Miscarriage is the most frequent pregnancy outcome, occurring when a pregnancy terminates before the fetus reaches viability. Consequently, all pregnancy losses that occur between the time of conception and the 24th week of pregnancy are encompassed within this term. Although 15% of clinically recognized pregnancies terminate in miscarriage, the overall reproductive losses approach 50%. Miscarriages are classified as sporadic or recurring. About 1% of couples experience recurrent miscarriage. 4 Conversely, one or more sporadic miscarriages affect at least 25% of women, and the number may even reach 50%. These losses are often brought on by haphazard fetal chromosomal abnormalities and rise with maternal age [1–4]. The age of the mother and her previous reproductive history are both significant risk factors for miscarriage. The chances of a woman over 40 becoming pregnant are low. Anticoagulant therapy for the treatment of recurrent miscarriages and the significance of hemostatic pathways in early pregnancy development have been underscored by the finding that some women with recurrent miscarriages are prothrombotic outside of pregnancy. Patients who have experienced recurrent miscarriages should be enlisted in placebo-controlled studies with adequate power, particularly in light of the numerous studies that have established the significance of psychological support in enhancing pregnancy outcomes [5]. The human cytomegalovirus (HCMV) infects the majority of individuals worldwide and generates no visible symptoms. When the rare owl’s eye inclusions were found in stillbirths in 1910 and again in 1964 among patients receiving cutting-edge organ transplantation, they garnered medical interest. According to progressively better diagnostic technologies, HCMV is a common opportunistic infection in fetuses, allograft recipients, bone marrow transplant patients, and AIDS patients. Statistical evidence links it to both direct consequences (overt end-organ illness) and indirect results (graft rejection, cardiovascular disease after transplantation). This virus’s inability to generate an effective cell-mediated and humoral immune response has been related to higher mortality in the general population, suggesting that it is not as harmless as it appears [6,7]. LncRNA MEG8 is an understudied lncRNA. MEG3 (Maternally Expressed 3) is a tumor-suppressive imprinted lncRNA that is transcribed from chromosome 14q32.2. It is a polyadenylated lncRNA that’s overexpressed in the pituitary gland of humans but downregulated in cancer cells. Overexpression of MEG3 has been shown to activate autophagy and increase cell proliferation in bladder cancer cells. MEG3 is involved in tumor suppressor p53 accumulation as well as the control of TGF-pathway genes involved in cell invasion, immunological modulation, and other functions. It also interacts with PRC2 to suppress MDM2 (murine double minute 2), which aids in the accumulation of p53 [8–10]. The aim of this study was to investigate the possible relationship between two genes expressing long non-coding and recurrent miscarriage. The results show that there is a significant relationship between the expression of these two genes in people with recurrent and healthy abortions.

Sample collecting from patients.

Tissue samples were taken from women who had experienced at least two miscarriages before the 20th week. One hundred tissue samples were taken by endometrial biopsy by a gynecologist and provided to us inside RNase-free tubes. Also, 100 healthy people who had at least two healthy pregnancies and had been referred to a specialist for a checkup volunteered to have a biopsy performed on them so that their uterine tissue could also be provided to us. These samples were taken from March 2022 to October 2022 patients in Bint Al Huda Maternity & Children Hospital, Dhi Qar, Iraq. Saliva samples from patients with recurrent miscarriages were also sent to a pathologist immediately after removal for diagnosis of cytomegalovirus infection. Tissue samples were stored in a laboratory refrigerator at -80 ° C until RNA extraction. Other patient information was received and archived in accordance with ethical principles and privacy.

Cytomegalovirus infection status

In order to evaluate the patients for cytomegalovirus, saliva samples taken from these patients were sent to the pathologist. The pathologist reported the status of each patient to us by performing immunological tests(CMV ELISA test kit , Biokit Barcelona, spain) as well as PCR.

RNA extraction

RNA is a sensitive and unstable organic matter. Therefore, its extraction must be done in a completely sterile and RNase-free environment. All surfaces and equipment used in the extraction were sterilized with 70% ethanol. Also, in order to prevent damage to tissues by intracellular enzymes, extraction was performed in a 4 ° C cold room with repeated use of liquid nitrogen.

Evaluation of the quantity and quality of extracted RNA

Agarose gel electrophoresis and spectrophotometry can be used to evaluate the quality and quantity of extracted RNA.

Primers

In this study, the primers were designed by the NCBI database with the help of primer3plus.com. After selecting the appropriate primer, BLAST software was used to control its specificity, and finally, the approved primers were ordered to Sinaclon for sequence synthesis. The table below shows the information related to the primers used in this research.

Table 1. Designed Primers

cDNA synthesis

Zistfanavaran Rena synthesis kit was used for cDNA synthesis. In order to reduce the possibility of DNA contamination in the extracted RNA, a certain amount of RNA was transferred into the RNase Free microtubule by the nanodrop device according to the amount of its light absorption in the quantitative evaluation. The DNase enzyme and its buffer were each added to the microtube containing the extracted RNA in the amount of one microliter and incubated for 30 minutes at 37 ° C in a thermocycler.

Statistical analysis

After completing the Real Time-PCR reaction and measuring the Ct of the samples, the Ct of each reaction was adjusted in a table to measure the expression of the gene and the values of 2-∆Ct for each sample were calculated by Excel software. Then, in order to compare the levels of gene expression between the two groups of patients with recurrent abortions and healthy individuals and the significance of MEG8 and TCL6 gene expression, Mann-Whitney and Independent Sample t-test were performed by GraghPad PRISM and SPSS software. Graphpad software was used to examine the ROC diagram to examine the potential of this gene to be considered as a biomarker.

Samples description and Pathologist results

Samples collected from patients and individuals between March 2021 and October 2022 were used for this study. Based on the references, we considered at least two consecutive abortions without cause (unintended) as recurrent abortions. One hundred samples of healthy individuals and 100 samples of individuals with recurrent abortions were collected. These samples were collected by a specialist using a biopsy of the endometrial wall of the uterus and provided to us. The results submitted by the pathologist showed that 56% of people who had recurrent miscarriages were infected with cytomegalovirus. We used this data in statistical analysis to see if there is a significant relationship between infection with this virus and the expression of both genes.

Real-time PCR reaction results for the studied genes Amplification curve obtained from Real-time PCR

Evaluating the expression level of MEG8, MCL6 and β-actin genes selected as internal control, after cDNA synthesis for each sample, Real-time PCR for 100 pairs of samples, including 100 tissue samples of recurrent miscarriage and 100 samples A healthy person was performed with least 2 replications, an example of the resulting proliferation curve is given below.

Figure 1. Amplification curve of MEG8

Figure 2. Amplification curve of TCL6

Determine results

Comparison of MEG8 gene expression in two tissues prepared from women with recurrent abortions and healthy individuals Statistical analysis of MEG8 gene expression in the two tissue samples shows a significant difference in the expression level of this gene in the two tissues. Mann-Whitney test was performed on samples (p-value <0/0001). As shown in the figure, the expression level of this gene in people with recurrent miscarriage has increased significantly.

Figure 3. Comparison of MEG8 gene expression in two tissues prepared from women with recurrent abortions and healthy individuals

Comparison of TCL6 gene expression in two tissues prepared from women with recurrent abortions and healthy individuals

Mann-Whitney test was performed on the expression of TCL6 gene in two tissue samples. The results show a significant difference in the expression level of this gene in the two groups of tissue samples. The image below clearly shows this rate has increased in people with recurrent miscarriages.

Figure 4. Comparison of TCL6 gene expression in two tissues prepared from women with recurrent abortions and healthy individuals

Detection Results of statistical analysis to measure the ability of MEG8 gene as a biomarker

After analyzing the data for the ROC curve, it was found that MEG8 with a value of 0.69 for the area under the curve (AUC), sensitivity and specificity percentages of 61% and 76% to further studies for function as a biomarker for recurrent abortion is needed.

Figure 5. ROC curve of MEG8

Determine Results of statistical analysis to measure the ability of TCL6 gene as a biomarker

After analyzing the data for the ROC curve, it was found that TCL6 with a value of 0.69 for the area under the curve (AUC), sensitivity and specificity percentages of 62% and 76% to further studies for function as a biomarker for recurrent abortion is needed.

Figure 6. ROC curve of TCL6

Determine Results of statistical study of the relationship between age and expression of MEG8 gene in people with recurrent miscarriage

The statistical results of this analysis show that there is a significant relationship between gene expression and age.  In this test, people were divided into two groups: over 37 years old and under 37 years old. (p-value=0.011)

Figure 7. statistical study of the relationship between age and expression of MEG8 gene in people with recurrent miscarriage

Detection Results of statistical study of the relationship between age and expression of TCL6 gene in people with recurrent miscarriage

The statistical results of this analysis show that there is a significant relationship between gene size and age. In this test, people were divided into two groups: over 37 years old and under 37 years old. (p-value= 0.045)

Figure 8. statistical study of the relationship between age and expression of TCL6 gene in people with recurrent miscarriage

Determine Results of statistical study of the relationship between cytomegalovirus infection and MEG8 gene expression in patients with recurrent miscarriage

Statistical analyzes did not show a significant relationship between infection or non-infection of aborted individuals and MEG8 gene expression. Mann-Whitney test was used in this statistical analysis.

Figure 9. statistical study of the relationship between cytomegalovirus infection and MEG8 gene expression in patients with recurrent miscarriage

Results of statistical study of the relationship between cytomegalovirus infection and TCL6 gene expression in patients with recurrent miscarriage Statistical analyzes did not show a significant relationship between infection or non-infection of aborted individuals and TCL6 gene expression. Mann-Whitney test was used in this statistical analysis.

Figure 10. statistical study of the relationship between cytomegalovirus infection and TCL6 gene expression in patients with recurrent miscarriage

A miscarriage occurs when a pregnancy is terminated prior to gestation. An approximated 23 million miscarriages transpire annually on a global scale, which equates to 44 lost pregnancies per minute. Miscarriage occurs in 153% of all recognized pregnancies (95 percent confidence interval: 125%-187%). Women who have had one miscarriage make up 108 percent of the population (103-114%), two miscarriages make up 19 percent (18-21%), and three or more losses make up 07% (05-08%). Risk factors for miscarriage include

Black ethnicity, smoking, drinking, stress, working night shifts, air pollution, pesticide exposure, having a very low or very high body mass index (BMI), being very young or old (below 20 and over 35), being older (over 40), and having had previous miscarriages. The physical effects of a miscarriage, such as bleeding or infection, can have an impact on a woman’s mental health. Anxiety, depression, PTSD, and even suicide can all be psychological repercussions. Recurrent miscarriage is a risk factor for obstetric complications like preterm birth, fetal growth restriction, placental abruption, and stillbirth in subsequent pregnancies, as well as venous thromboembolism and cardiovascular disease. Miscarriage costs people, healthcare institutions, and society money. The cost of miscarriage to the UK economy in the near term is projected to be £471 million per year. Women who have experienced recurrent miscarriages should seek assistance from preconception and obstetric clinics that specialize in high-risk patients because this is a warning indication for a wide range of obstetric issues in future pregnancies. Because miscarriage causes psychological morbidity, diagnostic tools and treatment options for mental health concerns related to miscarriage are needed [11]. lnc-SLC4A1-1, and lncRNA MEG8 have been shown to modulate trophoblast cell activities and influence miscarriage. We recently discovered that a new lnc-HZ03 might enhance trophoblast cell death and cause miscarriage by upregulating the p53/SAT1 signaling pathway. They also discovered a new lnc-HZ01 that creates a feedback loop with the MXD1. Lnc-HZ01 modulates miscarriage by regulating BPDE-inhibited trophoblast cell growth. However, a large number of new lncRNAs remain unidentified. It is unclear how these novel lncRNAs cause miscarriage by regulating BPDE-inhibited trophoblast cell proliferation, migration, or invasion [12]. lncRNA-TCL6 was shown to be substantially expressed in placental tissues of threatening abortion pregnancies by Liu et al. in 2018. Furthermore, lncRNA-TCL6 expression in placenta tissues of spontaneous abortion pregnancy was substantially greater than that of induced abortion pregnancy. Through the EGFR signaling pathway, knocking down lncRNA-TCL6 increased trophoblast cell growth and prevented abortion [13]. Wu et al. (2018) investigated the potential role of long noncoding RNA (lncRNA) TCL6 in the pathogenesis of preeclampsia (PE) and elucidated the underlying mechanism at play. An abundance of TCL6 was detected in the placental tissues of patients with PE. TCL6 overexpression decreased trophoblast cell proliferation while promoting PE formation by targeting PTEN [14]. The imprinted lncRNA Rian, discovered by Sheng et al. in 2019, may play a key function during placental development. The human spontaneous abortion villi homologous sequence MEG8 (RIAN) was expressed at an excessively high level. Upregulating MEG8 expression in trophoblast cell lines inhibited cell invasion and proliferation while downregulating MEG8 expression had the opposite effect. Moreover, methylation analysis of the MEG8 promoter region was shown to be elevated in spontaneous abortion villi. During placental development, imprinted lncRNAs showed dynamic spatiotemporal expression. MEG8, an imprinted lncRNA, controls trophoblast cell function. Anomalies in promoter methylation can result in trophoblastic cell deficiencies, which could be one of the causes of early unexplained spontaneous miscarriage [15]. In 2021, Ye et al. discovered that lnc-HZ07 suppressed trophoblast migration by dephosphorylating AKT and downregulating matrix metalloproteinase 2 (MMP2) expression. These findings revealed a novel regulatory mechanism whereby BaP enhanced lnc-HZ07, which in turn decreased MMP2 production and downregulated AKT phosphorylation. This suggests that lnc-HZ07 could be employed as a pathological marker and therapeutic target for BaP-induced RPL [16]. Liang et al. found in 2021 that BPDE-induced human trophoblastic cell death and miscarriage can be enhanced by the p53/SAT1 pathway, which is raised by lnc-HZ03 and miR-hz03. These results add new insight into the reasons for miscarriage. Recurrent miscarriage is regulated by miR-hz03 and the genes Lnc-HZ03 (RM). The expression of miR-hz03 in human trophoblastic cells is increased by lnc-HZ03. ln-HZ03 and p53 mRNA RNA stability are enhanced by miR-hz03. P53 causes cell death by increasing SAT1 transcription while decreasing cellular spermine content. Normal pregnancy is achieved by downregulating the lnc-HZ03/miR-hz03 and p53/SAT1 pathways. BPDE activates the lnc-HZ03/miR-hz03 and p53/SAT1 pathways, causing miscarriage [17].

The findings show a link between the expression of these two genes in people who have recurrent abortions and those who do not. Statistical findings also reveal a significant relationship between differences in gene expression in people over 37 and under 37 years old. The findings of this study could aid in a better understanding of the molecular process that causes recurrent miscarriage. The results of this study will give us a broader view of the physiology of this problem, and thus may suggest new treatments for abortion in the future.

1. Conducting repeated tests for women who have had a miscarriage once for the CMV virus
2. Using real time PCR for CMV as diagnostic method in clinical laboratories.

Acknowledgments: The author Thank the Bint Al Huda Maternity & Children Hospital, Dhi Qar, Iraq.

Author Contributions: All authors contributed equally to the writing of this paper. All authors read and approved the final manuscript.

Conflicts of Interest: The authors declare that they do not have any conflict of interests.

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