 mol/L KCL solution and mix well, hypotonicity for 30 min, then add 1 ml of fixative (glacial acetic acid mixed with methanol at 1:3) to each tube for 5 min. Repeat this step 3 times in a cycle. Finally, 0.6ml~1.5ml of fixative was added to the centrifuge tube according to the amount of cell precipitation, and 3~4 slides of each specimen were dried and placed in the oven at 76℃ for 6h~8h. After that, 0.01% trypsin solution was used to shift the enzyme, and Jimsa staining solution was used for 3min to show the band.
Karyotype analysis:150～200 split phases were scanned for each specimen with a Leica GSL-120 high-throughput automatic chromosome scanner into the computer analysis end, 50 chromosome split phases were counted, and 20 karyotype maps were analyzed.
The karyotype results of the husband were 46,XY,t(13;16)(q34;q12.1), 46,XY,t(13;16)(13pter→13q34::16q12.1→16qter;16pter→16q12.1::13q34→ 13qter); the wife's karyotype results were 46,X,t(X;3)(p22.1;q26.2), 46,X,t(X;3)(3qter→3q26.2::Xp22.1→Xqter;3pter→3q26.2::Xp22.1→Xpter). No relevant reports were found after checking the authoritative Cytogenetics Database, Borgaonkar Online Database, and China Human Chromosome Abnormalities Karyotype Database. The karyotype was included in the China Human Chromosome Abnormalities Karyotype Database with 4199 and 4198, respectively. It was found that the husband inherited the chromosomal translocation from his mother and the wife inherited the chromosomal translocation from her father. The couple's mother also had a history of adverse pregnancy and delivery.
Given that the couple's balanced chromosomal translocation involves 4 chromosomes and the probability of producing abnormal gametes is extremely high, the couple selected normal or translocated embryos for transfer with the help of PGT technology to avoid the implantation of chromosomal abnormalities (aneuploidy, deletions, duplications, etc.) in the uterus leading to pregnancy failure or neonatal defects, and finally successfully conceived a male child with identical karyotype to his father. The child is now two years old, and his growth and development are not significantly different from those of children of the same age.
Polycystic ovary syndrome is one of the most common gynecological, endocrine disorders and is the most common cause of menstrual disorders in women during their reproductive years. According to a recent study[3], patients with polycystic ovary syndrome are characterized by bilateral polycystic ovarian changes, often associated with menstrual disorders, hirsutism, obesity, acne and infertility, and significantly elevated androgen levels. In this paper, the female patient showed significantly elevated testosterone and ovulatory dysfunction, typical of this disease. It is assumed that polycystic ovary syndrome is one of the causes of the patient's failure to conceive after 5 years of marriage.
46,XY,t(13;16)(q34;q12.1) and 46,X,t(X;3)(p22.1;q26.2) balanced translocations each form one tetrasomy in meiosis, theoretically resulting in at least 18×18 gametes[4], of which only one is normal, and one is a carrier, the others are partially monosomic or partially trisomic, which can cause miscarriage, abnormal fetuses, etc. The probability of having a normal fetus is theoretically 1/324 (1/18×1/18); the probability of having a phenotypically common carrier is 1/324 (1/18×1/18), and the rest 322/324 (1/18×1/18). /324 were all chromosomally unbalanced abnormal individuals. Therefore, we believe that one of the important causes of infertility in this paper's patients is positional effects or mutations in genes near breakpoints resulting from chromosomal translocations. In recent years preimplantation genetic diagnosis has been widely used in people with a high risk of chromosomal abnormalities in pregnancy to achieve normal pregnancy by screening chromosomally abnormal embryos at the preimplantation stage[5]. The application of high-throughput sequencing in PGT can reduce the miscarriage rate, increase the pregnancy rate, and effectively avoid the vertical transmission of disease-causing genes in patients with genetic disorders[6].
It has been shown that two structurally normal X chromosomes are necessary to maintain follicle numbers. Two structurally normal X chromosomes are essential for maintaining typical follicular stores. xp11.2-p22.1 is a common region of X short arm deletion in patients with early-onset ovarian insufficiency[7], suggesting that this region has a vital role in ovarian function. The chromosomal breakpoint Xp22.1 in our patient falls within this region, so it is also the leading cause of follicular dysplasia in the patient.
Ethical approval and Consent to participate
All experiments were performed in accordance with the Declaration of Helsinki. This work was approved by Ethics Committee of the Affiliated Hospital of Putian University. Informed consent was obtained from all subjects involved in the study.
Consent for publication
The patients/participants of the study approve to publish their identifiable data in an online, open-access journal.
Competing interests
The authors declare that they have no conflict of interest in the publication of this article. This study is original research that has not been published previously, and not under consideration for publication elsewhere.
Authors' contributions
Kangying Wang: Conceptualization, Methodology, Investigation, Formal analysis, Software, Data curation, Writing - original draft. Hua Lin、Fan Wu: Conceptualization, Methodology, Investigation, Formal analysis, Software, Data curation, Writing - review & editing, Funding acquisition. Ben Liu、Yongzhi Lun: Project administration, Supervision, Conceptualization, Writing - original draft. All authors read and approved the final manuscript.
Funding
This work was supported by the Putian University Educational Teaching Reform Research Project (JG2022035).
Availability of data and materials
The data that support the findings of this study are available from the corresponding author upon reasonable request.
Acknowledgements
Not applicable.