The Karyotype Analysis of Sex Chromosome Mosaicism in Prenatal Diagnosis and Their Clinical Outcomes

Objective To analyze the karyotype of sex chromosome mosaicism in our prenatal diagnosis of 14034 pregnant women in their second trimester, and report the rate of sex chromosome mosaicism and their clinical outcomes. Methods A retrospective analysis of cytogenetic studies of 14043 cases of pregnant women from the Genetic Counseling Clinic from May 2017 to January 2020 by amniocentesis, were performed. Results A total of 46 cases of sex chromosome mosaicism were found, and the sex chromosome mosaicism rate was 0.328%, mainly including four types of mosaicism: mos45,X/46,XX(12); mos45,X/46,XY (11); mos47XXX(or XXY or XYY)/46XX(or XY)(11); and other types of complex abnormal karyotype mosaic(12). Among the 46 fetuses with sex chromosome mosaicism, the indications of prenatal diagnosis includes the numerical abnormality of sex chromosome by NIPT(23/46) (cid:0) the high risk of trisomy 21 by serum screening(12/46) (cid:0) abnomal ultrosound(4/46), the advanced maternal age(age ≥ 35)(4/46), and the histories of abnormal pregnancy(3/46). According to the results of cytogenetic analysis and genetic counseling, the pregnant women would decide to continue or terminate their pregnancy. Conclusion Prenatal cytogenetic diagnosis by amniocentesis is an accurate and convenient method and helps to avoid the delivery of fetuses with chromosomal diseases and reduce the risk of fetal malformation.


Introduction
It has been reported that sex chromosome mosaicism involving a numerical abnormality is the most common type of mosaicism 1 . The phenomenon of chromosomal mosaicism often occurs in prenatal diagnosis, including autosomal and sex chromosomal mosaicism. Sex chromosome abnormality may cause fetal sexual organ hypoplasia, usually accompanied by other organ structure aberrant and dysfunction 2 , or with mental retardation, mental and neurological disorders 3 . Therefore, it is very important to diagnosis fetus with chromosome aberrants.
Nowdays, it is a prevailing trend to perform prenatal screening and diagnosis, which could effectively reduce the born of children with abnormal chromosome. The karyotype analysis of amniotic uid cells was the main strategy for detecting fetal chromosomal abnormalities, and was regard as the gold standard for cytogenetic diagnosis currently.
Here in our study, we retrospectively collect 14034 cases of pregnant women with speci c prenatal diagnostic indications or of their own accord. Karyotype analysis of the amniotic uid cells were performed. Of them, karyotype of 46 fetuses were sex chromosome mosaicism. Different proportions of mosaicism may have different effects on fetal prognosis, which makes increased di culty in prenatal genetic counseling 4, 5 .
This study was approved by the Medical Ethics Committee in the First A liated Hospital of Zhengzhou University. All of the analysed samples were obtained with signed informed consent.

Subjects
Our study recruits 14034 pregnant women with the need of prenatal cytogenetic diagnosis who camed to the clicnic of the prenatal diagnosis center of the First A liated Hospital of Zhengzhou University between May 2017 and January 2020. Then amniotic uid samples were successfully extracted and cultured, at last, all of the fetuses received karyotype diagnosis. All of the pregnant women performed amniocentesis with speci c prenatal diagnostic indications or of their own accord. There are a variety of indications for genetic amnioticcentesis, including abnormal ultrasound nding, the numerical abnormality of sex chromosome by NIPT(Non-Invasive Prenatal Testing), the high risk of trisomy 21 by serum screening, abnormal ultrasound nding, the advanced maternal age(age ≥ 35), and the histories of abnormal pregnancy. The pregnant women were at 17-24 weeks' gestation undergoing amniocentesis to get amniotic uid samples.

Methods
Amniotic uid specimens (15 ml) was collected by amniocentesis under the guidance of ultrasonography, then centrifuged at 1500r/min for 8 minutes. After discarding the supernatant, the remaining liquid was mixed and incubated with two 5 ml of amniocyte culture medium (Gibco,USA, and Isreal). The cells grew in an incubator at 37 °C and 5% CO2 for about 9 days. When cell colonies reached more than 15, these cells were collected followed by making sections and conventional Giemsa banding, then scanned by Zeiss Automated Nuclear Scanning System. For each sample, 5 mitotic gures were analyzed with 30 counts (abnormal karyotype with 100 counts). For these abnormal karyotypes, the second line cells were also managed as the rst line.

Results
Out of the 14034 pregnant women recruited in our research, a total of 46 cases of sex chromosome mosaicism were found, and the sex chromosome mosaicism rate was 0.328%, mainly including four types of mosaicism: mos45,X/46,XX(12); mos45,X/46,XY (11); mos47XXX(or XXY or XYY)/46XX(or XY) (11); and other types of abnormal karyotype mosaic (12). The detailed information is in the Table 1.

Discussion
Chromosome abnormality are common genetics disorders causing spontaneous abortion, fetus aberration or birth defects 6,7 . However, there are no effective methods to treat this kind of abnormal chromosome disorders. Nowdays with the rapid development of technology, amniocentesis and subsequent karyotype analysis is the gold standard for prenatal disgnosis, which is an important way to con rm and prevent aberrant pregnancies 8,9 . Chromosome mosaicism would cause varying degrees of fetal abnormalities according to the mosaic propotion. Sex chromosome mosaicism will lead to hermaphroditism and psychiatric disorders 10 .
Early work indicated that sex chromosome mosaic is the most common type of mosaicism, which involved either a loss or gain of one sex chromosome, with the proportion of nearly a half of all kinds of mosaicism, including sex chromosome mosaicism, autosomal mosaicism and marker chromosome mosaicism 1 . Out of the 14046 cases of fetal karyotype analysis, 46 fetus were found sex chromosome mosaicism, the sex chromosome mosaicism proportion is 0.327%, which is a little lower than that of others' research in China reported in 2018 11 .
For all these 46 cases, their prenatal diagnostic indications are different, a half of them were the numerical abnormality of sex chromosome by NIPT, which could increase the sex chromosome mosaicism proportion among general pregnant women, because of the high accuracy rate of NIPT. 26% of the 46 pregnant women were grouped into high-risk serum screening, other high risk indications are less than or only 8.7%. In lights with these ndings, we would summarize that of the proportion of sex chromosome mosaicism is less than 0.327% of all the pregnant women in China. So when they had high risk in the numerical abnormality of sex chromosome by NIPT, they must perform the amniocentesis to get accurate cytogenetic diagnosis to exclude sex chromosome abnormality due to its de nite pathogenicity.
Among these mosaic cases, 45,X(Turner Syndrome) is the most common chromosome abnormality. 31 out of 46 were 45, X mosaicism, which usually causes infertility, cardiac and kidney malformation, and ear and hearing problems 12  , pregnant women usually choose to induce labor,but when the proportion of normal karyotypes are higher than 80%,pregnant women would continue the pregnancies. When the karyotypes of the chimeras are all abnormal karyotypes, that is, either the number or structure of sex chromosomes are abnormal or the structure of autosomes is abnormal, pregnant women would choose to induce labor. When there are three types of karyotypes in the chimera, pregnant women choose to induce labor. Three pedigrees lost contact, and we could not know their choices.
In lights with these ndings, we would suggest the fetus with mosaic sex chromosome karyotype abort their pregnancies if the abnormal karyotype was higher than 80%.
However, there are many potential factors associated with parental decisions regarding abnormal sex chromosome pregnancy in our research, we just did a simple follow-up, more studies need to be explored.
Concluding, if the pregnant women have clear incidences of prenatal diagnosis, they had better to follow the doctors' advices to conduct prenatal cytogenetic diagnosis. Prenatal cytogenetic diagnosis by amniocentesis is an accurate and convenient method and helps to avoid the delivery of fetuses with chromosomal diseases and reduce the risk of fetal malformation.

Declarations
Ethics approval and consent to participate: This study was approved by the Medical Ethics Committee in the First A liated Hospital of Zhengzhou University. All of the analysed samples were obtained with signed informed consent.