A Potential Mechanism of Kidney-Tonifying Herbs Treating Unexplained Recurrent Spontaneous Abortion: Clinical Evidence From the Homogeneity of Embryo Implantation and Tumor Invasion

Background: Kidney-tonifying herbs (KTHs) are widely used to treat unexplained recurrent spontaneous abortion (URSA) based on the theory of traditional Chinese medicine (TCM). However, there is still a lack of systematic evaluation and mechanistic explanation for these treatments. Objective: The purpose of this study was to assess the clinical efficacy, and to investigate the potential mechanisms, of KTH based on TCM for the treatment of URSA. Methods: A systematic literature search was conducted within PubMed, Embase, China Biomedical Literature database, Web of Science (WOS), China National Knowledge Infrastructure (CNKI) database, and the Wanfang database to find articles reporting on the Chinese herbal formula based around KTH for treating URSA, which were published between January 2010 and June 2021. A full bibliometric analysis was carried out; in addition, randomized controlled trial (RCT) articles were selected for systematic evaluation and meta-analysis. The drugs with the highest frequency of KTHs were screened for meta-analysis. Finally, network analysis and molecular docking were used to study the key components and potential pathway of KTHs in the treatment of URSA. Results: The meta-analysis included nine RCTs involving 1,054 subjects. Compared with the control groups, the clinical efficacy of TCM-based KTHs in the treatment of URSA patients significantly improved outcomes. Additionally, a component target pathway network was identified, which included 32 potential blood activating components and 113 main targets. Japonine, sopranol, lysine, and matrine were considered the most important bioactive molecules for KTHs. The key potential therapeutic pathway for URSA was a tumor-related signaling pathway. The target genes for URSA regulated by KTHs were highly similar to tumor biological processes such as the regulation of apoptotic signaling pathways, inflammatory responses, angiogenesis, and epithelial metabolic transition. Conclusion: KTH has great potential for treating URSA. Because the maintenance of pregnancy has a high similarity with tumor invasion, the research relating to tumor mechanisms should also be followed up as it may lead to new ideas and breakthroughs for research into URSA. At the same time, embryonic and decidual cells share a high degree of cellular heterogeneity and spatial structural complexity with tumor cells, and a single cell combined with spatial omics may be the best future approach for validating KTH mechanisms.


INTRODUCTION
Recurrent spontaneous abortion (RSA) is challenging to diagnose and treat. Its classic definition is two or more clinical continuous abortions before 20 weeks of pregnancy in fertile couples (Practice Committee of the American Society for Reproductive Medicine, 2012;Colley et al., 2019). The etiology of RSA includes genetic abnormalities, endocrine disorders, anatomical abnormalities, infectious, prothrombotic state, and immune factors (Li et al., 2002;Qian et al., 2018). Its incidence rate ranges approximately from 1% to 5% in women during their childbearing years (Nigro et al., 2011;Ewington et al., 2019;Homer 2019). However, about 50% of RSAs are still undiagnosed and/or untreated. This condition is often referred to as unexplained recurrent spontaneous abortion (URSA) after exclusion of diagnosis and is considered an early spontaneous abortion within the first 12 weeks of pregnancy (Pereza et al., 2017;ESHRE Guideline Group on RPL et al., 2018). The disease has serious physical and mental impacts on the patients and their families Tavoli et al., 2018). Therefore, it is necessary to study effective treatment methods to reduce pregnancy loss and to help maintain pregnancy in URSA patients (Zhang et al., 2021). The current therapeutic options for URSA mainly include preimplantation genetic screening (PGS), suppression of alloimmunity, and anticoagulant therapy (Rey et al., 2003;Qin et al., 2016;Zhao et al., 2017;Ding et al., 2019;Feng, 2019;Qin et al., 2020;Zhang et al., 2021). Cyclosporine A (Zhou et al., 2007), intravenous immunoglobulin Muyayalo et al., 2018), lymphocyte active immunity, and glucocorticoids are the main regimens used to suppress alloimmunity, but the effectiveness and safety of these regimens have not been fully validated using large sample clinical studies. Therefore, until now, there has been a lack of unified diagnostic criteria and efficient treatments for URSA (Mekinian et al., 2016;. In recent years, traditional Chinese medicine (TCM) has been accepted as a mainstream of medical care, and has become a popular supplement to Western medicine for the treatment of URSA (Fujii et al., 2000;Li et al., 2014;Yang et al., 2018). Kidneytonifying herbs (KTHs) are the most commonly used prescription for TCM-based treatment Li et al., 2016). In the past few years, the data accumulated from personal clinical experience, case reports, noncontrolled trials, animal experiments, and randomized controlled trials (RCT) show that when treating URSA, KTHs alone and KTHs combined with Western medicine have similar effects regarding the improvement of pregnancy outcomes and symptoms (Liu et al., 2009;Zhao et al., 2018;Feng 2019;Zhang et al., 2019). Although KTHs are widely used in patients with URSA, it is difficult for KTHs to be recognized internationally due to the complexity of its ingredients and a lack of pharmacological mechanisms.
At present, it is known that embryo implantation is highly similar to cancer invasion. The phenotypes of apoptosis, inflammation, proliferation, invasion, adhesion, and angiogenesis in the interactions between embryonic trophoblast cells and endometrial epithelial cells at the maternal-fetal interface are highly similar to cancer processes (Murray and Lessey, 1999;Hannan and Salamonsen, 2008;Perry et al., 2009;Zhang et al., 2020). According to previous studies, we hypothesized that the mechanism of TCM for tocolysis may be similar to that observed in tumor activation. A key difference between them lies in the gene targets and biological process affected by the medicine. The goal of this study was to test this hypothesis. Specifically, bibliometric analysis, meta-analysis, network pharmacology, and molecular docking were conducted to examine the efficacy of the published TCMbased KTHs preparations and the control groups for treating URSA. The research also identified the potential pathways of TCM kidney-tonifying prescriptions in the treatment of URSA, and provided evidence-based scientific support for URSA treatment in clinical practice ( Figure 1).

DATA AND METHODS
Bliometric Analysis of Unexplained Recurrent Spontaneous Abortion Treated by Traditional Chinese Medicine in the Last 10 Years by KTHs based on the PICOS strategy (Detailed strategies are provided in Supplementary Data Sheet S1), and then collated the screened literature using ENDNOTE software.
We also established a bibliometric analysis strategy for the CNKI and WOS databases (Supplementary Data Sheet S2). Between ("2011-01-01," "2021-06-01") and (literature classification was limited to traditional Chinese medical science, Chinese herb and integrated traditional Chinese and Western medicine); Search scope: general database. After downloading the refworks format, the synonyms were converted into Chinese and English, and the synonyms were clustered uniformly, including KTHs. The drug was defined as "KTH"; the selection used a modified g-index in each slice: To include more or fewer nodes, increase or decrease the scale factor k 25. The keywords co-occurrence analysis, cluster analysis, mutation term analysis, as well as time line graph analysis were performed using CiteSpace (Version 5.8.R1).

Clinical Evidence of Kidney-Tonifying Herbs in the Treatment of Unexplained Recurrent Spontaneous Abortion
The systematic review and meta-analysis were conducted in accordance with the PRISMA guidelines (Moher et al., 2009;McInnes et al., 2017). The detailed research process is illustrated in the Supplementary Materials. All randomized controlled trials (RCTs) were included to study the efficacy of KTHs alone or combined with modern drugs, in the treatment of URSA. Studies reported in languages besides Chinese and English were excluded. Nonrandomized controlled trials or animal experiments were excluded.

Participant Inclusion
Patients included were in their first 3 months of pregnancy, had a pregnancy confirmed by serum human chorionic gonadotropin (hCG) or ultrasound, and had a history of URSA diagnosis, which was defined as two or more spontaneous abortions. The following four causes were excluded: infection, abnormal parental karyotype, endocrine imbalance, and anatomical abnormalities, regardless of the maternal or gestational age, race or nationality, educational level, or economic status. The inclusion criteria also ensured that no treatment was received before pregnancy or before entering the trial. Trials with recurrent spontaneous abortion and nonpregnant URSA participants with a definite etiology were also excluded.

Measurement of Treatment Outcomes
The primary outcome was the clinical response rate and pregnancy outcome reported in the trials. The secondary outcomes included reported hormone levels, serum immunological parameters, and incidence of adverse events during treatment.

Search Strategy
The search strategy was the same as the bibliometric analysis. The reference list of all identified articles was also manually searched to find possible related studies to supplement the relevant literature.

Data Extraction and Quality Assessment
Two reviewers (Hang Zhou and Yi Yang) extracted the general information from eligible studies through a predesigned standardized data extraction table: first author, year of publication, TCM syndrome difference, sample size, age, abortion frequency, definition of abortion and live birth, intervention time, treatment intervention and control group, treatment time, and results. Any inconsistencies were resolved by a third reviewer (Yongqing Yao). The methodological quality of each individual study was independently evaluated by two researchers (Yi Yi and Hang Zhou) referring to the Cochrane Handbook (Cumpston et al., 2019;Propadalo et al., 2019) for systematic review of interventions. We used the following criteria for evaluation: random sequence generation, allocation concealment, blinding of participants and personnel, blinding of outcome evaluation, incomplete outcome data, selective reporting, and other biases. Each study was classified as either low-risk, high-risk, or unclear. If there were differences in opinion, the third researcher was referred to (Yongqing Yao).

Statistical Analysis
A meta-analysis was conducted using the Review Manager (Revman) (computer program; version 5.3, Copenhagen: Nordic Cochrane Center, Cochrane Collaboration, 2014). The relative risk (RR) of 95% confidence interval (CI) was used for binary variables, while weighted mean variance (WMD) and 95% confidence intervals were used for continuous variables. Cochrane's p-value and I2 were used to test the heterogeneity of the study.
tcmsp.php) and the Batman database (http://bionet.ncpsb.org/ batman-tcm/). The structures of the above components were obtained from the PubChem database and imported into the Swiss target prediction database (http://www. swisstargetprediction.ch/). The targets with prediction scores greater than 0 were selected as drug targets. OMIM (https:// omim.org/) and the Genecards database (https://www.genecards. org/) were also searched with the keywords "unexplained recurrent spontaneous abortion" and the disease targets were obtained. The drug targets and disease targets were integrated, and then gene intersection was performed.

PPI Network Construction and Core Target Analysis
We searched the above drug disease common targets using the string database (https://string-db.org) (Von Mering et al., 2003). The protein type was set as "Homo sapiens." The minimum interaction threshold was set at 0.4. After the construction of the PPI network of protein interaction, Mcode module was used to analyze gene clusters and screen core targets.

Molecular Docking
According to the CAS number of small molecules, the 3D structure of small molecules in SDF format was downloaded from the PubChem database, imported into chembio3d ultra 14.0 for energy minimization, and autodock tools-1.5.6 for hydrogenation, charge calculation, charge distribution, and rotatable key setting. The key target proteins were downloaded from the database PDB (http://www.rcsb.org/), and the crystal water and original ligands were removed using pymol2.3.0. They were then hydrogenated, the charge was calculated and distributed, and the atomic type was specified. AutoDock Vina1.1.2 was used for molecular docking, and PyMOL2.3.0 was used to analyze the interaction mode of the docking results.

Enrichment Analysis of GO and KEGG
In metascape and R software, the Bioconductor bioinformatics software package was used to analyze the function enrichment of key target genes GO and KEGG with a p-value <0.05 and a Q-value <0.05, the results were output in the form of bar and bubble charts. A heatmap was plotted using http://www. bioinformatics.com.cn, an online platform for data analysis and visualization. According to the results of the enrichment analysis, the network diagram for traditional Chinese medicine-components-targets-pathways-phenotypes-diseases was constructed using Cytoscape.

Results of Bibliometric Analysis
A total of 1,012 articles were obtained through the literature searches, and the number of published articles increased annually ( Figure 2A). Additionally, Citespace was used for keyword Frontiers in Pharmacology | www.frontiersin.org January 2022 | Volume 12 | Article 775245 8 colinear analysis ( Figure 2B), and the polar coordinate histogram was calculated according to the count value ( Figure 2C). Analysis from the publications over the past 10 years showed that the research hotspots for traditional Chinese medicine in the treatment of RSA mainly focused on the following key words: recurrent spontaneous abortion, TCM treatment, tonifying kidneys, and promoting blood circulation, KTH, integrated traditional Chinese and Western medicine, clinical research, experience of famous doctor, prethrombotic state, progesterone, kidney deficiency and blood stasis, the damage of pre culture, and deficiency of spleen and kidney (Yang et al., 2013;Obstetrics;Subgroup et al., 2019;Cao et al., 2021;Li et al., 2021). The formula was used to show the keyword saliency map ( Figure 2D), with a total of seven keywords. Finally, a clustering time line chart was constructed. The results showed that the main related research areas were divided into one of four categories: TCM treatment, USRA, KTH, and famous doctor experience ( Figure 2E).

Clinical Evidence Search Results
After excluding duplicate studies, 562 studies were examined according to their abstracts and titles, resulting in 41 articles in the final evaluation. Finally, nine studies (Liu et al., 2015;Xu et al., 2018;Feng, 2019;Feng et al., 2019;Han et al., 2019;Feng et al., 2020;He, 2020;Niu and Hao, 2020;Xu et al., 2020) were included in the present systematic review. Table 1 summarizes the basic information pertaining to the randomized controlled trials included. All of these trials were published in China. These studies included 1,054 URSA patients, of which 629 participants were assigned to the treatment group, and 425 participants were assigned to the control group. There was no significant difference in the baseline parameters of all trials.

Research Characteristics
The patients in the treatment group were treated with KTHs or combined with Western medicine, while the patients in the control group were treated with Western medicine alone. Of Frontiers in Pharmacology | www.frontiersin.org January 2022 | Volume 12 | Article 775245 9 those, six randomized controlled trials used natural progesterone, including injection, capsule, or desgesterone (Liu et al., 2015;Feng, 2019;Feng et al., 2019;Han et al., 2019;He, 2020;Niu and Hao, 2020). One study used natural progesterone in combination with other treatments (Mekinian et al., 2016), and one used active immunotherapy . The treatment duration ranged from 4 to 20 weeks of gestation.

Bias Risk Assessment
Overall, the methodological quality of the included trials was poor. All nine studies were designed with two arms and were declared randomized controlled trials. None of the trials reported any details relating to the blinding of patients and researchers. No trials showed the number and reasons of dropouts. The evaluation results are shown in Figure 3.

Secondary Outcomes
Six of the nine studies enrolled (Liu et al., 2015;Xu et al., 2018;Han et al., 2019;Feng et al., 2020;He, 2020;Niu and Hao, 2020) reported estradiol and progestins before and after KTH treatment in the URSA patients, as detailed in Figure 5. The conclusions were consistent across the studies.

Tumor Necrosis Factor Alpha
Combination therapy of KTHs and Western medicine could decrease the level of TNFa compared with Western medicine

Publication Bias and Sensitivity Analysis
Although the funnel plot of early pregnancy loss rate was asymmetrically distributed, Egger's test analysis showed only marginally significant publication bias (p 0.09). The sensitivity analysis of early pregnancy loss rates showed that the effect evaluation remained unchanged, indicating no strong publication bias of the combined results.

Coagulation-and Anticoagulation-Related Outcome Indicators
Three studies Niu and Hao, 2020;Xu et al., 2020) included changes in indicators related to coagulation and anticoagulation, such as PT, APTT, TT FIB, t-PA, and PAI-I in URSA patients before and after treatment with KTHs. Unfortunately, due to the large variations in detection methods, modes of intervention, and modes of comparison among the studies, a quantitative analysis of this data was not possible. The wide variation in results across the studies precludes formation of a consistent conclusion. Finally, we developed a grade assessment for the meta-analysis results based on the Cochrane recommendation (Supplementary Data Sheet S3), but the quality of clinical evidence and grade of recommendation for these indicators are of concern.

Drug Screening and Data Intersection
A drug network was constructed for the nine articles screened ( Figure 6A). Statistics were also performed on the drug doses and frequencies used ( Figure 6B). The drugs with the top four tastants of frequency, which were set at ob ≥ 30% and DL ≥ 0.18 in the tcmsp database, were selected to screen the active ingredients of the screened drugs acanthopanax, Sambucus parasitica, and Cuscuta cuspida sequestration. Additionally, the ingredients of the four herbs in KTHs were also searched in the Batman database ( Figure 6D), resulting in a total of 32 potential active ingredients after removal of any duplicates from both databases. Furthermore, 546 drug targets were screened using the Swiss target prediction database with strict criteria of the species as "human" and a probability greater than 0.6. A total of 708 disease targets for URSA were obtained after dereplication. Finally, 546 drug targets and 708 disease targets were established, and a Venn diagram was drawn using the venny2.1 online software mapping tool platform, and 113 drug disease common targets were obtained after intersection of the two sets ( Figure 6E).

PPI Network Construction and Core Target Analysis
The 113 intersection targets were brought into the string database to construct the PPI network conditional on the species "human" and a confidence score ≥0.4. The results were imported into Cytoscape. Figure 7 shows the intersection target PPI network. The larger the node, the larger the degree value was. Next, the PPI network of URSA was further analyzed by mcode module with score cutoff 0.2 and K core 2. The clustering analysis was performed conditional on maximum depth 100 and degree cutoff 2. A total of 16 DEGs were obtained, and the top five DEGs with the highest score were taken ( Figure 8B). In total, five DEGs ( Figures 8C-G) and four core genes were obtained, and these five foundation clusters were scored. After screening, the four core genes obtained were mitogen-activated protein kinase (MAP1), matrix metalloprotinase1 (MMP1) (Zhang et al., 2021), ATP-binding cassette subfamily G member 2 (ABCG2), and recombinant caspase 1 (CASP1).

Molecular Docking Verification
Using different drug complexes in KTH as active compounds, the core proteins analyzed by mcode were used as targets for the molecular docking validation ( Figure 9A), and the best binding compounds were selected to construct the docking map. The binding energy of Sylvestroside III small molecules with MAPK1 protein was −7.6 kcal/mol, which proved to have a good binding interaction ( Figure 9B). Sylvestroside III small molecules interact with the MAPK1 protein, mainly  FIGURE 11 | Tumor signaling pathways interact with a variety of signaling pathways, which are closely related to embryo implantation. Orange represents the core target of KTHs.

Enrichment Analysis of BP and KEGG
After running mcoed core clustering through Metascape, GO analysis selected biological processes for analysis. The BP results showed that the intersection gene set was enriched to 2,045 biological process pathways ( Figure 10A), mainly including: positive regulation of kinase activity, cellular responses to organic cyclic compound, responses to inorganic substance, cellular responses to nitrogen compound, phosphatidylinositol 3-kinase signaling, responses to oxygen levels, protein kinase B signaling, responses to molecules of bacterial origin, inflammatory responses, epithelial cell proliferation, and others. Core genes were selected by Metascape, with parameters min overlap 3, p-value cutoff 0.01, and min enrichment 1.5. Twenty KEGG pathways were screened out, and the results of the top 20 formed a bar graph of KEGG functional enrichment ( Figure 10B). Padjust represented the significance of enrichment; the more the red the color is, the more significant. Finally, based on the core target screening and molecular docking, together with the results of BP and KEGG enrichment analyses, the 32 potentially active ingredients from the lifespan pill were the inputs with the 113 drug disease cotargets into Cytoscape software to remove isolated ingredients with no intersection with targets. A network diagram of "drug ingredient target disease" interactions was drawn ( Figure 10C). Using the score of 30 potentially active ingredients, and the average of the degrees as the screening criterion, we selected six compounds including Sylvestroside III, Japonine, sophranol, Gentisin, Lysine, and Matrine as core ingredients that were either large at the network nodes or showed high binding to protein targets (The screening process and criteria are detailed in Supplementary Data Sheet S4). The most important active compounds that were selected are listed in Table 2.

DISCUSSION
At present, there is a lack of targeted diagnostic and treatment options for URSA, all of which provide suboptimal clinical outcomes (Pierce, 1983;Youssef et al., 2019;. The question on how to find better interventions to achieve good pregnancy outcomes for more URSA patients is currently a hot topic and a challenging point in the field of reproductive medicine. Although progesterone was used as the primary method of treatment in most of the studies included in this research, it has limited clinical efficacy and can cause side effects such as dizziness, nausea, and vomiting (Yan et al., 2013;Coomarasamy et al., 2020a;Coomarasamy et al., 2020b). Potential risks include allergies, infectious diseases, and bleeding, as well as an ever-increasing financial burden on individuals and society. In the future, new methods for treating URSA will be necessary. As a supplement, traditional Chinese medicine, based on four main diagnostic methods, which regulate the human body as a whole using an inspection method, auscultation, olfaction method, and pulse diagnosis method, looks promising (Zhou et al., 2007). In TCM, KTHs are considered as a treatment, which pays more attention to the syndrome of patients and infers localized treatment from the whole, which may be the result of multichannel and multitarget approaches. However, modern medicine emphasizes the specific etiological treatment affecting URSA, such as focusing in on improving the uterine blood supply and endometrial immune status of the patient. Although KTH has a complex composition and a presently unclear mechanism of action, its comprehensive effects on diseases, especially on health care and prevention, are  3 | The main biological processes of cancer progression and embryo implantation, and the targets of related components in traditional Chinese medicine.

Cell biological processes
The significance in embryonic development The significance in cancer progression The related genes obtained in this study

Regulation of epithelial cell proliferation
Promote the uterus into the receptive state, decidualization of uterine stromal cells and the occurrence of placenta (Zhu et al., 2019) It can make tumor proliferate rapidly, compress tissue, and promote angiogenesis (Zhong et al., 2020) AR , TNF, XDH, PPARG, PGR, VDR,  STAT3, JUN, MTOR, EGFR, KDR,  AKT1, SHH, ITGB3, SCN5A,  HIF1A, AGTR1, ITGA4, GLUL,  FLT1, FGFR1, TEK Response to oxidative stress Excessive ROS can cause mitochondrial damage, DNA damage, lipid peroxidation, and even cell apoptosis in embryos, and then lead to embryonic development arrest (Ivanov et al., 2021) High level and long-term oxidative stress can directly damage tissues through this redox system, and also lead to oxidative modification of amino acid residues and DNA mutation, thus promoting the occurrence of tumor (Shrivastava et al., 2021) PTGS2

Regulation of mitochondrion organization
In the process of embryo implantation, the number, distribution, and activity of mitochondria are regulated strictly and orderly, and affect the embryo implantation potential at the same time (Moher et al., 2009) The malignant phenotypes of tumor cells, such as unlimited proliferation, abnormal metabolism, inhibition of apoptosis, strong invasion, and easy metastasis, are also closely related to mitochondrial dysfunction (Roth et al., 2020;Suldina et al., 2018) BCL2L1

Regulation of inflammatory response
The balance of pro-inflammatory factors and anti-inflammatory factors promotes endometrial receptivity, and appropriate inflammatory environment promotes embryo implantation and pregnancy maintenance (Quirke et al., 2021) The infiltration of inflammatory cells and the production of ROS are necessary and sufficient conditions to accelerate the carcinogenesis (Rossi et al., 2021) NOS2 temporarily irreplaceable by modern medicine (Li et al., 2016;Luo et al., 2012;Liu et al., 2009). Oncology is an important crossover area for reproduction. Based on the evidence from this meta-analysis, a network pharmacological approach was used to investigate which targets are important, and how these targets and signaling pathways play a role in RSA. Since the concept of an embryonal origin of cancer was proposed in 1982 by Lobstein, the resemblance between the biological behaviors of embryo implantation and tumor invasion and metastasis has been increasingly recognized (Figure 11) (Murray and Lessey, 1999;Perry et al., 2009). Especially given that "pseudo malignant" blastocyst trophoblast cells and malignant cells (Soundararajan and Rao, 2004;Fest et al., 2008) exhibit defects in cell proliferation and differentiation, invasion signaling pathways, vascular erosion, and neovascularization. There are additional striking similarities in many aspects of both processes, such as immune escape and apoptosis. The most fundamental biological process during embryo implantation is the invasive properties of trophoblast cells (Hanahan and Weinberg, 2011), which is regulated by a network of extracellular matrix, matrix degrading enzyme, cell adhesion molecules, and growth factors ( Figure 12A).
The results of this study suggest that the main signaling pathway of KTHs when used to treat URSA is a cancer signaling pathway, which confirms our conjecture. This signaling pathway dominates many biological processes. Therefore, we identified several main biological processes and related targets, which are regulated by KTH active components, and compared this biological process between embryo implantation and cancer progression, as shown in Table 3, in order to obtain more insights and beneficial information.
In conclusion, tumor cells and early embryos show similar mechanisms to each other, which gives us enlightenment as to how the microenvironment of embryo growth can guide and activate the potential of tumor development and reverse its phenotype. The process of embryonic development and differentiation is almost a reversion of tumor differentiation.
Further study on the regulation mechanism and important regulation steps during embryonic development can provide important indicators and cues for the study of tumor differentiation reversal.
The combination of embryo implantation and tumor invasion and metastasis not only helps understand the internal mechanism of complex life phenomena but also may provide new concepts for clinical treatment. Antitumor invasion and metastasis therapy may ultimately find the answer from embryo implantation, and repeated embryo implantation failure or repeated abortion may also provide new evidencebased answers toward understanding and treating tumors.

LIMITATION AND OUTLOOK
Although we have undertaken a comprehensive analysis and evaluation of all of the published studies, this research still has some limitations, which are worthy of recognition. The quality of the included studies was low due primarily to unclear allocation concealment, selective bias, consumption bias, and the blinding methods, or lack of thereof. The one major limitation within all of the included studies was that they were all conducted in China and have major methodological flaws, which greatly reduced the reliability and validity of study results as a whole.
The approach taken helps map cell types, cell subpopulations, and even cell states in a spatial context. The similarity of pregnancy maintenance to cancer invasion allows us to recognize the high complexity of subcellular species and spatial architecture at the maternal-fetal interface (Srivatsan et al., 2021). This complexity is often accompanied by spatiotemporal dynamic changes, so the study of KTHs for URSA cannot be satisfied by employing traditional single ex vivo experiments with animal experiments ( Figure 12B). Therefore, how KTHs could map the cell types, cell subsets, and even cell states and gene expression profiles of each class of cells regulated at the maternal-fetal interface of URSA patients in a spatial context, is a very 3 | (Continued) The main biological processes of cancer progression and embryo implantation, and the targets of related components in traditional Chinese medicine.

Cell biological processes
The significance in embryonic development The significance in cancer progression The related genes obtained in this study

Components of KTHs
Epithelial-mesenchymal transition, EMT It can make endometrial epithelial and stromal cells more invasive and mobile, promote embryonic organ formation, embryonic differentiation, and nervous system differentiation (Ran et al., 2020) EMT plays an important role in the invasion and metastasis of tumor in situ and the formation of new metastasis. It is an important way of invasion and metastasis of epithelial cell carcinoma, which accounts for more than 90% of malignant tumors in adults (Thiery, 2002) urgent matter, which provides an important area of future research. It is widely known that single-cell RNA sequencing (scRNAseq) identifies cell subpopulations within tissue but does not capture their spatial distribution nor reveal local networks of intercellular communication acting in situ. Fortunately, a suite of recently developed techniques that localizes RNA within tissues, including multiplexed in situ hybridization and in situ sequencing (here defined as high-plex RNA imaging) and spatial barcoding, could help address this issue (Longo et al., 2021). We believe that this method of single-cell sequencing, combined with spatial transcription, will bring about a more profound insight into how KTHs acts on URSA.

CONCLUSION
In conclusion, the TCM-based KTH formula provides good efficacy and safety for the treatment of URSA, and has great potential. According to the results of the network pharmacology analysis, we predicted that the main pathway of the KTH effective component in URSA is a tumor signaling pathway. With the in-depth study of the mechanisms of tumor formation, as well as single-cell spatial transcriptome in the future, will bring forth breakthroughs in the pathogenesis research and treatment of USRA.

DATA AVAILABILITY STATEMENT
The datasets presented in this study can be found in online repositories. The names of the repository/repositories and accession number(s) can be found in the article/ Supplementary Material.

AUTHOR CONTRIBUTIONS
LD was responsible for the overall research framework of this project. HZ was responsible for the clinical data analysis and all figure designs. YiY and YoY were responsible for the data collection. XL provided evidence-based idea guidance.

FUNDING
This work is supported by the Science and Technology Department of Sichuan Province (No. 2021YJ0257).