Correlation of BDNF Gene Polymorphism and Psychological Nursing Intervention in Patients with Recurrent Spontaneous Abortion

Objective: To investigate the correlation between recurrent spontaneous abortion patients and brain derived neurotrophic factor BDNF gene polymorphism and the mechanism of BDNF and recurrent spontaneous abortion under stress state in order to provide theoretical basis for nursing psychological intervention of patients with recurrent abortion. Methods: Medical coping Questionnaire (MCMO) and post-traumatic stress Diagnostic Scale (POST-traumatic stress Scale) were adopted Dissorder (PTSD) (PCL-c) scale was used to diagnose the psychological stress of recurrent spontaneous abortion in our hospital, and the BDNF gene polymorphism and the correlation factors of METHYLation in BDNF promoter region were studied in 30 cases (control group) and normal control group. Results: The MCMQ score of the case group and the control group was significantly lower than that of the conventional group (P < 0.05), and the scores of “avoidance and submission” were higher than that of the conventional group, with statistical significance (P < 0.05). The distribution of BDNF gene g-712A genotype was significantly different between the case group and the control group. Conclusion: There are significant differences in PTSD between the case group and the control group, which are related to methylation in the PROMOTER region of BDNF and SNP g-712A of BDNF gene. AG patients in the case group are more sus-ceptible to anxiety and depression, and GG PTSD is more severe in the case group. BDNF promoter methylation and G-712A were independent risk factors for PTSD in the case group.


Introduction
The probability of one abortion in pregnancy is 15% -20%, and 1% -5% of pregnant women have recurrent spontaneous abortion (RSA) [1], among which 40% -55% RSA has no definite cause at present [2]. Pregnancy itself is a complex psychological process, there are many factors to form the source of stress, which results in highly maternal stress state, causing many psychological barriers but at present the causes and treatment of repetitive abortion research mainly from the Angle of biology [3], this special group of patients with repetitive abortion the psychosocial characteristics and their influence on abortion, There are few studies at home and abroad, and they are not systematic and comprehensive [4]. This study explored the relationship between psychological stress and BDNF (rs6265) gene polymorphism in patients with unexplained habitual abortion and scientific psychological nursing intervention, systematically explored the psychosomatic symptoms, psychosocial influencing factors and genetic factors of patients with recurrent spontaneous abortion [5] [6]. From the perspective of psychology and genetics, the pathogenesis of unexplained habitual abortion high-risk groups was studied [7] [8] [9] [10], in order to provide a theoretical basis for the psychological nursing intervention of patients with habitual abortion, and to find out the method of individualized prevention and treatment of unexplained habitual abortion.

Cases
Thirty patients with recurrent spontaneous abortion who visited the department of gynecology of the fourth people's hospital of haikou from l to August 2021 were selected aged between 30 and 38. The inclusion criteria were: 1) two or more consecutive spontaneous abortions without a history of live birth; 2) No restrictions on age or occupation, primary school education or above; 3) Patients with serious physical diseases and mental disorders were excluded.

In the Control Group
Thirty healthy women were selected aged between 30 and 38. The inclusion criteria were: 1) they had children and had no history of abortion, stillbirth or stillbirth; 2) General demographic data such as age, occupation and education level were matched with case group; 3) except those suffering from serious physical diseases, mental disorders and serious mental frustration within the past 1 year.
This study has been approved and supervised by the Ethics Committee of our hospital. All subjects in this study have obtained informed consent and signed informed consent by themselves and their family members.

PTSD Diagnostic Scale
The PTSD Checklist-Civilian Version (PCL-c) [11] [12] [13] [14] was developed by the Behavioral Science Branch of the Post-Traumatic Stress Disorder Re-Health search Center in November 1994 according to DSM-W. Pcl-c combined with the dSM-IV 17-item self-report Scale for PTSD symptoms. Respondents rated each item on a scale of one (not quite) to five ("very") to indicate how much they had been bothered by that particular symptom in the past month, with a total score of 17 -85. Therefore, the PCL-c scale is a very good diagnostic tool. In the United States, THE PCL-c scale is used to evaluate the diagnosis of PTSD symptoms, the effect of intervention and treatment of PTSD, and the prognosis of PTSD. According to the unified diagnostic criteria (DSM-IV, DSM-V, AND PTSD), the patients with PTSD with habitual abortion were diagnosed by 2 psychiatrists with intermediate professional title and above with clinical experience [15]

Medical Response Questionnaire
MedicaI Coping Modes Questionnai Re (MCMO) was developed by Feifel H et al., which is suitable for evaluating patients' medical Coping styles. The scale divides coping styles into 20 items, including three coping styles of "facing", "avoiding" and "yielding", which are in line with the basic coping styles that people tend to adopt when facing stressful events.

Gene Polymorphism Detection
After admission, 5 ml of fasting blood was taken from the anterior elbow vein of all subjects in the morning, and 2 ml of the blood was slowly injected into the anticoagulant tube containing heparin along the tube wall, and immediately frozen at −70˚C. Genomic DNA was extracted by centrifuge column method using whole blood genomic DNA extraction Kit (OMEGA, USA), and the concentration and purity tests met the requirements of PCR amplification.

Methylation Detection
Genomic DNA was extracted using TIANamp Blood DNA Kit whole Blood Genomic DNA Extraction Kit (Tiangen, Beijing, China) and stored at −80˚C for future use. Methylation of BDNF promoter region was determined by MS-HRM (Methylation-Sensitive High Resolution Melting) method. Strictly follow the instructions of the kit (QIAGEN methylation Kit) and conduct the blood genome DNA sulfite test. After the reaction, the product was purified and recycled using the kit's own centrifugal column, and stored at −20˚C for future use. Ms-hrm primers (Table 1) are designed and synthesized by Shanghai Sangong Bioengineering Company and carried out on rotor-Gene 6000 (Corbett, Sydney, Australia). Each sample is in duplicate, and each run includes 100%, 50%, 10%. And 3% methylated and completely unmethylated standard substances were used to detect the methylation level of samples. 20 μL reaction system consists of 1 × PCR buffer, 1.5 to 3.0 mM MgCl2200 μMdNTP mixture, 200 -400 nM forward and reverse primers, 1 × SYTO9 embedded dye (Thermo Fisher Scientific, Waltham, MA, USA), 0.5 U Hotstar Taq polymerase and 10 ng bisulphite conversion of DNA.

T-Test Was Used for Comparison between the Two Groups
One-way ANOVA was used for comparisons between multiple groups, and Tukey's Multiple post test was used for pairwise comparisons after ANOVA analysis. The count data were compared using Fisher's exact test. P was a two-sided test, and P < 0.05 was considered statistically significant. Odds rarios (Odds rarios) and 95% confidence intervals (95% confidence intervals) were calculated by nonlinear logistic regression analysis. The χ 2 test was used to test the existence of Hardy-Weinberg equilibrium of genotype distribution.

Medical Response Questionnaire
Psychological stressors, facing difficulties, different coping styles will have different effects on the psychological stress response of patients. MCMQ includes three subscales: face, avoid and yield, which has good reliability and validity, and can be applied to the clinical research of psychological stress in Chinese patients. The "face" score of the MCMQ score comparison study group was significantly lower than that of the conventional group (P < 0.05), and the "avoidance and yield" scores were higher than that of the conventional group, with statistically significant differences between the two groups (P < 0.05) ( Table 2).

Gene Polymorphism Detection
All subjects received 5 to 10 ml of pre-elbow venous blood, anticoagulated with 0.5 mol/LEDTA (ethylenediamine tetraacetic acid) l mL, and immediately sent to the Central Laboratory of Hainan Hospital Affiliated to Hainan Medical College for −20˚C freezing. Genomic DNA was extracted by centrifugation column method with whole blood genomic DNA extraction kit (OMEGA, USA), and the concentration and purity test met the requirements of PCR amplification. Primer Premier5.0 Primer design software was used to design BDNF gene primers. The primers were synthesized by Shanghai Shenggong Bioengineering Technology Service Co., LTD. (Table 3). The design and synthesis of the primers were provided by Kingdomain Biological Co., LTD. The BDNF ELISA kit was operated according to the instructions. PCR kit was purchased from Shanghai Jerui Bioengineering Co., LTD. The PCR products were sent to Shanghai Shenggong Bioengineering Technology Service Co., LTD for sequencing.

Genotyping of BDNF Gene G11757C and RS6265 Polymorphisms
After the PCR amplification product of G11757C site of BDNF gene was di-  (Figure 1(a)). After the PCR ampli-  (Figure 1(b)).

Comparison of Genotype rs6265 and G-712A of BDNF Gene between the Case Group and the Control Group
There was no statistical difference in the distribution of rs6265 genotype of BDNF gene between the case group and the control group, while there was a significant difference in the distribution of G-712A genotype between the case group and the control group (Table 4).

Genotype and Allele Frequency Distribution among Groups
There was no significant difference in genotype and allele frequency distribution of BDNF gene G11757C between the case group and the PTSD group, and be-  Table 5 and Table 6).

Discussion
BDNF has been found to be associated with several neuropsychiatric diseases in studies [20]. According to previous studies, BDNF may help relieve fear of PTSD patients through BDNF signaling and serve as a potential target for PTSD treatment outcomes [21]. The aim of this study was to investigate the effect of BDNF gene polymorphism on patients with recurrent spontaneous abortion. Our results suggest that the GG genotype of BDNF RS6265 polymorphism may be a protective factor for PTSD in patients with recurrent spontaneous abortion.   First, we found that the MMSE score of patients with recurrent spontaneous abortion was significantly lower than that of the control group, and the level of BDNF in patients with recurrent spontaneous abortion was positively correlated with the neurocognitive function of PTSD. As previous studies have found, patients with recurrent spontaneous abortion perform less well in neurocognitive function than healthy controls [22]. BDNF has been reported to be involved in higher neurocognitive function as well as mental disorders, including depression [23]. There is evidence that BDNF can play a role in neurocognitive function through its expression, secretion and action [24]. In addition, the expression level of BDNF is also inhibited in depression and bipolar disorder [25]. The BDNF-TrKB pathway produces a ligand receptor system for synaptic plasticity and has been implicated in PTSD in fear states and inhibitory learning, and BDNF can be directly associated with PTSD through peripheral plasma and serum studies [26].
An important finding of our results was that GA + AA allele of RS6265 BDNF polymorphism caused lower MMSE scores, in contrast to GG allele and GG genotype of RS6265 BDNF polymorphism, and may be a potential contributing factor to the increased risk of recurrent spontaneous abortion. MMSE is a short examination designed to test mental state, and a lower MMSE score means poorer neurocognitive performance [27]. It has been reported that a variant of the BDNF allele may mediate anxiety disorders characterized by learning cues that suggest safety and risk [28]. In addition, BDNF variants can alter hippocampal neurocognitive function by mediating intracellular transport and actiitydependent BDNF release (e.g. Rs6265) [29]. Met66 variants can affect the pro-Health cessing of BDNF peptides, and also affect the release of BDNF peptides when neurons are activated [30]. According to previous studies, BDNF Val66Met (RS6265) carriers of (A/A or G/A) genotype showed A significantly larger hypo perfusion hippocampus compared with G/G genotype carriers, suggesting that G/G genotype carriers showed moderated neurocognitive function, which is consistent with our results [31]. In addition, RS6265 is associated with methamphetamine-induced psychosis, and its GG genotype shows low frequency in this psychosis, which can reduce the occurrence of this psychosis in the Thai population [32].
Our results also suggest that haplotype GA of BDNF G-712A and RS6265 may increase the risk of PTSD in patients with recurrent spontaneous abortion, while haplotype gene CG may reduce this risk. Previous findings suggest that individuals with a certain type of BDNF haplotype may overcome functional deficits because they are likely to benefit from corresponding therapeutic interventions that help restore neuronal plasticity [30]. Rs6265 and G-712A are considered to be two common SNPs in THE BDNF gene, and are believed to be clinically relevant to schizophrenia and depression [33]. G-712a polymorphism may affect the volume of white matter and gray matter in cerebellar hemispheres, and BDNF gene variation was found to be related to brain morphology [34]. Consistent with our results, BDNF gene polymorphisms including G-712A may be the cause of Alzheimer's disease-related depression [35]. In addition, in a similar study, C-C-G-G haplotype was considered to be the most common non-highrisk C-C-G-G haplotype in g-712A and RS6265 of BDNF gene [36].
After psychological intervention nursing, the psychological stress score of patients with recurrent spontaneous abortion was significantly improved, and the distribution of G-712A locus genotype was significantly different between the case group and the control group. It has an important influence on post-traumatic stress disorder in patients with recurrent spontaneous abortion. At the same time, it also shows that psychological intervention nursing can relieve the stress emotional distress of patients with recurrent spontaneous abortion.

Conclusion
Therefore, this study found that BDNF gene polymorphism was associated with PTSD, and GG genotype at rs6265 was a protective factor for PTSD in patients with recurrent spontaneous abortion. These findings may provide a new target for the prevention and treatment of PTSD. However, it is not clear why different haplotypes of BDNF G-712A and RS6265 polymorphism have opposite effects on PTSD. Therefore, further research on BDNF gene polymorphism in recurrent spontaneous abortion and the genetic mechanism of psychological intervention nursing is needed in the future.

Foundation Project
Hainan Health Commission Science and Technology Project (No. 20A200187).