Analysis of Parental Abnormal Chromosomal Karyotype and Subsequent Live Births in Chinese Couples With Recurrent Miscarriage

The frequency and distribution of chromosomal abnormalities and the impact of parental chromosomal aberration on the pregnancy outcomes of couples with recurrent miscarriage remains controversial. 3235 RM couples who experienced two or more miscarriages before 20 weeks were diagnosed in our tertiary referral hospital during 2008 to 2018 and included in the single-centre retrospective cohort study covering a 10-year period. Chromosome aberration was detected in 121 (3.74%) among 3235 RM couples which included 75 female and 46 male cases at an individual level. 101 cases were structural aberrations including balanced translocations in 46(38.0%) cases, Robertsonian translocations in 13(10.7%) cases, inversions in 42(34.7%) cases and 20(16.5%) cases were numerical aberrations. 121 carriers and 428 non-carriers were followed up for two years (cid:0) 55 carriers and 229 non-carriers were subsequent pregnant after diagnosis by natural conception or intrauterine insemination. The frequency of carriers to have a health newborn was not signicantly different with non-carriers (72.7% vs. 71.2%, adjusted p=0.968). This study described the majority of carriers were balanced translocations and chromosome aberrations had a limited inuence on live birth rate from the present data. The results of the study also remind us that natural conception may be also a good alternative rather than PGD (Pre-implantation Genetic Diagnosis) which is common in many other reproductive centers for such patients.


Introduction
Recurrent miscarriage (RM) is defined by the ESHRE guidelines in November 2017 as the loss of two or more pregnancies [1]. According to the history of live birth, it can be divided into primary and secondary recurrent miscarriage [2]. The causes of recurrent miscarriage are very complicated. In addition to anatomy, endocrine, thrombophilic, immune and other factors embryo chromosomal abnormalities are often considered an important cause of miscarriage [3,4]. The embryo chromosomal abnormality rate in the general population is 60% [5], while the rate in the recurrent miscarriage is 29%-60%[6-8]. Embryonic chromosomal abnormalities may occur during the mitosis of embryo development, or come from parental abnormal ovum or sperm. For example, parental balanced chromosomes lead to unbalanced gametes which might cause abortion [9]. Therefore, the chromosomal karyotype of both parents is considered to be an important examination in the cause of recurrent miscarriage recommended by American Colleges of Obstetricians and Gynecologists [10]. However, the evidence that parental chromosomal abnormalities lead to miscarriage is still unclear, a considerable percentage of couples with chromosomal abnormalities have successfully given birth [11]. In addition to chromosomal factors, other factors may cause miscarriages that coexist with chromosomal aberrations. Due to the limited number of samples in couples with abnormal chromosomes, other known and unknown pathological factors were studied as homogeneous, and chromosome aberrations could hardly completely solely studied with removal of other pathological factors. Therefore, it is more di cult to judge and analyze the cause of miscarriage due to parental chromosomal abnormalities, which often makes clinicians' understanding of parental chromosomal abnormalities leading to miscarriage not accurate enough. Page 3/19 5680 couples with recurrent miscarriage were diagnosed in our reproductive center from 2008 to 2018.
The study attempts to summarize the frequency of abnormal chromosomal karyotype couples, the topography of abnormal types, and the frequency of the male and female carriers in the recurrent miscarriage population. The coexistence of other causes of miscarriage and respective pregnancy outcomes were further evaluated. We hope that the results of the study will provide more scienti c evidences for genetic counseling among RM couples.

Study population
The study was approved of the Ethic Committee of The First A liated Hospital of Xi'an Jiaotong University according to the declaration of Helsinki. All the participates were informed and signed consent for the study. From January 2008 to December 2018, a total of 5680 couples who had two or more spontaneous miscarriage before the 20th week of pregnancy came to our reproductive center for outpatient treatment. The included patients must provide objective evidence of past birth history, including HCG testing, or the histology report after curettage and evacuation of uterine or the gestational sac under ultrasound, all the clinical data bring to the study was carefully recorded and checked.

Peripheral blood karyotype analysis
Chromosome analysis was performed on routinely cultured peripheral blood lymphocytes as described previously [12]. Brie y the sections were treated with trypsin using standard techniques, the slides were Giemsa stained and then G-banding analysis was performed. Add colchicine 4 hours before cytology preparation. For each sample, at least 20 cells from two independent cultures were used for microscopic observation and analysis in metaphase.

Follow-up
All patients had been followed up for at least two years to get the subsequent rst pregnancy outcomes. The details of each individual were entered into a computerized database with clinical features and miscarriage history recorded. Data of the present study was retrieved from medical records and telephone interviews.
Statistical methods t test was used for measurement data between the two groups, and chi-square test or Fisher's exact test was used for count data. Binary logistic regression analysis was used to evaluate risk factors for pregnancy outcomes. P<0.05 was considered statistically signi cant. The statistical software of SPSS 20.0 was applied in the study.

Results
The frequency and distribution of aberrant chromosomal RM couples The First A liated Hospital of Xi'an Jiaotong University is a tertiary referral teaching hospital. 5680 recurrent miscarriage couples came to the Reproductive Medicine Center from January 2008 to December 2018 as shown in Figure 1. The ow chart shows that 954 couples had not completed the etiology screening evaluation and 1491 couples had not peripheral karyotype test of both female and male, so the remaining 3235 couples had complete karyotype analysis results. There are 121 couples of abnormal chromosomal karyotypes in 3235 couples with complete results (including abnormalities of either the female or the male and excluding chromosomal normal polymorphisms) with the abnormality rate of 3.74% (121/3235). Among 121 couples with abnormal chromosomal karyotypes, 101 cases were structural abnormalities (3.12%, 101/3235), and 20 cases were abnormal numbers (0.62%, 101/3235).
As shown in Figure 2A, 101 structural abnormal cases included 46(38.02%) with balanced translocation, 42(34.71%) with inversion, 13(10.74%) with Robertsonian translocation, and 20(16.53%) cases had the numerical chromosome aberrations. In order to further illustrate whether the chromosomal abnormality comes from the female or the male, we noticed that 75 female and 46 male were with chromosomal abnormality among 121 RM couples, and the distribution of abnormal chromosome types in female and male respectively can be shown in the Figure 2B. During the following-up of 121 chromosomal abnormal couples with recurrent miscarriage, 55 couples were pregnant and 66 couples were not pregnant merely by medical expectant management through natural conception or intrauterine insemination without IVF/PGD as in the owchart of gure 1. The proportions of the four types of chromosomal abnormalities among pregnant and non-pregnant carriers was shown in Figure 2C and 2D. The two groups had no statistical difference in the four-type abnormal distribution by chi-square test (p=0.31).

Comparison of the etiological results and live birth rates between 55 carriers and 229 non-carriers
Because it is di cult to achieve the complete the pregnancy results from thousands of patients from 2008 to 2018, 428 RM couples with normal chromosomes who came to our outpatient department in the whole year of 2018 were selected and followed up for 2 years. They completed all etiological screenings and 229 of them were pregnant in the follow-up period as in the gure 1. Comparison of 55 carriers and 229 non-carriers showed that female carriers were younger at the time of consultation, while other clinical characteristics and combined pathological factors were not statistically different in Table 1. The outcome after the pregnancy, namely the live birth rate (LBR), was also not statistical different (p=0.87). Among the 55 carriers, 51 carriers with primary recurrent miscarriage (no previous live birth history), and 4 were secondary recurrent miscarriages. 6 females were diagnosed with polycystic ovary syndrome (PCOS, according to the Rotterdam criteria [13]), and 3 females were diagnosed with decreased ovarian reserve (DOR, according to the hormonal markers and ultrasound parameters [14]). To further analyze of other etiological screening results in the 55 carriers, 8 cases (14.6%) were positive for infection factors (including male or female genital tract Mycoplasma and Chlamydia infection), 2 cases (3.64%) were with abnormal uterine anatomical structure, 14 cases (25.5%) were with imbalance of peripheral blood lymphocyte subsets, 9 cases (16.4%) were with endocrine disorders (including ovarian hormone abnormalities, thyroid hormone abnormalities and hyperprolactinemia), 7 cases (12.7%) were with nutritional element de ciency (including folic acid and vitamin B12). Among the combined autoimmune antibodies, 13 cases (23.64%) were positive for anti-phospholipid antibodies, and 9 cases (16.4%) were positive for connective tissue antibodies. During the follow-up period 40 in 55 pregnant RM carriers gave birth to healthy babies in the way of natural conception or intrauterine insemination without IVF/PGD, the live birth rate (LBR) in the carriers (72.7%) was similar to that in the non-carriers (71.2%). It could be seen that, apart from age, the above-mentioned combined pathological factors and the nal LBR was not statistically different between carriers and non-carriers in the Table 1. The results were still consistent after using binary logistic analysis to adjust the age factor. Table 2 showed the details of every patient number, the age of the female, the karyotypes of the female and the male, the number of miscarriages and the outcome of pregnancy of 55 carriers. 40 of 55 carriers gave birth to healthy newborns at the end with the LBR of 72.73%. Among the 40 cases, 7 cases were numerical abnormalities (LBR of 87.5%) and 33 cases were structural abnormalities (LBR of 70.21%). The structural abnormalities included 14 cases with balanced ectopic (LBR of 60.87%), 17 cases with inverted position (LBR of 80.95%), and 2 cases with Robertsonian translocations (LBR of 66.67%). As shown in Figure 4, there was no statistical difference in the LBR in the four types of chromosomal abnormalities (p=0.35).
Among the 55 pregnant couples, 34 were female and 21 were male carriers. In the Table 3 we analyzed the women's age, number of miscarriages, distribution of karyotype abnormalities and the total LBR in female and male carriers respectively. There was no statistical difference in all items and showed gender of carrier had no effect on the pregnancy outcome (p=0.428).
In order to rule out the in uence of other etiological factors on the pregnancy outcomes, we analyzed the female age, the number of abortions, infection factors, anatomical uterine abnormalities, autoimmune antibodies positive rate, blocking antibody de ciency, peripheral blood lymphocyte subset disorders, endocrine disorders and nutritional elements de ciency between 40 carriers with live birth and 15 carriers with miscarriage again in the Table 4. All the differences in above items between the two groups were not statistically signi cant and no one showed huge in uence to alter pregnant outcomes.

Discussion
The cause of spontaneous abortion is generally attributed to two sources, namely seed problems and environmental problems. Seed problems are often considered to be abnormal parental chromosomes or abnormal fetal chromosomes. The results of this study showed that the incidence of chromosomal abnormalities in couples with recurrent miscarriage was 3.74%(Figure1). The present results consistent with previous studies have shown that the incidence of chromosomal abnormalities in the general population is less than 1% [15,16] and RM population is 2%-5% [17,18], indicating that parental chromosomal abnormalities rate increased assuredly in the miscarriage couples.
Balanced translocation was the most common type, accounting for 38.02% and consistent with other ndings [11]. A meta-analysis from Zouhair reported that frequency of chromosomal abnormalities in couples with RM was 5.16% and the most common reciprocal translocation accounts 48.4% in the worldwide literature review [12]. The balanced translocations and inversions will not affect the parents themselves in phenotype, but their unbalanced gametes during meiosis may indeed be part of the cause of miscarriage. Similarly, Robertsonian translocation of parental chromosomes can also cause miscarriage, birth defects or mental retardation of offspring [19]. However, all these studies could not demonstrate the explicit causality between aberrant chromosome and abortions.
Additionally, the LBR of RM carriers in our reproductive center have reached more than 70%, indicating that the proportion of miscarriage caused by chromosomal abnormalities in RM couples was very slight. A retrospective study from Howard et al. concluded that no statistically signi cant was found in the LBR between RM couples with chromosomal abnormality (45.2%, 33/73) and the normal couples was (55.3%, 325/588), regardless of number of miscarriages and rearrangement types of chromosomal abnormalities [20]. In Goddijn's study the screening results of 1324 RM couples showed that all the 41 couples with abnormal structure chromosomes did not yield an unbalanced fetal chromosome pattern [21]. It is also consistent with Franssen's study, the LBR of RM carriers was equivalent to the normal couples after six accumulated gestations, and had no relevance with the type of abnormal chromosome (83% vs. 84%) [22]. However, Sugiura's study showed pregnancy prognosis was worsened with either maternal or paternal reciprocal translocations than normal couples (63% vs. 78.7% of LBR) [23]. Pregnancy outcomes for RM couples with chromosomal abnormalities were still very satisfactory generally, although the decrease in the live birth rate may not have been detected due to insu cient sample size in our study. In addition, inversion of chromosome 9(inv(9)) is also considered as normal polymorphism in other reports [24,25]. The live birth rate of inv(9) in our study is 77.8%(14/18) which is not signi cantly different with the LBR in the other groups.
Preimplantation genetic testing (PGD) has been proposed as a controversial method in the worldwide for selecting normal chromosome embryos in the IVF to lower risk of miscarriage for patients with unexplained RM and balanced translocations carriers. However, well-designed trials comparing EM (expectant management) to PGD have not been performed. Several previous cases indicated bene ts of PGD including fewer miscarriages and shorter time to successful pregnancy without taking into account the emotional and nancial cost of a failed or canceled cycle. More recent reports suggested clinical outcomes including pregnancy rate, live birth rate (53% vs. 67%) and clinical miscarriage rate were similar between PGD and EM among recurrent miscarriage patients [26]. Even in the parental carriers of structural chromosomal rearrangement and history of RM, no signi cant difference with regards to reproductive outcomes such as miscarriage rate, time to live birth, or live birth rate was observed between couples who pursued PGD compared with EM [27,28]. These data combined with our results allow us to re ect on the actual bene ts of PGD to these patients, so clinicians can be more cautious when making an alternative of PGD in clinical work. Natural conception is also recommended as a good alternative for these aberrant chromosomal carriers.
50%-60% of spontaneously aborted product of conception have been detected with chromosomal abnormality [29]. The abnormal chromosomes of the fetus are derived from the parental abnormal chromosomes or produced in the process of gamete meiosis and mitosis of the fertilized egg by mistake randomly. The types of fetus abnormality were often mainly manifested as trisomies of chromosome 13, 18, 21 and X monosomy (45, X) [30], but not consisted with the translocation chromosomes of the parents showed as in the Figure 3. According to Howard Carp's study, parental karyotyping was not particularly predictive of a subsequent miscarriage, 43.5% of abortus from parental carriers were euploidic and the parental aberration was passed on to the abortus in only 10% of cases [31]. The phenotypes are inconsistent that parental karyotyping prefers balanced translocations (No. 8, 2, 6) and inversions (No. 9, 1, 6) rather than the more common numerical aberrations such as trisomies (No. 13,18,21) and polyploidy in fetus. Most aberrant chromosomes in the fetus are generated randomly and only a small percent derives from their parents.
One of the most important results in our data is the in uence of parental chromosomes on live birth rate (LBR). In our study, the LBR of both carriers and non-carriers can reach about 70% without relationship of gender, female age, chromosome abnormal type, abortion times and other pathological factors. Amounts of non-genetic pathological causes related to endocrine, infection, immune and nutrition were detected not only in aberrant chromosomal carriers but also in non-carriers, while these factors have a strong impact on the pregnancy outcomes. After effective treatments such as anticoagulation and immunotherapy, the LBR of re-pregnancy after two recurrent miscarriages has reached more than 70% internationally. A prospective study showed that closely following management and treatment of other high-risk factors can increase the LBR of RM couples with chromosomal abnormalities from 25% to 70% [32] or from 20% to 71% without the addition of assisted reproductive technology [11]. The differences of LBR in RM carriers in previous reports may due to the different management of non-genetic pathological factors that are usually more important in fetal survival.
From these data, we strongly recommend that RM carriers should still undergo comprehensive and systemic etiological screening. Therefore, it is necessary to actively deal with other causes of miscarriage in order to improve the chances of successful pregnancy for RM patients with chromosomal abnormalities. In order to improve the live birth rate, our treatment included surgical correction of the anatomically abnormal uterus, male lymphocyte treatment, anticoagulation aimed at anti-phospholipid antibody and immunosuppressive therapy were strongly recommended besides chromosome abnormality in our opinions. Male lymphocyte treatment and immunosuppressive therapy were done according to our experience and suggestions from some published reports [33][34][35]. The formation frequency of abnormal gametes theoretically is not equal to the birth rate of abnormal babies in practice, we still recommend that the chromosome test or next-generation sequencing analysis of the amniotic uid through puncture should be performed around 18 weeks of gestation in the natural pregnancy of RM patients with chromosomal abnormalities, although the deletion or duplication of smaller fragments still cannot be detected.
The present study did not detect the karyotype of aborted fetuses and not achieve complete amniocentesis results from the pregnancy carriers. So we could not assess the impact of fetal chromosomal problems came from parents. In the study of embryo chromosome analysis of abortion tissue, trisomy and polyploidy are the majority which account for 65% and 17% respectively, a considerable proportion of fetus with aberrations include trisomy, structural abnormality and lowfrequency mosaic could survive after birth [30]. The phenotypes are inconsistent that parental karyotyping prefers balanced translocations and inversions rather than the more common numerical aberrations such as trisomy and polyploidy in fetus. In addition, G-banding karyotype analysis used in this study can only detect a part of patients with abnormal numerical and structural chromosomes. Conventional karyotype analysis identifies balanced and unbalanced chromosomal rearrangements and copy number variants (CNVs) to a∼5 Mb resolution. Due to the limitations of the detection method itself, it could not exclude some other types of genes or chromosome abnormalities related to miscarriage problems, such as deletion, insertion, duplication and point mutation of some gene fragments. In 2019, Chen et al. used low pass genome sequencing (GS) to detect the chromosomes of RM couples with abnormalities rate increased to 11.7% compared to traditional karyotyping with 5.7%. However, inversions and copy-number variants detected by GS additionally had not been con rmed to directly related with miscarriage. 10 carriers observed in follow-up observations and ve of them miscarried again (miscarriage rate of 50%). The small sample size did not indicate that the risk of miscarriage of abnormal chromosome couples was higher than that of couples with normal chromosomes [36].
Finally, the lack of samples even in this 11-year study and other combined known and unknown nongenetic factors are shortcomings in the present data. The etiology of recurrent miscarriage is complicated and there are many controversies in the treatment. The coexistence of these other pathological factors and chromosomal abnormalities makes the results confused and controversial.

Conclusions
In conclusion, balanced translocation is the most common phenotype in RM carriers, and LBR of subsequent rst pregnancy is similar to the non-carriers. The present studies can help to provide more scienti c clinical consultation, such as more accurate diagnosis and the prognostic outcome of subsequent pregnancy, and help doctors to raise awareness of miscarriage-related chromosome problems and foster a theoretical basis for reasonable treatment.

Declarations
Study funding: This work was supported by a grant to M.D. Li Shan from the National Natural Science Foundation of China (No. 81901497) Con ict: All authors declare that they have no con ict of interest in the article.   Figure 1 The ow chart presents the process of collecting abnormal chromosomal carriers from 5680 RM couples in our Outpatient service.

Figure 3
The percents of aberrant chromosome No. in the balanced translocation and inversion RM patients.

Figure 4
The live birth rates in the non-carriers and carriers of four aberrant types.