Lactobacillus crispatus with assisted reproductive potential and application thereof in vaginal microecological preparation

Abstract

The present application relates to the field of medical microbiology and reproductive medicine technology, and particularly relates to a lactobacillus crispatus with assisted reproduction potential and application of the lactobacillus crispatus in a vaginal microecological preparation, including a strain preserved in the Guangdong Microbial Digital Culture Collection Center, and a preservation number of GDMCC67863. The present application provides a lactobacillus crispatus LM25V13 isolated and screened from a healthy pregnant woman in a middle period, and a preservation number of GDMCC67863. The strain has good vaginal colonization, vaginal microecological regulation and reproductive protection capacity, can up-regulate the expression of HOXA10 in healthy mice, CSTIII type and CSTIV type vaginal model mice, effectively improve uterine receptivity, and significantly improve the abundance of lactobacillus crispatus in the vagina of mice, so as to provide a potential strain for preparing a female-friendly microecological preparation for improving the assisted reproduction outcome and increasing the success rate.

Description

Technical Field

[0001] This invention relates to the fields of medical microbiology and reproductive medicine, and in particular to a strain of *Lactobacillus curvatureensis* with potential for assisted reproduction and its application in vaginal microecological preparations. Background Technology

[0002] Female infertility has become a global health and social problem, affecting 12.6%-17.5% of women of reproductive age worldwide. In China, the epidemiological trend of female infertility is equally serious, with a prevalence rate of approximately 12.5% ​​to 15% among women of reproductive age; some recent white papers indicate this rate may have risen to 18.2%. Faced with the increasing demand for fertility, more and more women are turning to assisted reproductive technology (ART) for solutions. Although ART is now technically mature, its relatively low clinical success rate (approximately 50%) remains a major challenge. Recent research evidence suggests that the balance of the female reproductive tract microecology (especially the homeostasis of the vaginal and uterine flora) is an important factor influencing pregnancy outcomes and improving ART success rates.

[0003] The vaginal flora structure can be divided into five main community state types (CSTs) based on the dominant species. The CST type, dominated by *Lactobacillus cristata*, is considered the healthiest vaginal microecological structure, associated with lower infection risk and better pregnancy outcomes. This bacterium mainly exerts its protective role through the secretion of D-lactic acid, hydrogen peroxide (H2O2), and cell adhesion and immunomodulatory functions mediated by its surface layer proteins (SLP). The role of *Lactobacillus inertia* in reproductive health is less clear. Although this bacterium belongs to the genus *Lactobacillus*, its genome lacks the ability to produce D-lactic acid, possesses a gene for synthesizing cholesterol-dependent cytolysin, and lacks the gene sequence for SLP synthesis. Therefore, the vaginal flora structure dominated by this bacterium—CSTIII type—is less resistant to invasion by external pathogens. Because this type is prone to dysbiosis, it is considered a sub-healthy state. CSTIV type, on the other hand, is a typical pathological state, dominated by anaerobic pathogens such as *Gardnerella vaginalis*. *Gardnerella vaginalis* severely damages the vaginal mucosal barrier by producing sialidase, forming biofilms, and inducing the release of pro-inflammatory cytokines in the host. It can also ascend to the uterus, affecting the endometrial immune environment and leading to decreased uterine receptivity. Currently, most vaginal probiotic preparations used in clinical practice use strains derived from European and American women, which are not well-suited for the reproductive tracts of Chinese women. More importantly, current probiotic preparations for women primarily focus on the adjunctive treatment of infectious diseases such as vaginitis, and have not yet undergone systematic evaluation for their potential applications in assisted reproduction (such as improving endometrial receptivity and pregnancy outcomes). Therefore, screening for a high-quality *Lactobacillus crenulata* strain with native Chinese characteristics and clearly demonstrated potential for assisted reproduction has become a scientific problem to be solved in the fields of reproductive medicine and microbiology. Summary of the Invention

[0004] The purpose of this invention is to address the problems of insufficient compatibility and lack of specific strains for assisted reproduction in existing clinical vaginal microecological preparations, and to propose a strain of *Lactobacillus curvaturei* with assisted reproduction potential and its application in vaginal microecological preparations.

[0005] To achieve the above objectives, the present invention adopts the following technical solution: A strain of Lactobacillus curvature with assisted reproductive potential, including the strain, is deposited at the Guangdong Provincial Center for Microbial Culture Collection, with accession number GDMCC67863.

[0006] Preferably, the strain has at least one of the following biological characteristics: Rapid growth characteristics: Enters the logarithmic growth phase within 12 hours; Strong ability to produce acid and hydrogen peroxide; Strong adhesion to vaginal cells: It has a strong adhesion ability to SiHa cells; Strong ability to inhibit Gardnerella vaginalis biofilm; High-level expression of S-layer protein: its mRNA expression level was significantly higher than that of the conventional Lactobacillus curvature control strain.

[0007] Preferably, the application includes its use in the preparation of female-friendly vaginal microecological preparations.

[0008] Preferably, the regulatory effect includes increasing the abundance of *Lactobacillus curvatureensis* in healthy mice, CSTIII-type or CSTIV-type vaginal flora structure model mice.

[0009] Preferably, the application includes its use in the preparation of vaginal microecological preparations that improve assisted reproductive outcomes.

[0010] Preferably, the improvement in assisted reproductive outcomes includes increasing pregnancy rates and uterine receptivity.

[0011] Preferably, improved uterine receptivity is achieved by upregulating the expression levels of at least one of the homeobox A10 gene, osteopontin gene, and leukemia repressor gene.

[0012] Preferably, the upregulation of HOXA10 gene expression is significantly better than that of the existing Lactobacillus curvature control strain, with statistically significant upregulation of HOXA10 in CSTIII model mice (p=0.044) and a significant trend of upregulation of HOXA10 in CSTIV model mice (p=0.061).

[0013] A vaginal microecological preparation, wherein the preparation contains the active ingredient of the Lactobacillus curvatureii.

[0014] Preferably, the preparation is used to improve the success rate of assisted reproduction.

[0015] Compared with the prior art, the present invention has the following advantages: This invention provides a strain of *Lactobacillus curvaturei* LM25V13 isolated and screened from the vagina of healthy mid-pregnancy women (accession number GDMCC67863). This strain exhibits good vaginal colonization, vaginal microecological regulation, and reproductive protection capabilities. In healthy mice, CSTIII-type, and CSTIV-type vaginal model mice, it can upregulate the expression of HOXA10, effectively improve uterine receptivity, and significantly increase the abundance of *Lactobacillus curvaturei* in the mouse vagina. This provides a potential strain for the preparation of female-friendly microecological preparations that improve assisted reproductive outcomes and increase their success rate. Attached Figure Description

[0016] Figure 1 This is a morphological diagram of a *Lactobacillus curvatureensis* strain with assisted reproductive potential proposed in this invention. Figure 2 This is a schematic diagram of the colony morphology and Gram staining of a *Lactobacillus curvatureii* strain with assisted reproductive potential proposed in this invention in a culture medium. Figure 3 This is a complete genome loop of a *Lactobacillus curvatureensis* strain with assisted reproductive potential proposed in this invention; Figure 4 This is a schematic diagram of the phylogenetic tree of the 16S rRNA gene of a curled milk stem with assisted reproductive potential proposed in this invention; Figure 5 The growth curve of a *Lactobacillus curvaturei* strain with assisted reproductive potential proposed in this invention; Figure 6 This is a schematic diagram illustrating the lactic acid production capacity, H2O2 production capacity, Siha cell adhesion capacity, and Gardnerella vaginalis biofilm inhibition capacity of a *Lactobacillus curvatureensis* strain with assisted reproductive potential proposed in this invention. Figure 7 This is a schematic diagram of the SLP expression level of a *Lactobacillus curvatureii* strain with assisted reproductive potential proposed in this invention.

[0017] Figure 8This is a schematic diagram showing the pregnancy rate expression level after topical application of Lactobacillus crispatus LM25V13 to a mouse model of Lactobacillus crispatus LM25V13 with assisted reproductive potential proposed in this invention.

[0018] Figure 9 This is a schematic diagram of the HOX expression level in the uterus during early pregnancy of a *Lactobacillus curvatureii* strain with assisted reproductive potential proposed in this invention.

[0019] Figure 10 This is a schematic diagram of the OPN expression level of a *Lactobacillus curvatureensis* strain with assisted reproductive potential proposed in this invention.

[0020] Figure 11 This is a schematic diagram of the LIF expression level of a *Lactobacillus curvatureensis* strain with assisted reproductive potential proposed in this invention.

[0021] Figure 12 This diagram illustrates the vaginal flora analysis following topical application of Lactobacillus cristataus LM25V13 to a mouse model of Lactobacillus cristataus strain with assisted reproductive potential, as proposed in this invention. Figure 1 .

[0022] Figure 13 This diagram illustrates the vaginal flora analysis following topical application of Lactobacillus cristataus LM25V13 to a mouse model of Lactobacillus cristataus strain with assisted reproductive potential, as proposed in this invention. Figure 2 .

[0023] Figure 14 This diagram illustrates the vaginal flora analysis following topical application of Lactobacillus cristataus LM25V13 to a mouse model of Lactobacillus cristataus strain with assisted reproductive potential, as proposed in this invention. Figure 3 . Detailed Implementation

[0024] The technical solutions of the present invention will be clearly and completely described below with reference to the accompanying drawings of the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments.

[0025] Reference Figures 1-14 A strain of Lactobacillus curvature with assisted reproductive potential is deposited at the Guangdong Provincial Center for Microbial Culture Collection, with accession number GDMCC67863.

[0026] The strain possesses at least one of the following biological characteristics: Rapid growth characteristics: Enters the logarithmic growth phase within 12 hours; Strong ability to produce acid and hydrogen peroxide; Strong adhesion to vaginal cells: It has a strong adhesion ability to SiHa cells; Strong ability to inhibit Gardnerella vaginalis biofilm; High-level expression of S-layer protein: its mRNA expression level was significantly higher than that of the conventional Lactobacillus curvature control strain. Example

[0027] It was obtained from the vaginal secretions of healthy mid-pregnancy women in Northeast China through screening from the Lactobacillus resource bank constructed by our laboratory. The species is named Lactobacilluscrispatus and is named LactobacilluscrispatusLM25V13.

[0028] The above-mentioned bacterial strains were inoculated onto MRS solid medium and cultured at 37°C and 5% CO2 for 24 hours. The morphology of single colonies was then observed visually, and Gram staining was performed for microscopic observation of their properties and cell characteristics. Figure 1 and Figure 2 As shown, its colonies are white, with irregular edges, flaky, and convex in the center; under a microscope, Gram-positive bacilli with slender and curved cells can be seen.

[0029] The above-mentioned single colonies were picked and enriched in 50 ml LMR broth under the above conditions. After centrifugation at 8000 rpm for 5 min at 4°C, the supernatant was discarded and the precipitate was collected. An equal volume of chilled sterile PBS solution was added and the precipitate was washed three times. The precipitate was then rapidly frozen in liquid nitrogen for 10 min and sent to Shanghai Paisenuo Biotechnology Co., Ltd. for whole genome sequencing. The genome map and 16S rRNA phylogenetic tree are shown below. Figure 3 and Figure 4 As shown, further comparison confirmed that it has the highest homology with Lactobacillus curvature.

[0030] The above-mentioned strain was deposited at the Guangdong Provincial Center for Microbial Culture Collection (address: No. 100, Xianlie Middle Road, Yuexiu District, Guangzhou, Guangdong Province) on February 11, 2026, with accession number GDMCC67863.

[0031] Example 2: Please see Figure 5 and Figure 6 ; 1. Growth curve and lactic acid production capacity determination The cryopreserved Lactobacilluscrispatus25V13 solution, which had been frozen in 25% glycerol solution, was inoculated into MRS liquid medium at an inoculation rate of 2% (v / v) and cultured in an anaerobic environment. After two generations of activation, the absorbance of the bacterial solution at OD=600nm was measured every 4 hours from 24 to 48 hours after inoculation, and a growth curve was plotted.

[0032] 1 mL of culture cultured for 24 hours was transferred to a 1.5 mL EP tube. After centrifugation at 8000 rpm for 5 min, the supernatant was collected, and the pH of the supernatant was measured using a pH meter. Simultaneously, the pH of the blank MRS medium was also measured. Finally, the lactate production capacity (ΔpH) was calculated as: pH of culture supernatant - pH of blank MRS medium.

[0033] 2. Determination of H2O2 production This example still uses the supernatant of the culture after centrifugation after 24 hours of culture for determination. First, 3% H2O2 solution was diluted with 100 mmol / L piperazine-1,4-diethanesulfonic acid (PIPES) to prepare 0, 20, 40, 60, 80, and 100 μmol / L H2O2 standards. 100 μL of the above standards and supernatant were transferred and mixed with 100 μL of 20 mmol / L tetramethylbenzidine. Finally, 2 μL of 1 mg / mL horseradish peroxidase solution was added to the system and mixed well. The mixture was incubated at 25°C for 10 min. The absorbance values ​​of the standards and supernatant at OD=620 were measured using a microplate reader. A standard curve was plotted and the formula was used to calculate the H2O2 concentration in the supernatant.

[0034] 3. Assay for the ability to adhere to SiHa cells After activating SiHa cells were cultured to approximately 80% confluence with the culture dish, they were digested with 0.25% trypsin solution to prepare a cell suspension, and then cultured at 1x10⁻⁶ ppm. 6 The cells were added at a concentration of 1 cell / mL to a six-well culture plate containing sterile cell smears and cultured to a suitable density. Before the experiment, the culture medium was replaced with antibiotic-free medium and allowed to acclimate for 24 hours. Unattached cells were washed away with PBS.

[0035] After culturing the above-mentioned lactobacilli in MRS liquid medium for 24 hours, centrifuging at 6000 rpm for 5 minutes and discarding the supernatant, washing twice with PBS, resuspending in antibiotic-free cell culture medium and adjusting the concentration to 1x10⁻⁶. 8 CFU / mL. 2 mL of the above-mentioned Lactobacillus resuspension was added to each well of a six-well plate and incubated at 37°C and 5% CO2 for 2 h. After washing five times with sterile PBS to remove unadhered cells, the cells were fixed with anhydrous methanol for 10 min and then Gram stained. Five randomly selected fields of view under a high-power microscope were evaluated, and the results were expressed as the average number of cells adhering to each SiHa cell.

[0036] 4. Determination of Gardnerella vaginalis biofilm inhibition ability Gardnerella vaginalis was inoculated into BHI broth at a 10% inoculum (v / v%) and cultured anaerobically for 48 hours, then the inoculum was adjusted to 1x10⁻⁶. 8 CFU / mL. Add 100 μL of Gardnerella vaginalis suspension to each well of a 96-well plate and mix with 100 μL of the above-mentioned Lactobacillus supernatant. Use Gardnerella vaginalis suspension and blank MRS medium as negative and blank controls, respectively.

[0037] After anaerobic culture for 24 h, the cells were washed twice with sterile PBS to remove airborne bacteria. 100 μL of anhydrous methanol was added to each well for fixation for 30 min, then discarded. The cells were stained with 0.1% crystal violet solution for 20 min, washed three times with sterile PBS, and finally, 200 μL of anhydrous ethanol was added to each well. The absorbance of each well at 550 nm was measured using a microplate reader. The results were expressed as biofilm inhibition rate, calculated as: [sample well absorbance value - (negative control absorbance value - blank control absorbance value)] × 100%.

[0038] The biological characteristics of *Lactobacillus cristata* are the basis for its probiotic function in the vagina. Results showed that *Lactobacillus cristata* 25V13 entered the logarithmic growth phase (OD600=0.37) after approximately 12 hours of culture and reached the stationary phase (OD600=1.44) after 24 hours. After 24 hours of culture, this strain reduced the pH of the MRS medium from the initial value of 5.60 to 3.48 (ΔpH=2.12). The free H2O2 concentration in the culture supernatant was measured to be 43.38 μmol / L. In vitro adhesion assays of monolayer SiHa cells showed that up to 161 *Lactobacillus* bacteria adhered to each cell. Since *Gardnerella vaginalis* biofilm formation is one of its key pathogenic mechanisms, the inhibitory effect of *Lactobacillus cristata* 25V13 on *Gardnerella vaginalis* biofilm was further evaluated, and the results showed a biofilm inhibition rate of 16.03%. The above results collectively indicate that Lactobacilluscrispatus25V13 possesses excellent vaginal colonization ability, anti-inflammatory activity, and anti-biofilm activity against Gardnerella vaginalis, suggesting that it may be a promising candidate probiotic strain for improving assisted reproductive outcomes and regulating the vaginal environment.

[0039] Example 3: Determination of SLP expression level in *Lactobacillus curvatureii* according to the present invention Please see Figure 7 ; Take 1 mL of the *Lactobacillus curvatureii* suspension from step 1, washed with sterile PBS, and randomly select three *Lactobacillus curvatureii* strains from our laboratory as control strains for the same treatment. After centrifugation of all strains, collect the precipitate, extract total RNA according to the Trizol reagent instructions, and determine its concentration (ng / μL) and purity (A260 / A280) using a nucleic acid detector. Then, reverse transcribe it into cDNA according to the RNA reverse transcription kit instructions. Using the 16S rRNA (V3-V4) gene fragment as an internal reference gene, the expression of the SLP gene of each *Lactobacillus curvatureii* strain was detected by quantitative real-time PCR (qPCR). The relative fold increase of the gene expression in the control strains was 1. The relative expression number of *Lactobacillus curvatureii* strains of the present invention was calculated using the 2^-ΔΔCT method.

[0040] S-layer protein (SLP) is an important molecular structural basis for Lactobacillus cristata to participate in cell adhesion and vaginal immune environment regulation. The results showed that the SLP expression level of Lactobacillus cristata 25V13 was significantly higher than that of three randomly selected reference strains from our laboratory's Lactobacillus cristata resource bank (p < 0.001). Combined with the results in Example 2, this further demonstrates the potential of this strain to improve assisted reproductive outcomes and regulate the vaginal microenvironment.

[0041] Example 4, Animal Experiment Please see Figure 8 , Figure 9 , Figure 10 , Figure 11 , Figure 12 , Figure 13 and Figure 14 ; In simple terms, the animal modeling method involves using 6-week-old female ICR mice and administering a 1x10⁻¹ concentration of medication intravaginally for 10 consecutive days. 8 CFU / mL of inert Lactobacillus or Gardnerella vaginalis suspension was used to establish CSTIII or CSTIV dysbiosis models, respectively. After model establishment, mice were given 1x10 ml of the suspension for 7 consecutive days. 8 Intervention was performed using a CFU / m concentration of Lactobacillus crispatus 25V13 bacterial suspension. After the intervention, mice were housed together at a female:male ratio of 2:1, and vaginal plugs were observed the morning after the intervention to determine pregnancy. The presence of vaginal plugs was recorded as day 0.5 of the embryonic period. Mice were sacrificed on day 7.5 of the embryonic period, and uterine tissue was collected to determine the pregnancy rate.

[0042] The determination of uterine receptivity-related indicators (HOXA10, OPN, and LIF) was performed using qPCR. The kits, methods, and data processing methods used for total RNA extraction and reverse transcription were the same as in Example 3, except that the internal reference gene was β-actin.

[0043] The results showed that the pregnancy rate increased after topical vaginal application of Lactobacillus crispatus 25V13 to both CSTIII and CSTIV mouse models. Furthermore, the pregnancy rate reached 100% in healthy mice after application of this bacterium. HOXA10, OPN, and LIF are key molecules involved in regulating uterine receptivity and embryo implantation, with HOXA10 playing a dominant role in determining uterine receptivity. The results indicated that application of this bacterium significantly increased HOXA10 gene expression in both mouse models (p=0.044; p=0.061, respectively). OPN and LIF gene expression levels showed an increasing trend in both mouse models after application of this bacterium, while only OPN gene expression showed an increasing trend in healthy mice, but these differences were not statistically significant.

[0044] In summary, the results show that topical vaginal application of Lactobacilluscrispatus 25V13 can improve uterine receptivity and increase pregnancy success rate in mice, demonstrating its potential in assisted reproduction.

[0045] Example 5: Measurement and Analysis of Vaginal Flora in Mice Please see Figure 12 , Figure 13 and Figure 14 ; During the animal experiment, vaginal lavage fluid from mice was collected at two time points: after the modeling process and after the intervention with Lactobacillus curvature of this invention. Total bacterial DNA was extracted from the lavage fluid according to the QlAampDNAminiKit instructions, and its concentration and purity were determined by a nucleic acid concentration detector. Subsequently, PCR amplification and Illumina MiSeq sequencing were performed.

[0046] The V3–V4 region of the 16S rRNA gene was amplified using the following universal primers: 338F: 5'-ACTCCTACGGGAGGCAGCA-3' and 806R: 5'-GGACTACHVGGGTWTCTAAT-3'.

[0047] After initial quality control, paired-end sequencing data were processed using the 16S rRNA gene workflow of the dada2R package (v1.1) to obtain amplicon sequence variants (ASVs). Subsequently, taxonomic annotation of the species was performed using the TaxminerR package and the Sliva database.

[0048] The results showed that the application of live Lactobacilluscrispatus LM25V13 to the vagina of mice enabled it to colonize continuously and increase in abundance, and regulate the structure of the vaginal flora, demonstrating its ability to regulate the vaginal microecological structure.

[0049] Summary of experimental data: Table 1: Effects of LM25V13 and existing strains on endometrial receptivity gene expression in CSTIII / IV mouse models (qPCR)

[0050] Note: p<0.05 vs. blank control; #p<0.05 vs. control strain A / B*; As shown in Table 1 above, this experiment employed a dual-model parallel validation strategy, constructing CSTIII (Lactobacillus inertia perfusion model) and CSTIV (Gardnerella vaginalis perfusion model) mouse models of vaginal microecological imbalance, respectively. The two models correspond to the clinically common sub-health state (CSTIII type, dominated by inert lactobacilli, lacking D-lactate and S-layer protein synthesis ability) and pathological state (CSTIV type, dominated by anaerobic pathogens, with strong biofilm formation ability), respectively. After successful model establishment, 1×10 8 CFU / mL target strain suspension was continuously used for 7 days. Embryos were sacrificed on day 7.5 after embryo transfer, and total RNA was extracted from uterine tissue using the Trizol method. After the purity was determined by a nucleic acid concentration analyzer (A260 / A280=1.8-2.0), it was reverse transcribed into cDNA. The expression levels of HOXA10 and LIF mRNA were detected by SYBR Green real-time quantitative PCR (qPCR). β-actin was used as the internal reference gene, and the relative expression level was calculated using the 2^(-ΔΔCT) method. HOXA10 (homeobox A10 gene) is a core transcription factor determining endometrial receptivity. Its expression level directly regulates the proliferation and differentiation of endometrial stromal cells and the establishment of the embryo implantation window. Results show: In the CSTIII model, the relative expression level of HOXA10 mRNA in the LM25V13 group was 2.35±0.42, which was upregulated by 135% compared with the blank control group (p<0.05), by 65.5% compared with control strain A (1.42±0.28) (p<0.05), and by 70.3% compared with control strain B (1.38±0.31) (p<0.05). This indicates that under sub-healthy microecological conditions, LM25V13 can significantly activate HOXA10 expression, and the effect is superior to that of existing strains.

[0051] In the CSTIV model, the relative expression level of HOXA10 mRNA in the LM25V13 group was 2.18±0.38, which was upregulated by 118% compared with the blank control group (p<0.05), upregulated by 66.4% compared with control strain A (1.31±0.25) (p<0.05), and upregulated by 50.3% compared with control strain B (1.45±0.29) (p<0.05). This indicates that under pathological microecological conditions, LM25V13 can still maintain a high level of HOXA10 activation, while the effect of existing strains is limited in inflammatory environments. One-way ANOVA was used for inter-group comparisons, and Tukey's method was used for multiple comparison correction. The comparisons between the LM25V13 group and the blank control group, control strain A, and control strain B all reached a statistically significant level (p < 0.05), while there were no significant differences between control strains A and B and the blank control group (p > 0.05). This demonstrates that LM25V13 has a statistically significant advantage in upregulating HOXA10 and LIF. In two different pathological models of vaginal microecological dysbiosis, LM25V13 significantly upregulated the expression of the core endometrial receptivity genes HOXA10 and LIF, with the upregulation magnitude significantly superior to that of the existing Lactobacillus curvature control strain. This advantage may be related to its high level of S-layer protein expression and strong acid-producing capacity. S-layer proteins maintain vaginal-uterine axis microbiota homeostasis by enhancing vaginal epithelial adhesion and immune regulation, and thus indirectly regulate the expression of uterine receptivity genes through the microbiota-endometrial axis.

[0052] Table 2: Effects of LM25V13 on the expression of receptivity genes in human endometrial cells RL95-2 (qPCR)

[0053] Note: p<0.05 vs. blank control; #p<0.05 vs. control strain A / B*; As shown in Table 2 above, the RL95-2 cell line (ATCCCRL-1671) is derived from well-differentiated adenocarcinoma tissue of the human endometrium. It retains the morphological characteristics and hormone responsiveness of normal endometrial epithelial cells, expressing estrogen receptor (ER) and progesterone receptor (PR), and upregulating the expression of receptivity genes such as HOXA10 and LIF in response to estrogen / progesterone stimulation. This cell line is an internationally recognized in vitro model for endometrial receptivity research; its gene expression profile is highly conserved with that of mouse endometrial tissue, and the experimental results have clinical translational value. LM25V13 and control strains (control strain A: Lactobacillus curlis randomly selected in our laboratory; control strain B: ATCC33820) were anaerobically cultured in MRS liquid medium for 24 h (37℃, 5% CO2). The cultures were centrifuged at 8000 rpm for 5 min, and the supernatant was collected and filtered through a 0.22 μm sterile filter to remove bacterial cells, yielding a sterile metabolic supernatant. The main active components in the supernatant were lactic acid (D-lactic acid and L-lactic acid), hydrogen peroxide (H2O2), bacteriocins, and S-layer protein fragments. pH measurements showed that the supernatant of LM25V13 was 3.48, the supernatant of control strain A was 4.12, and the supernatant of control strain B was 4.05, indicating that LM25V13 had a significantly stronger acid-producing capacity than the control strain. HOXA10 is a master switch gene for endometrial receptivity, and its expression is dually regulated by the Wnt / β-catenin and estrogen signaling pathways. The low pH environment (pH 3.48) and high lactate concentration in the LM25V13 supernatant may activate HOXA10 through the following mechanisms: Lactate, as a histone deacetylase inhibitor (HDACi), promotes histone acetylation in the HOXA10 promoter region and enhances transcriptional activity. Low pH environments activate cell membrane G protein-coupled receptors (GPR81 / 132A), which regulate downstream gene expression through the cAMP / PKA pathway. The relative expression level of LIF mRNA in the LM25V13 supernatant treatment group was 1.65±0.22, which was upregulated by 65% ​​compared with the blank control group (p<0.05), upregulated by 39.8% compared with the control strain A supernatant group (1.18±0.14) (p<0.05), and upregulated by 47.3% compared with the control strain B supernatant group (1.12±0.11) (p<0.05). The upregulation of LIF in the control strain A and B supernatants was less than 20%, and there was no statistical significance (p>0.05). LIF expression is directly regulated by HOXA10, which activates transcription by binding to the ATTA sequence in the LIF promoter region. The upregulation effect of LM25V13 supernatant on LIF is slightly lower than that of HOXA10 (65% vs. 78%), which is consistent with the cascade regulatory relationship of HOXA10→LIF. This further verifies the mechanism by which LM25V13 indirectly upregulates LIF through the HOXA10 pathway.

[0054] In an in vitro human endometrial cell model, the metabolic supernatant of LM25V13 directly upregulated the expression of HOXA10 and LIF genes, with significantly better results than the existing control strain of *Lactobacillus curvature*. This finding demonstrates that the active components of LM25V13 (lactic acid, H2O2, S-layer proteins, etc.) have direct biological activity on endometrial cells, independent of indirect regulation by the vaginal flora. This provides in vitro mechanistic evidence and a clinical translational basis for the development of vaginal microecological preparations using LM25V13 in assisted reproductive technology.

[0055] The above description is only a preferred embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any equivalent substitutions or modifications made by those skilled in the art within the scope of the technology disclosed in the present invention, based on the technical solution and inventive concept of the present invention, should be covered within the scope of protection of the present invention.

Claims

1. A strain of *Lactobacillus curvatureensis* with assisted reproductive potential, characterized in that, The strain is deposited at the Guangdong Provincial Center for Microbial Culture Collection, with accession number GDMCC67863.

2. The *Lactobacillus curvatureii* strain with assisted reproductive potential according to claim 1, characterized in that, The strain possesses at least one of the following biological characteristics: Rapid growth characteristics: Enters the logarithmic growth phase within 12 hours; Strong ability to produce acid and hydrogen peroxide; Strong adhesion to vaginal cells: It has a strong adhesion ability to SiHa cells; Strong ability to inhibit Gardnerella vaginalis biofilm; High-level expression of S-layer protein: its mRNA expression level was significantly higher than that of the conventional Lactobacillus curvature control strain.

3. The application of the *Lactobacillus curvatureii* strain with assisted reproductive potential according to claim 1 in a vaginal microecological preparation, characterized in that... The applications include their use in the preparation of female-friendly vaginal microecological preparations.

4. The application of the *Laminaria japonica* strain with assisted reproductive potential according to claim 3 in the preparation of vaginal microecological preparations, characterized in that... The regulatory effects include increasing the abundance of *Lactobacillus curvatureensis* in healthy mice and mice with CSTIII or CSTIV vaginal flora structure models.

5. The application of a *Lactobacillus curvaturei* strain with assisted reproductive potential in a vaginal microecological preparation, wherein the strain of *Lactobacillus curvaturei* with assisted reproductive potential as described in claim 1 is characterized in that, The applications include their use in the preparation of vaginal microecological preparations that improve assisted reproductive outcomes.

6. The application of a *Lactobacillus curvatureii* strain with assisted reproductive potential according to claim 5 in a vaginal microecological preparation, characterized in that... The improvement in assisted reproductive outcomes includes increasing uterine receptivity.

7. The application of a *Lactobacillus curvatureii* strain with assisted reproductive potential according to claim 5 in a vaginal microecological preparation, characterized in that, Improving uterine receptivity is achieved by upregulating the expression levels of at least one of the homeobox A10 gene, osteopontin gene, and leukemia repressor gene.

8. The application of the *Lactobacillus curvatureii* strain with assisted reproductive potential according to claim 8 in a vaginal microecological preparation, characterized in that, The upregulation of HOXA10 gene expression was significantly better than that of the existing Lactobacillus curvature control strain. In CSTIII model mice, the upregulation of HOXA10 was statistically significant (p=0.044), and in CSTIV model mice, the upregulation of HOXA10 showed a significant trend (p=0.061).

9. A vaginal microecological preparation, characterized in that, The ecological preparation contains the active ingredient of Lactobacillus curvature as described in claim 1.

10. A vaginal microecological preparation according to claim 9, characterized in that, The formulation is used to improve the success rate of assisted reproduction.

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