3.3.1. B Cell-Related Genes

One study [Reference Danaii, Ghorbani and Ahmadi40] showed a significant decrease in mRNA expression of B-cell-associated factors IL-10 and PD-L1 and increased expression of genes BLIMP1, IRF4 and XBP-1 in patients with RPL. An abnormal increase in PD-1/PD-L1 is detrimental to pregnancy and increases maternal immune rejection, leading to miscarriage [Reference Li, Chen, Lai, He and He41]. The result [Reference Esteve-Solé, Luo and Vlagea42, Reference Liang, Diao and Huang43] of one study showed that the levels of IL-10-synthesizing B cells in the stimulated total B cell population isolated from the peripheral blood of RPL patients were markedly lower compared to those of normal pregnant women, unraveling that a decrease in the number of these cells may contribute to RPL. The decrease in the peripheral blood IL-10-synthesizing B cells may prompt RPL pathogenesis [Reference Guzman-Genuino and Diener44].

3.3.2. NK Cell-Related Genes

Natural killer cells (NKs) are the most pivotal cells in fetal-maternal immune tolerance induced by the interaction of maternal killer cell immunoglobulin-like receptors (KIR) with fetal leukocyte antigens (HLA). IL-10 may negatively regulate the cytotoxicity of uterine NK (uNK) cells affecting pregnancy [Reference Bonifacino, Dasso, Harford, Lippincott-Schwartz and Yamada45]. In RPL women, elevated levels of NK cells and increased NK cytotoxicity are relative to an increased T helper 1 immune response. It has been shown that the suppressor gene KIR3DL1 is a protective factor and the activator genes KIR2DS2 and KIR2DS3 are risk factors for RPL [Reference Akbari, Shahsavar, Karami, Yari, Anbari and Ahmadi46].

NK cells are related to the decidual immune microenvironment, where the meconium immune cells at the maternal-fetal interface are predominantly composed of NK cells, macrophages, T cells, and a few other cell types (e.g., dendritic cells, NKT cells, etc.) [Reference Guo, Cai and Jin47]. It is suggested that abnormalities in the metaplastic immune microenvironment may be involved in the pathogenesis of RPL [Reference Triggianese, Perricone, Chimenti, De Carolis and Perricone48].

NK cells are also pertinent to TLR3, a type I transmembrane protein consisting of 904 amino acids and composed of four parts, namely, an extracellular region containing 23 LRRs, N- and C-terminal cysteine-rich flanking regions, a transmembrane region, and a cytoplasmic tail region containing TIR. TLR3 recognizes “non-self” origin of nucleotide derivatives [Reference Liu, Botos and Wang49]. TLR3 activates NK cells, which participate in the maintenance of pregnancy tolerance by regulating fertilized egg implantation and uterine vascular alterations, probably through the association with poly (I-C), but excessive NK cell activity may lead to embryonic resorption and thus induce abortion [Reference Lin, Zeng, Zeng and Wang50].

3.3.3. HLA-Related Genes

The embryo derives half of its genetic inheritance from the father and develops in the uterine environment, similar to a hemizygote. Thus, the fetus may be rejected by the maternal immune system, and one of the most essential immune factors is HLA-G. HLA-G is a nonclassical HLA class I antigen highly expressed on embryonic trophoblast cells in the meconium [Reference Jin51].

HLA expression in trophoblast cells has been shown to play an important role in maternal-fetal interface immune tolerance, with specific KIR in women with RPL and HLA ligands in couples causing susceptibility to RPL. One study found a prevalence of HLA-DQ2/DQ8 haplotype positivity in 51.58% of the women with RPL included in their trial, which is 1.5–2 times higher than the general population, which is in the range of 25%–40%, resulting in a higher prevalence of HLA-DQ2/DQ8 polymorphism and poorer pregnancy outcomes [Reference Feng, Liu and Wang52]. A report exploring the relationship between KIR2DL2 and its cognate ligand HLA-C1, found that a decrease in inhibitory KIR (inhKIR) ligands may be responsible for insufficient trophoblast inhibition by maternal uterine NK cells, resulting in RPL pathogenesis. Specific KIR and HLA-C genotyping may also be used to predict reproductive outcomes in women with RPL [Reference Yang, Yang and Wang53].

3.3.4. Genetic Polymorphisms in Interleukin Genes

Many interleukin cytokines play a role in human conception [Reference Jin51]. Variations in genes alter the corresponding protein expression levels. SNPs in promoters are suspected to affect transcription factor binding, which may affect interleukin production and therefore be associated with RPL [Reference Barbaux, Poirier and Godefroy54]. IL-1β (−511C/T) polymorphism leads to an increase in IL-1β production and the proportion of NK cells in the lymphocyte population [Reference Hefler, Tempfer and Bashford55, Reference Kim, Lee and Lee56], producing a pro-inflammatory effect, which is elevated in women with RPL. IL-6 plays a role in trophoblast function [Reference Galazios, Papazoglou, Giagloglou, Vassaras, Maltezos and Anastasiadis57], and IL-6 (−634) promoter mutations directly reduce IL-6 transcription and expression, and this nucleotide alteration also provides a potential for NF-1 transcription factor binding sites [Reference Lee, Na and Kim58]. Variants in the IL-18 promoter region affect IL-18 transcription and translation, and IL-18 protein expression is lower in patients with RPL [Reference Wilson, Jenkins and Miller59]. Interleukins and the corresponding immune cells work cooperatively to maintain the immune homeostasis of the mother and fetus; an imbalance of interleukin cytokines may lead to miscarriage [Reference Erlebacher60]. The relationship between some interleukin gene polymorphisms and RPL is consistent in studies, such as IL-1β (−511C/T), IL-6 (−634C/G), IL-10 (−1082G/A, −819T/C), IL-18 (−137G/C) and IL-18 (−105G/A) [Reference Zhang, Xu and Bao61]. However, in a small number of papers, interleukin genes have been linked to RPL, which may be influenced by factors such as race.

3.5.1. Metalloproteinase Gene Polymorphisms

The regulation of matrix metalloproteinase proteins (MMPs) during embryo and placental implantation is pivotal for a successful pregnancy. In humans, 23 MMPs have been identified. MMPs are calcium-dependent zinc-containing endopeptidases that mediate ECM degradation, tissue remodeling, shedding of cell surface receptors, and processing of various signaling molecules [Reference Page-McCaw, Ewald and Werb73]. A meta-analysis by Yan [Reference Yan, Fang and Li74] showed that the MMP2 −735T allele and the MMP9 −1562T allele were closely integrated with the risk of RPL.

3.5.2. ATP 6V1G3 Gene

The ATP 6V1G3 protein was predominantly expressed in the cytoplasm and stained brown. In the study of Chen [Reference Chen and Hu75], high expression of ATP 6V1G3 protein was found in placental villi and metaphase tissues, respectively. High expression of ATP 6V1G3 protein in women with RPL. However, its molecular mechanism in the development of RPL remains unclear.

3.5.3. Genetic Polymorphisms of Cytoplasmic GST Genes

Oxidative stress (OS) [Reference Peter Stein, Scholl and Schluter76] refers to the state of oxidative and antioxidant imbalance in the body. An essential prerequisite for normal metabolism, growth and development, is the provision of adequate oxygen during the embryonic, fetal and postnatal periods. The production of ROS due to hypoxia or hyperoxia, inflammation, or infection causes oxidative stress and changes in cell structure and function [Reference Torres-Cuevas, Parra-Llorca and Sánchez-Illana77]. Defects in the maternal detoxification system may lead to RPL because the embryo is more exposed to exogenous and endogenous compounds. Many studies have shown that genetic polymorphisms in the cytoplasmic GST gene are associated with the risk of RPL [Reference Parveen, Faridi, Das, Tripathi and Agrawal78–Reference Zusterzeel, Nelen, Roelofs, Peters, Blom and Steegers82]. It has been proposed that a genetic variant of the GSTA1 gene, the GSTA1-69C/T polymorphism (rs3957357), is significantly associated with the risk of RPL in Italian women with RPL [Reference Polimanti, Piacentini and Lazzarin83]. However, some studies have also reported that the GSTA1-69C/T polymorphism is not significantly associated with the development of RPL in the Chinese Han Chinese population [Reference Zong, Sha and Xiang84]. Therefore, the relationship between GST gene polymorphisms and RPL may also be related to the ethnic. In addition, sperm DNA is susceptible to oxidative damage, and increased sperm DNA fragmentation (SDF) may also lead to abnormal embryonic development [Reference Jayasena, Radia and Figueiredo85].

3.5.4. Genetic Variation in the CLOCK Gene

There is growing evidence that circadian rhythms affect a large number of physiological systems, including reproduction [Reference Boden and Kennaway86, Reference Sukumaran, Almon, DuBois and Jusko87]. Recent animal evidence unravels that disruption of synchronized clock activity relates to the pathogenesis of pregnancy complications. Repeated shifts in the light-dark cycle disrupt endogenous circadian rhythms and dramatically decrease the success rate of pregnancy in mice [Reference Summa, Vitaterna and Turek88]. In addition, impaired reproductive capacity in humans has been closely linked with night work [Reference Gamble, Resuehr and Johnson89]. In humans, night shift workers have been shown to have increased rates of reproductive abnormalities and adverse pregnancy outcomes in terms of miscarriage, low birth weight and preterm birth [Reference Knutsson90]. Genetic variants in the circadian genes ARNTL and NPAS2 are thought to contribute to fertility, with genetic variants in the ARNTL gene being closely related to a higher number of miscarriages and specific genotypes of the Npas2 gene being associated with a reduced number of miscarriages [Reference Kovanen, Saarikoski, Aromaa, Lönnqvist and Partonen91]. Genetic variants in the circadian genes ARNTL2, CRY2, DEC1, PER3 and RORA have also been conjoined with an increased risk of premature placental abruption [Reference Qiu, Gelaye and Denis92]. Additionally, it has been proved that low levels of CLOCK expression in pregnant women may lead to spontaneous abortion [Reference Li, Cheng and Wang93], and a study provided evidence that genetic variants in the CLOCK gene may be connected with IRSA [Reference Hodžić, Lavtar, Ristanović, Novaković, Dotlić and Peterlin94].

3.5.5. Mucin-Related Gene Polymorphisms

A recent study showed that MUC4 polymorphism correlates with RPL susceptibility in Korean women [Reference Kim, Park and Lee95]. In this study, MUC4 rs882605 C > A and MUC4 rs1104760 A > G were strongly associated with an increased risk of RPL in Korean women. Mucin is secreted by the epithelial cells of the reproductive tissues to produce mucus of the cervix and endometrium, which plays an important role in reproductive processes [Reference Koscinski, Viville and Porchet96]. Mucin 4 (MUC4) is the major mucin in the endometrial epithelium [Reference Koscinski, Viville and Porchet96]. A study has found that MUC4 promotes cell migration, alters the endometrial environment, and creates weak spots in the epithelium, thereby prompting the failure of embryo implantation [Reference Koscinski, Viville and Porchet96].

Thus, some genetic mutations and genetic polymorphisms are risk factors for RPL (Table 1), and it can be speculated that genetic mutations and genetic polymorphisms may occur in multiple concurrently, increasing the complexity of RPL etiology.

TABLE 1: Summarize the possible factors affecting RPL in gene mutations and gene polymorphisms.

4.1.1. Chromosome Number Abnormalities in Embryos

Numerical abnormalities of chromosomes are classified as aneuploidy (trisomy, haploidy) and polyploidy, and chromosomal aneuploidy abnormalities are the most common, accounting for 70%, of which 60% are trisomic [Reference Rai and Regan97], followed by polyploidy and haploidy, 16-trisomy (12%–19%), 22-trisomy (4%–10%), and X-haploidy (6%–10%) are the most common [Reference Eggenhuizen, Go, Koster, Baart and Galjaard98]. Genetic risk factors for embryonic aneuploidy include meiotic errors, mitotic errors, and abnormal parental chromosome structure. Trisomies are usually the result of chromosome non-separation in maternal meiosis and commonly involve chromosomes 13, 16, 18, 21 and 22. At the same time, autosomal haploids are less common in monosomal abortions and are mostly X-sex chromosomes that occur as a result of the loss of the couple’s X chromosome. Polyploidy, such as triploidy or tetraploidy, is usually caused by double spermatozoa or eggs that do not separate during maternal meiosis and are directly fertilized; tetraploidy may result from mitotic non-separation of the fertilized egg [Reference Yatsenko, Quesada-Candela and Saller99]. Maternal age was also found to be a primary risk factor for embryonic aneuploidy [Reference Saravelos and Li100]; the proportion of aneuploid embryos increased from 25–35% in women under 35 years of age to 55–85% in women aged 40–45 years [Reference Gruhn, Zielinska and Shukla101, Reference Capalbo, Hoffmann, Cimadomo, Maria Ubaldi and Rienzi102].

4.1.2. Embryonic Chromosomal Structure Abnormalities

Embryonic chromosomal abnormalities originate from two sources: first, chromosomal aberrations caused by internal and external factors during gamete formation or fertilized egg division; second, chromosomal abnormalities in either spouse that are inherited to the fetus, thus causing embryonic abortion or spontaneous miscarriage. Theoretically, embryos with unbalanced translocations cannot survive, while chromosomes with balanced translocations can survive with essentially preserved genetic material and no apparent abnormalities. However, most clinical studies have found that a few embryos with balanced translocation chromosomes can also miscarry, and other causes of miscarriage cannot be excluded [Reference Klimczak, Patel, Hotaling and Scott103].

4.2.1. Translocation

Reciprocal balanced translocation (RBT) is formed by a mechanism in which two chromosomes break simultaneously and the broken fragments are exchanged to form two derived chromosomes, generally without increasing or decreasing in genetic material. Thus, the individual usually has no phenotypic alterations. Reciprocal balancing translocations (RBT) can occur between homologous or non-homologous chromosomes. Still, balancing translocations between homologous chromosomes cannot produce gametes, so we will only discuss the case of balancing translocations arising between non-homologous chromosomes. (Figure 1(a)). It has been reported that 18 gametes can be produced during gamete formation, only one of which is normal, and the rest are unbalanced gametes. Segregation was performed by five possible modes: alternate, adjacent-1, adjacent-2, 3 : 1 or 4 : 0 (Figure 1(b)). Alternating segregation produces only balanced gametes. Adjacent-1, Adjacent-2, 3 : 1 and 4 : 0 segregation will produce unbalanced gametes. Reciprocal balanced translocations occur in 0.195% of the general population, and the frequency of translocations is about 1.3% in infertile males [Reference Elghezal, Hidar, Braham, Denguezli, Ajina and Saâd109]. In 3% to 6% of RPL, one of the two parents carries a chromosomal balanced translocation [Reference Zhao, Zhao, Ping, Chen and Feng37]. When an abnormal gamete binds to a normal egg or sperm, an imbalance in genetic material can induce monosomies or trisomies. Thus resulting in miscarriage and stillbirth.

FIGURE 1: (a) Balanced translocations on nonhomologous chromosomes. (b) Possible gametes in patients with balanced translocations. During meiosis I, the translocated chromosome combines with its normal homologous chromosome to form a tetrad. Balanced gametes containing normal non-homologous chromosomes or two translocated chromosomes resulting from alternate segregation are designated by green border, and unbalanced gametes by red border. Chromosome segregation patterns for tetrad are shown: 2 : 2 (two non-homologous or two homologous chromosomes segregate together in an adjacent-1 or adjacent-2 segregation, respectively), 3 : 1 (three chromosomes segregate into one cell and one into the other), and 4 : 0 (all chromosomes segregate together).

Robertsonian translocation occurs in acrocentric chromosome and refers to the process in which two proximal chromosomes break at the trophectodomain to form a long-arm chromosome. Robertsonian translocations can occur between homologous or non-homologous chromosomes, but Robertsonian translocations between homologous chromosomes also fail to produce gametes. Therefore, we shall only summarize the case of non-homologous chromosome equilibrium translocations (Figure 2(a)). It is a specific form of translocation with an incidence of 0.1% in the general population. After translocation, the two long arms fuse with each other to form a larger chromosome, while the two short arms are often lost. The chromosomes in which translocations occur are classified as homozygous Robertsonian translocations or non-homozygous Robertsonian translocations. Non-homologous Robertson translocations can produce six types of gametes when forming germ cells, one normal, one balanced and the other four unbalanced (Figure 2(b)). Unbalanced gametes can cause abortions, malformations and stillbirths due to an imbalance of genetic material. In the case of homozygous Robertsonian translocations, the general offspring only have the possibility of forming translocated trisomies or monosomies.

FIGURE 2: (a) Robertsonian translocations on non-homologous chromosomes. (b) Possible gametes in patients with robertsonian translocations. Non-homozygous robertsonian translocations can produce six types of gametes in the formation of germ cells, one normal, one balanced, and the remaining four unbalanced gametes. Normal and balanced gamets are designated by green border, and unbalanced gametes are designated by red border. The probability of normal and robertsonian translocation carrier are both 1/8.

Balanced translocations and inversions do not affect the phenotype of the parents themselves, but their unbalanced gametes during meiosis may indeed be partially responsible for abortion. Likewise, Robertsonian translocations of parental chromosomes may cause miscarriages, congenital disabilities or mental retardation in the offspring [Reference Li, Chen and Zheng110]. Chromosomes 11, 6, 4, 1 and 18 are the most common translocated chromosomes [Reference Verdoni, Hu and Surti111].

4.2.2. Inversion

An inversion is a rearranged chromosome formed when a chromosome breaks in 2 places, forming 3 segments. The middle segment is inverted by 180° and then joined to form a rearranged chromosome, which is divided into inter-arm inversion and intra-arm inversion (Figures 3(a) and 3(b)). Inverted chromosomes form an inversion loop during meiosis, and homologous chromosomes undergo recombination to produce four types of gametes, one normal, one inversion carrier, and the other two unbalanced gametes with partial duplication and partial deletion of no or double mitosis (Figures 3(c) and 3(d)), which, when combined with normal gametes, cause an imbalance of genetic material, resulting in abortion or stillbirth. Interarm inversions are most common on chromosomes 1, 9 and 11, with a prevalence of 1.0% in the population and 2.28% in RPL patients, observably higher than in the general domestic population. There are some controversies regarding the effect of the inversion of chromosome 9 on RPL. Some studies have illustrated that Inv (9) is the least common polymorphic variant in infertile couples [Reference Hong, Zhou, Tao, Wang and Zhao112], while Jeong et al. [Reference Jeong, Kim and Yu113] also suggested that inter-arm inversions of chromosome 9 are normal variants and generally do not affect individual health. Most scholars believe that interarm inversion of chromosome 9 is a polymorphism and that carriers do not have an abnormal phenotype. However, an increasing number of studies clarify that it is closely related to abnormal clinical conditions such as infertility and RPL.

FIGURE 3: Chromosomal inversions: two breaks in the same chromosome, causing the resulting fragments to reconnect after 180 degrees of reversal. (a) Paracentric inversion: the inverted segments do not contain chromosomes. (b) Pericentric inversion: the inverted segments contain chromosomes. (c) Possible gametes of paracentric inversion. In meiosis, a crossover between a normal chromosome and an inverted chromosome results in the loss or duplication of a segment of the gametophyte chromosome, leading to chromosome abnormality and abnormal traits in the offspring. Balanced gametes are designated by the green border. Unbalanced gametes are designated by the red border. (d) Possible gametes of pericentric inversion. Balanced gametes are designated by the green border. Unbalanced gametes are designated by the red border.

4.2.3. Duplicates and Deletions

Chromosomal deletions and additions, called copy number variants (CNV) [Reference Popescu, Jaslow and Kutteh105], are classified as large CNV (≥10 Mb) and submicroscopic CNV (<10 Mb). Nucleotide microarray technology was used to detect chromosomes in recurrent flow products, and small deletions of chromosome X were found in up to 6% of RPL women. Chromosome 16 duplications were the most common, followed by X chromosome deletions and triplet chromosome abnormalities, and again by chromosome 21 and 22 duplications. Minor deletion duplications of chromosomes, such as chromosome 2, 4, 9, 13, 14, 15, 17, 18 and 20 duplications were also found [Reference Hong, Zhou, Tao, Wang and Zhao112]. Larger deletions and increases in CNVs involving online human genetics (OMIM) genes and CNVs not found in large databases of normal individuals are likely to be associated with pregnancy loss, and pathological smaller CNVs (<400 kb) are of uncertain significance and may not be closely linked with pregnancy loss [Reference Popescu, Jaslow and Kutteh105].

4.4.1. Closed Placental Chimerism (CPM)

Restrictive placental chimerism occurs when all or part of the genetic makeup of the placenta differs from that of the fetus. Genetically abnormal placentas inextricably linked to placental insufficiency, fetal growth restriction and death [Reference Popescu, Jaslow and Kutteh105]. Fetal growth restriction (FGR) was reported in 71.7% of CPM cases, and preterm birth (<37 weeks) was reported in 31.0% of cases. A high percentage of structural fetal malformations of 24.2% was also found in cases of CPM.

4.4.2. Skewed X Inactivation

In females, partial or complete inactivation of one X chromosome in a particular cell during the embryonic period is called X chromosome inactivation [Reference Popescu, Jaslow and Kutteh105]. The X chromosome inactivation (XCI) process begins at the preimplantation stage of human embryonic development, probably around the eight-cell stage [Reference van den Berg, Laven and Stevens115]. The extreme skew of XCI (when defined as greater than 90%, the incidence of XCI is significantly higher) is associated with RPL. The essentiality of RPL is diminished when it is defined as two or more losses [Reference Sui, Chen and Sun116]. In Korea, skewed X chromosomes were not bound up with patients with RPL of unknown cause [Reference van den Berg, Laven and Stevens115]. In a case-control study, curved XCI and shortened telomere length were found to be closely tied with idiopathic premature ovarian failure (POI) despite the absence of alterations in the androgen (AR) and FMR1 genes. Additionally, women with shorter telomeres tended to exhibit a skewed XCI [Reference Miranda-Furtado, Luchiari and Chielli Pedroso117]. In a study by Sharp et al. [Reference Sharp, Robinson and Jacobs118], the incidence of severe skewing was higher in women with idiopathic premature ovarian failure and increased with age, with an incidence of 7% in women younger than 25 years and 16% in women older than 60. Through Mark’s research [Reference Lanasa, Hogge and Kubik119], solid statistical evidence was provided that female carriers of X-linked recessive fetal lethal defects are at incremental risk of RPL.