Chromosomal integrity is vital to embryo survival, with chromosomal abnormalities typically resulting in spontaneous abortion or intrauterine fetal demise. Analyses of embryo chromosomal status in cases of early MA can provide insight into the causes of miscarriage. Conventional chromosome G-banding techniques have historically been used for the karyotyping of miscarriage products. This strategy, however, is only capable of detecting certain chromosomal abnormalities and it is subject to limitations including the need for extended cell culture, susceptibility to contamination with maternal cells, poor microscopic resolution, and high rates of failure.
Molecular genetic techniques have recently emerged as strategies that can overcome the limitations of chromosomal karyotyping in the clinic. Low-depth high-throughput whole-genome CNV-seq strategies can effectively detect chromosomal structural abnormalities, including microdeletions and microduplications under 100 kb in size. CNV-seq is also inexpensive, easy to implement, and rapid such that it is widely used for the chromosomal characterization of miscarriage products. Here, cases that had undergone CNV-seq analyses were retrospectively evaluated to characterize chromosomal abnormalities in cases of MA and to explore correlations between these abnormalities and patient clinical data.
3.1 Chromosomal Abnormality Distribution Frequencies
Over 50% of MAs result from chromosomal abnormalities, with previous studies estimating anywhere from 50-70% of MAs to be associated with these abnormalities [9-11]. Variations in these rates may be the result of gestational age, the detection techniques employed, and other variables. Here, 950 of the analyzed cases (63.76%) were found to harbor karyotypic abnormalities, with chromosomal aneuploidy being the most common finding [12].
Chromosomal aneuploidy is the most prevalent cause of MA or intrauterine fetal demise. These abnormalities entail the absence or presence of particular chromosomes owing to errors that arise during the first round of meiotic division giving rise to an egg cell [13]. Autosomal trisomy is the most common finding in cases of early MA, with trisomies 13, 16, 18, 21, and 22 being particularly frequent[14], followed by X monosomy and polyploidy [9,10]. For instance, one prior survey of 1,030 products of conception from MAs following single-embryo transfer detected aneuploidy in 80.6% of cases, of which 62.3% were trisomies, 7.8% were double trisomies, 0.5% were triploid or tetraploid, 1.3% exhibited 21-monosomy, 3.2% exhibited X-monosomy, 0.1% exhibited 47,XXY, 1.0% exhibited polyploidy, 1.0% presented with mixed abnormalities, 1.1% exhibited embryonic chimerism, and 2.4% exhibited structural abnormalities [15].
A separate cytogenetic analysis of embryos from 1,011 cases of early pregnancy loss revealed chromosomal abnormalities in 711 cases (70.3%) [16]. The most commonly reported abnormalities in these cases included autosomal trisomies (64.6%), triploidy (13.1%), and X monosomy (10.4%). In the present cohort, the most frequently detected abnormalities included trisomy 16 (16.85%), 45XO (10.84%), trisomy 22 (10.74%), trisomy 21 (4.42%), trisomy 15 (4.11%), and trisomy 13 (3.89%), in line with these past results. Triploidy/polyploidy was the second most frequent abnormality in this cohort (11.68%), including two cases of sex chromosome trisomy (one each of 47,XXX and 47,XXX with chimeric duplication and a chimerism ratio of 55%). X tetraploidy was also observed in one case (49,XXXXY). There were no instances of trisomy 19, while trisomy 1 was only detected in a single case of chimeric duplication (chimerism ratio: 19%). Chromosome 19 exhibits the highest gene density in the human genome, whereas the greatest number of genes is encoded on chromosome 1. Trisomy 19 or 1 may thus be lethal at an early stage of embryogenesis.
3.2 Chromosomal Abnormalities and Gestational Age
There have been multiple past reports indicating that the detection rates and types of cytogenetic abnormalities in cases of early MA before 12 weeks gestation vary markedly as a function of embryo/fetus size, emphasizing the important roles that particular chromosomal factors play during the early phases of embryonic/fetal development[17]. The yolk sac precedes pregnancy, and its absence is indicative of a blighted ovum, most often as a consequence of poor embryonic development and premature demise. The embryo develops early during fetal development, with the pulsation of the primitive heart tube being the first indicator of fetal cardiac development. The yolk sac tends to appear around week 5 of pregnancy, with the embryo and fetal heart first manifesting at approximately weeks 6-7. The frequencies of embryo and fetal heart rate detection have been reported to be higher in cases of viable trisomies, X monosomy, and triploid miscarriages as compared to cases of karyotypic normality or miscarriages with other karyotypic abnormalities [18]. A CRL of less than 15 mm is most commonly associated with other forms of trisomy or structural trisomy in 57.7% and 11.5% of cases, respectively, whereas a CRL ≥ 15 mm is instead associated with instances of monosomy (38.7%), triploidy (29%), and viable trisomy (12.9%)[19]. Postmortem embryo pole length also reportedly varies significantly across karyotype groups, with the longest lengths in cases of trisomy 21, X monosomy, and triploidy at 16, 15.3, and 11.6 mm, respectively [20].
Here, the lowest rate of chromosomal abnormality detection was evident in the group in which the yolk sac was empty, whereas this rate rose significantly after yolk sac detection. Trisomies were most often detected in the yolk sac and embryo groups, whereas their detection rates were comparatively lower in the groups exhibiting an empty sac or cardiac pulsation. The 45XO detection rate was highest in the group with cardiac pulsation, supporting the differing developmental potentials of embryos harboring particular chromosomal abnormalities, with trisomy being related to embryogenesis and fetal heart development[15]. Certain forms of trisomy, including trisomy 16, are potentially associated with early embryonic development[20], whereas 45XO appears to have no effect on the early stages of embryo growth.
Nobuaki et al[17] observed no variations in triploidy or chromosomal structural abnormality frequencies as a function of gestational age. Igher embryonic development rates have been reported for cases of triploidy, X monosomy, and trisomy 21 as compared to other karyotypic findings [19]. Segawa et al[15]posited that double trisomy and triploidy events are significantly more common when no fetal heartbeat is detected, and that higher karyotypic abnormality rates are related to blighted ovum[21]. In the present study, the distributions of double/multiple trisomies, triploidy, and CNVs varied as a function of gestational age. The highest rate of double/multiple trisomy detection was evident in the yolk sac group, whereas triploidy was most commonly detected in the empty sac and cardiac pulsation groups. CNVs were more common in the empty sac group, suggesting that while triploidy/multiploidy are more likely to result in very early or later miscarriage, CNVs are linked to higher odds of very early miscarriage. The proportion of karyotypic abnormalities in the very early MA group with an empty yolk sac was relatively low, whereas the abnormality detection rate rose after the yolk sac was evident. This supports the important role of non-genetic factors including infections, immune activity, clotting, and other environmental factors in very early pregnancy failure [12].
3.3 Chromosomal Abnormalities as a Function of Maternal Age and Ovarian Reserve
Primary oocytes develop from primordial germ cells at ~3 months of fetal development and enter into dormancy for decades after undergoing meiosis I and genetic recombination, only resuming meiosis I and entering metaphase II prior to ovulation. Errors that impact chromosomal segregation can occur at each stage of meiosis and become more common as maternal age rises [22]. Chromosomal aneuploidies in human gametes and pre-implantation embryos are a common cause of pregnancy failure or infertility. Aneuploidy impacts an estimated 20% of human oocytes, and this number rises exponentially from ages 30-35, reaching 80% as of age 42 [7]. Indeed, maternal age is firmly established as a factor that is closely associated with chromosomal aneuploidy [23].
Here, patients were grouped according to maternal age in 5-year intervals, revealing that rates of chromosomal abnormalities were lowest among women ages 25-34 (62.17%, 60.81%), whereas these rates rose with age to a peak of 80.23% for women ≥40 years of age. The chromosomal abnormality detection rate began increasing significantly from age 30 to 34, raising the question of whether ovary aging begins to manifest at 30. However, an analysis of the types of chromosomal abnormalities associated with these different age groups revealed that trisomy detection rates began rising from age 35-40 (74.85%) to even higher levels after age 40 (85.51%). In contrast, 45XO detection rates peaked in cases where the mother was < 25 years old (22.97%), gradually declining with age to 10.24% at 30-34 and further declining in the 35-40 age group. Women 35+ years of age thus face higher odds of trisomy[12,24], whereas 45XO is more common under the age of 35. Ozawa et al[17] also previously found that chromosomal abnormality rates for trophoblasts were higher for older (≥35 years) as compared to younger (< 35 years) women (70.59% vs. 51.48%), with autosomal trisomy rates rising with age, particularly in the case of trisomy 15 and 21. Rates of 45XO and CNV detection also declined with maternal age, while triploidy/multiploidy was more commonly detected among patients <34 years, declining sharply at ages above 35. Double/multiple trisomy rates also rose with age, thus supporting a model in which CNVs, sex chromosome abnormalities, and triploidy/multiploidy are more likely to impact women at a younger age, whereas double/multiple trisomies have greater odds of manifesting in women ≥35 years of age [12,17]. These differences may be attributable to the dynamics of chromosomal recombination, which tends to decline with age, resulting in a reduction in chromosomal segregation [25].
While maternal age is known to be closely associated with recombination rate, the corresponding effects of paternal age are less clear. This distinction is related to the sex-specific meiotic duration, timing, and outcomes [26]. Triploidy is a consequence of improper ploidy at the time of fertilization, which can arise from fertilization by two sperm or two eggs [27]. Sex chromosome monosomies are generally the result of the loss of the paternal X or Y chromosome, and they also present with a reverse age effect. One study previously demonstrated that the X chromosome source is related to maternal age, such that the average maternal age in the 45,XP group with a single X chromosome from the paternal side was significantly lower than that in the 45,Xm group with a single X chromosome from the maternal side[28].
Despite the strong evidence that advanced maternal age is related to the occurrence of trisomies and other chromosomal abnormalities and its status as an independent risk factor associated with miscarriage, the mechanistic basis for this relationship remains unclear. Ovarian reserve and function trend downward with age. To determine whether these changes may be associated with embryonic chromosomal abnormalities, correlations with ovarian reserve were thus examined at length. Serum AMH levels are positively correlated with ovarian reactivity in women of various ages[29], and both age and levels of AMH are related to miscarriage risk[30]. Clinical pregnancy rates and live birth rates for women with higher AMH levels tend to be lower, with a corresponding rise in the rate of miscarriage [31]. AMH levels were thus leveraged as an index for ovarian reserve function in this study [32]. When specifically focusing on patients who had undergone AMH testing within the past year, a decrease in ovarian reserve function was found to be associated with a gradual rise in the rate of chromosomal abnormality detection. Given the independent association between age and chromosomal abnormalities, age-stratified analyses were performed, revealing that the chromosomal abnormality detection rate continued to rise with declining ovarian reserve function among both younger and older women. CNV, 45XO, and triploidy/multiploidy detection became less frequent with declining ovarian reserve, whereas the opposite was true for trisomy detection. These trends aligned well with age-related trends, further supporting a link between reduced ovarian reserve function and the incidence of chromosomal abnormalities. Shahine et al. [33] found that relative to patients with normal ovarian reserve function who experienced unexplained recurrent spontaneous abortion (RSA), those with declining ovarian reserve function who experienced RSA exhibited higher rates of embryonic aneuploidy. In this study, A higher proportion of aneuploid chromosomes was observed among women exhibiting declining ovarian reserve function (57% vs. 49%), with this difference having been most pronounced among women < 38 years of age (67% vs. 53%). The present study aligns well with these prior results. Limitations of these analyses include the smaller sample size, the fact that ovarian reserve function was only assessed using a single index, and the absence of any corresponding analyses of paternal age, however. Shahine[33] suggested that, compared to unexplained recurrent spontaneous abortion (RSA) patients with normal ovarian reserve function, unexplained RSA patients with declining ovarian reserve function have a higher incidence of aneuploid chromosomal abnormalities in embryos. Women with declining ovarian reserve function have a higher proportion of aneuploid chromosomes (57% vs. 49%), and this difference is more significant in patients under 38 years of age (67% vs. 53%). Our study aligns with the findings of Shahine's research. The limitations of this study include a small sample size, the need for further analysis with a larger sample, the use of a single indicator for assessing ovarian reserve function, and the lack of analysis of paternal age.
3.4 The Relationship Between Chromosomal Abnormalities and ART
How ART relates to the incidence of embryonic chromosomal abnormalities has been a matter of some controversy. In some reports, ART was found to have no effect on such abnormalities [34-35], although others have described higher rates of chromosomal structural abnormalities for patients undergoing ART (13.2% vs. 4.2%, P < 0.05)[10]. With respect to the types of embryo transfer protocols employed, chromosomal abnormality rates are reportedly lower in cases of frozen embryo or frozen blastocyst transfer [36]. Deletions and microdeletions are reportedly more common in cases of intracytoplasmic sperm injection (ICSI) as compared to in vitro fertilization and embryo transfer(IVF-ET) protocols. The utilization of frozen embryos has been suggested to help protect developing embryos from the adverse effects of exposure to an estrogen-rich environment[10]. Some studies, however, have argued that ART can contribute to a greater risk of embryo chromosomal abnormalities and miscarriage [37]. While the general types of chromosomal abnormalities in cases of ART may be similar to those for natural conception[9], particular ART techniques also have the potential to preferentially give rise to certain abnormalities. ICSI, for instance, is linked to a greater risk of non-disjunction abnormalities, higher X monosomy rates [38], and lower polyploidy rates [39]. Kim et al.[40] reported that aside from a higher rate of sex chromosome abnormalities among patients undergoing ICSI in cases of male factor infertility, ART was not linked to any greater odds of chromosomal abnormalities. The differences that do exist also have the potential to be a consequence of parental factors, rather than any risks unique to the procedure of ICSI itself.
Here, a higher rate of trisomy detection was observed in the ART group, whereas CNV and triploidy/multiploidy detection rates were higher in the NC group. These trends remained intact when stratified according to age, supporting a link between ART and changes in the incidence of trisomies. ART has been reported to increase the risk of chromosomal numerical abnormalities, particularly in cases of trisomies[9]. One limitation of these analyses is that no effort was made to distinguish between different ART protocols when assessing chromosomal abnormality rates, underscoring a need for further studies aimed at clarifying the relationships between ART and these outcomes in order to guide the development of more optimal ART strategies.
3.5 The Link Between Prior Natural Miscarriages and Chromosomal Abnormalities
Recurrent spontaneous abortion(RSA) is a term used to refer to instances of two or more miscarriages. More than half of women experience at least one miscarriage in their lifetime, and this proportion rises among women 35+ years of age, but < 1% of women experience three consecutive natural miscarriages [41]. These recurrent miscarriages can be physically and emotionally taxing for couples such that efforts to improve pregnancy outcomes for RSA patients are desperately needed. Efforts to clarify the etiological basis for RSA are vital for the appropriate evaluation and targeted treatment of pregnancies for couples with a history of RSA.
Embryonic chromosomal abnormalities are in 64.8% of all cases of spontaneous abortion, whereas these abnormalities are only evident in 3.9% of unnaturally aborted embryos [9]. An estimated 76.2% of patients first found to exhibit embryonic chromosomal abnormalities have subsequent embryos with abnormal chromosomes [42]. A history of prior miscarriages may be linked to greater odds of embryonic chromosomal abnormalities [9], owing either to abnormalities in the sperm or eggs themselves or to the effects of external environmental factors during the early stages of pregnancy [43-44]. As these same factors are also relevant across multiple pregnancies, any previous diagnoses of embryo karyotypic abnormalities are considered indicative of a greater risk of subsequent miscarriage [45]. Embryogenetic testing is thus often recommended in cases of retained miscarriages in order to provide more information for future pregnancies. Some studies, however, contradict this and indicate that a history of prior miscarriage is not linked to greater odds of detecting embryonic chromosomal abnormalities. One meta-analysis of 55 studies published since 2000 reported no differences in embryonic chromosomal abnormality rates when comparing sporadic and recurrent miscarriages (46%, 95% CI: 39-53 vs. 46%, 95% CI: 39-52) [46]. However, significantly higher rates of sex chromosome and structural abnormalities are evident in cases of sporadic miscarriage relative to recurrent miscarriage[10, 12]. As the number of miscarriages increases, some studies have suggested that the odds of chromosomal abnormalities decrease such that they are detected less frequently in cases of RSA [47]. Indeed, women with a history of ≥ 2 miscarriages exhibit significantly lower rates of chromosomal abnormalities relative to those with a history of < 2 miscarriages, and viable trisomy seemed to be the main contributor to this trend[17]. Here, no significant increase in the frequency of embryonic chromosomal abnormalities was detected as a function of the number of prior miscarriages, nor were there any significant differences in abnormal chromosomal distributions across these groups. Miscarriages typically occur in cases where both parents have normal chromosomes. However, in instances where both spouses exhibit chromosomal abnormalities as in the case of balanced translocations and Robertsonian translocations, carriers often appear phenotypically normal and have the potential for reproduction. As chromosomal imbalance affects 50-70% of the gametes and embryos in these cases, this contributes to a greater risk of embryonic chromosomal abnormalities and miscarriages. Couples who suffer a MA in China often seek out peripheral blood chromosomal testing at the hospital. When this leads to the detection of chromosomal abnormalities, some opt for preimplantation genetic testing (PGT), which can provide an avenue to obtain karyotypically normal healthy embryos prior to implantation, contributing to better pregnancy outcomes.