Despite years of research, miscarriage, particularly when recurrent, continues to pose a medical challenge. An embryo chromosomal error is responsible for 50–60% of recurrent cases; however, up to 30–50% remains an enigma. Successful pregnancy involves different maternal physiologic changes and certain complex interactions between the fetus and the mother by cytokines, angiogenic mediators and hormones. To date, research lines have focused on genetic and epigenetic polymorphisms related mainly to immune response and inflammatory mediators, and have yielded a significant relationship between recurrent miscarriage and immune mechanisms. Thus, unknown causes of miscarriage could be due to an immune imbalance induced by T-helper Th1/Th2/Th17 cytokines and regulatory T cells. Furthermore, these genes and mediators have long been suspected of being blood markers for the clinical diagnosis and management of miscarriage; however, more evidence is required for them to be included in medical practice and obstetric guidelines.
Highlights
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Although fetal chromosomal errors is the major cause of early single miscarriage, their relationship with recurrent miscarriage is less common. Most of these abnormalities are numerical modifications that arise de novo. The higher the number of miscarriages, the lower the probability of their being related to chromosomal abnormalities will be.
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In 2–4% of couples with recurrent miscarriages, one partner has a structural chromosome abnormality, the most common of which is a balanced translocation. The prognosis for a subsequent live birth is more dependent on factors such as previous obstetric outcomes.
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Genetic immune polymorphisms involving immune-regulation pathways are gaining importance in recurrent miscarriage, and could provoke an immune imbalance towards Th1/Th2/Th17 profile that can eventually modulate T regulatory cell function.
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Some genetic polymorphisms related to coagulation or fibrinolysis are mainly associated with late recurrent miscarriage.
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No agreement exists on the systematic karyotyping of parents, since it is expensive and does not always yield useful information. Neither is there conclusive evidence to support prenatal genetic screening for recurrent miscarriage or the use of molecular techniques to identify fetal chromosomal abnormalities.
Introduction
Miscarriage also referred to in the literature as pregnancy loss or abortion , and the term to be used herein, is the most common complication of pregnancy. It poses a clinically-frustrating and emotionally-charged challenge for patients and providers.
Miscarriage may be single (SM) or recurrent (RM). Based on the idea that separate entities could represent different pathophysiologic mechanisms leading to recurrent miscarriage, several studies have classified it according to maternal reproductive history as: primary (no pregnancies to term), secondary (series of miscarriages after a live birth) and tertiary (three non-consecutive miscarriages) .
Miscarriage occurs in approximately 10 to 15% of pregnancies (4 to 5-fold higher if biochemical pregnancies are included), and is defined as the spontaneous loss of a clinically-established intra-uterine pregnancy before the fetus reaches viability, i.e. up to a maximum of 24 weeks of gestation .
RM, historically defined as three or more consecutive losses before 20 weeks of gestation and affecting 1 to 3% of pregnancies is currently considered by several researchers and clinicians to represented two or more losses ; this new definition raises the rate to 5% of sexual active couples trying to conceive .
Reproductive history is an independent predictor of future pregnancy outcome . Accumulating evidence has suggested that the risk of a further miscarriage increases after each successive pregnancy loss, reaching 45% after three consecutive losses . In addition, a previous live birth does not preclude a woman developing RM .
Genetic factors are the main cause of early miscarriage. Epidemiologically, the most common cause described is fetal chromosomal abnormalities , particularly numerical alterations, with the majority occurring de novo rather than being inherited . Risk increases with advanced maternal age, which is also an independent risk factor for both single and recurrent miscarriages . Other known risk factors for RM include non controlled endocrine disorders, luteal phase defects, infectious diseases, environmental factors, uterine diseases or thrombophilic disorders, particularly antiphospholipid syndrome.
Routine evaluation of a couple with RM determines a cause in less than 50% of cases , thus reflecting the heterogeneous nature of the condition. The aim of this review was to assess and synthesize the current data on genetic factors involved in miscarriage, with emphasis on novel research lines. Embryo chromosomal abnormalities, parental karyotype alterations, sperm alterations and gene polymorphisms will be discussed below. Moreover, the role of different management tools in pregnancy loss according to the literature will be proposed.
Search strategy and selection criteria
Data for this review were obtained through a comprehensive search of the literature using the relevant keywords: “recurrent miscarriage, genetic alterations, chromosome abnormalities, genetic polymorphisms” to identify articles published in English from MEDLINE, PubMed and The Cochrane Library (January 2000- December 2016). Study selection was based on existing clinical evidence. The included articles were: randomized controlled studies or meta-analyses when possible followed by prospective, matched or non-matched case-control, cohort studies and reviews. The initial search provided more than 1000 articles. Some were found in duplicate. Finally, only 78 studies regarding the genetics of recurrent miscarriage were collected and discussed, 62 of which were included in accordance with the Editorial requirements of the Journal.
Search strategy and selection criteria
Data for this review were obtained through a comprehensive search of the literature using the relevant keywords: “recurrent miscarriage, genetic alterations, chromosome abnormalities, genetic polymorphisms” to identify articles published in English from MEDLINE, PubMed and The Cochrane Library (January 2000- December 2016). Study selection was based on existing clinical evidence. The included articles were: randomized controlled studies or meta-analyses when possible followed by prospective, matched or non-matched case-control, cohort studies and reviews. The initial search provided more than 1000 articles. Some were found in duplicate. Finally, only 78 studies regarding the genetics of recurrent miscarriage were collected and discussed, 62 of which were included in accordance with the Editorial requirements of the Journal.
Chromosomal alterations
Abnormal embryonic karyotypes
The most common known cause of miscarriage is fetal chromosomal error, which occurs mainly before 10 weeks of pregnancy, and concerns about 50–70% of couples ; however, villous biopsies have demonstrated that this percentage might even reach 83% . In contrast, very low rates are described in later losses: miscarriages between 12 and 22 weeks constitute approximately 4% of pregnancy outcomes, and less than 4% of these exhibit chromosomal errors . This event is more frequent in SM than in RM: approximately 70% of SM samples studied showed abnormalities compared with a range of 29–50% described for RM , depending on the maternal age and number of previous miscarriages. In contrast to these findings, a recent study by Chan Wei et al. of 832 miscarriage samples from Chinese women, found no statistically-significant differences in the prevalence of aneuploidy between recurrent and sporadic or single miscarriage.
Cytogenetic studies of miscarriage samples have shown that most of these abnormalities arise de novo in the first trimester of pregnancy. The majority are numerical modifications (86% in SM vs 90% in RM) , mainly autosomal trisomy, followed by polyploidy such a triploidy or tetraploidy, and monosomy X . A minority of cases are caused by structural chromosomal abnormalities (6% in SM vs 8% in RM), chromosome mosaicism and other alterations (8% in SM vs 13% in RM) . Single gene defects, such as those associated with cystic fibrosis or sickle cell anemia, are seldom associated with RM .
Autosomal trisomies are the result of maternal meiotic errors with some studies showing the most frequent chromosome involved to be 16 followed by 22, 21, 15, 18 and 2 . In a case-control study with 420 specimens, Stephenson et al. showed the most frequent trisomy to be 15 followed by 16, 22, 21, 14 and 13, attributing these differences to higher mean maternal age in their data set and the inclusion of cytogenetically-defined preclinical miscarriages (<6 weeks). By contrast, in pregnancy trimesters, while the commonest kind of trisomy in the first was 16 (known as “miscarriage chromosome”) ( Fig. 1 ), the most common in the second was 21 ( Fig. 2 ). Trisomy of chromosomes 19 and 1 were the rarest .
Polyploidy generally originate from fertilization by polyspermy or postzygotic division error and are not compatible with life . The most common triploid arrangement was 69, XXY (4%) followed by 69, XXX (2.7%) ( Fig. 3 ).
Mitotic error may result in at least two cell lines (mosaicism) in the developing fetus . The degree of mosaicism depends on the timing of the error e.g if the error occurs very early in the zygote, the percentages of each cell line may be equal .
All that abnormalities, particularly trisomies but rarely monosomy X, are strongly associated with parental age. Maternal age is widely known to be an independent risk factor for miscarriages. Current data support this theory showing losses in up to 10% of women 20–24 years of age, 51% in those 40–44 and a loss risk of 75% in women 45 or older . However, the mechanisms involved have not been determined to date. Historically, one accepted hypothesis was the presence of diminished ovarian reserve in these women. A prospective cohort study recently conducted by Shahine et al. analyzing 239 women undergoing in vitro fertilization showed that those with diminished ovarian reserve had a higher percentage of aneuploid blastocysts; however, in contrast to what was expected, more significant differences were found in patients <38 years (67% vs. 53%).
On the other hand, the role of paternal age in the pathogenesis of miscarriage continues to be an unexplored area and little research has been carried out in this field.
Chan Wei et al. reported that paternal age could be involved in fetal aneuploidy, finding an association up to age of 40, after which this rate decreased. However, no other studies had found statistically-significant differences, thereby showing that more evidence is needed to confirm that association. Thus, the weight of that factor remains unclear.
Many reports have suggested that the abnormal embryonic karyotype predicts subsequent live birth . Ogasawara et al. described a cumulative live birth rate in women with miscarriage caused by an abnormal embryonic karyotype higher than that in women with recurrent miscarriage of truly unexplained cause (71.2% vs 52.5%). Moreover, embryonic karyotype abnormalities, whatever it were, tend to repeat in subsequent miscarriages and it is also a predictor of good prognosis for that partner, noticed for the comparison with live birth rate in both groups ; On the other hand, interestingly, it seems that the higher the number of miscarriages, the less probable it is that they are related to chromosomal abnormalities . Thus, patients with RM due to the abnormal embryonic karyotype might have gene mutations associated with aneuploidy which explain their poor outcomes and the increased number of miscarriages. For example, recent findings have shown that mutations in SYCP3, a gene encoding as essential component of the synaptonemal complex, could contribute to abnormal chromosomal behavior leading to RM .
Chromosome abnormalities in either partner
The majority of miscarriages occur in chromosomally-normal parents. One partner has a structural chromosome abnormality in only 2–4% of couples with RM . In addition, the prevalence of chromosomal aberrations appears to be independent of the number of previous miscarriages.
Various alteration types have been described: reciprocal translocation (balanced or unbalanced) caused by rearrangement of terminal parts from different chromosomes; centric fusion of two acrocentric chromosomes, known as Robertsonian translocation, being the most frequent t(13;14) and t(14;21) ; or inversion, in which a chromosome segment is reversed end to end. The most commonly reported human inversion is (p12;q13) .
Carp et al. , comparing chromosome abnormalities in a retrospective comparative cohort study, found that the most frequent chromosomal rearrangement to be balanced translocations (52%), followed by inversions (26%), with a significantly greater prevalence in the male partner. Mosaics were found at a frequency of 21%, with a significantly greater prevalence in the female partner.
Although carriers of balanced alterations are usually phenotypically normal and have a demonstrated possibility of giving birth, up to 50–70% of their gametes and hence embryos are unbalanced owing to of errors that occurred during segregation at meiosis . Thus, their pregnancies are at increased risk of miscarriage and may result in a live birth with multiple congenital malformations and/or mental disability secondary to an unbalanced chromosomal arrangement . The reproductive risk seems to depend on the type of rearrangement, the size and genetic content of the rearranged chromosomal segments, and the mode of ascertainment. However, there was no significant distinctive effect if the aberration was maternally- or paternally-derived .
Ogasawara et al. , who analyzed the karyotypes of 1284 couples, suggested that the presence of parental karyotype aberrations defines a group of high risk for miscarriages, since between 61% and 72% of their patients had subsequent miscarriages.
However, the studies by Goddijn et al. and a case-control study by Franssen et al. showed that the proportion of couples giving birth to one or more healthy newborns was similar in the various types of abnormalities, with no significant differences in birth rates for any kind of alterations. In the Carp et al. study outcomes were 83% for reciprocal translocations, 82% for Robertsonian translocations, 78% for inversions and 93% for other abnormalities. Furthermore, analyzing the reproductive outcome after chromosome analysis in couples with RM, they reported that couples whose carrier status was ascertained after two or more losses had a low risk of viable offspring with unbalanced chromosomal abnormalities, and their chances of having a healthy child were as good as non-carrier couples, despite a higher risk of miscarriage .
It seems more likely that the prognosis for a subsequent live birth depends more on factors such as the number of previous miscarriages, maternal age, karyotype of the previous miscarriage, and primary or secondary aborted status rather than parental karyotype .
Sperm implication in chromosomal abnormalities
As mentioned previously, the majority of chromosome abnormalities arise de novo from random errors produced essentially during three development stages: gametogenesis, fertilization and embryonic development . The current knowledge shows that those alterations are mostly derived from non-disjunction errors during the first meiotic division of the oocyte, with a significant association with advanced maternal age.
On the other hand, the male role in those errors remain uncertain, and sperm studies have demonstrated that paternal meiotic errors also occur and have the potential to cause abnormal fertilization. Sperm quality has been associated with the embryo’s ability to reach the blastocyst stage and progress to implantation . A meta-analysis conducted in 2012 showed a significant increase of miscarriage in patients with major DNA damage compared to those with minor DNA damage . Although no significant changes in sperm morphology, mean count and motility had been found in the past, a recent review reported a chromosomal abnormality frequency of 15.2% in men with azoospermia and in 2.3% of nonazoospermic men .
After analyzing 12 couples with RM, Rubio et al. reported a significant increase in disomy frequency for sex chromosomes and diploidy rates in sperm samples from RM couples compared to those from internal controls (0.84% vs 0.37%). This was confirmed by Carrell et al. (2.77% vs 1.19%), indicating that this may be an important etiologic factor in RM. Agarwal et al. suggested that microdeletions of the azoospermia factor located in the long arm of chromosome Y, and which are essential for normal spermatogenesis, may have a direct effect on the early prophase of mitosis decreasing the normal pairing rate in the pachytene stage of spermatocytes. This pairing failure may increase chromosomal abnormalities and could be related to RM.
In addition, a case-control study based in sperm samples of 11 partners affected with unexplained RM, revealed a statistically-significant increase in meiotic errors involving chromosome 16 which contributed to increased sperm disomy in more than 60% of their patients . Thus, these data suggest that among paternal meiotic errors the non-disjunction of chromosome 16 might exert similar relative influence on fetal aneuploidy compared with maternal chromosome 16 disomy .
The association of sperm quality with recurrent pregnancy loss emphasizes the importance of evaluating the male factor. Several different tests are available, but no consensus has yet been reached as to which tests are more predictive .
Genetic disorders
Certain genetic mutations, such as autosomal-dominant disorders leading to myotonic dystrophy, may predispose patients to infertility or even RM . Other presumed autosomal-dominant disorders associated with RM include lethal skeletal dysplasia, connective tissue disorders such as Marfan syndrome, Ehler-Danlos or Pseudoxanthoma elasticum, or hematologic abnormalities including fibrinogen alterations, factor XIII deficiency or sickle cell anemia .
Since 1990s, many genetic polymorphisms have been proposed as being implicated in pathogenesis of RM. It has been suggested that biologic processes for maintaining the pregnancy stability are mediated by the expression of a series of different genes related to inflammatory mediators, placental function regulators, thrombogenic factors and sex hormone receptors . For this reason, research lines had focused on principally seeking these related candidate genes. However, the results of these studies were usually inconsistent, especially when they were conducted in different populations.
In 2004, Baek at al described 30 genes showing different levels of expression between normal and RM patients which were involved in immunity, angiogenesis and apoptosis pathways. Later, other research groups also identified a large number of genes that are expressed aberrantly in pregnancy failure . Xiaonhan et al. recently published a large meta-analysis in which 53 polymorphisms of 37 genes were shown to be associated with RM. However, Pereza et al. reported in their meta-analysis that the results of all associations were modest, and the pathophysiologic mechanisms of how specific genetic variants might contribute to RM remain largely unexplored.
Of all RM etiologic factors, scientists are currently more interested in the field of reproductive immunology . T lymphocyte plays a central role in cell-mediated immunity and two main subsets of them are described depending on the presence of cell surface molecules: CD4 and CD8. T lymphocytes expressing CD4, also known as helper T cells (Th) can be further subdivided into Th1 (pro-inflammatory response) and Th2 (anti-inflammatory response). Since a close relationship between RM and immune mechanisms was observed in some studies, suggesting that the unknown causes of miscarriage could be explained by immune imbalance induced by Th1/Th2 cytokines towards mounting Th1 response .
Immune response ( Table 1 )
Pro-inflammatory cytokines are known to exert an adverse effect on pregnancy; many of them, which are mostly regulated by Th1/Th2 balance, have been reported to be associated with RM . During pregnancy, the normally-dominant Th-1 inflammatory immune response switches to a Th2 cytokine profile, thereby permitting induction of maternal immune tolerance in the allogenic fetus and assuring successful implantation and reproductive outcomes. If the Th1 response prevails beyond Th2 as a result of an immune imbalance induced by cytokines, the risk of RM is increased.
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