Lactobacillus reuteri and its use in alleviating perimenstrual syndrome

Abstract

The present application discloses Lactobacillus reuteri and its application in relieving perimenstrual syndrome, and relates to the technical field of biology. Limosilactobacillus reuteri ​ Lactobacillus reuteri (Lactobacillus reuteri) -NCU-37 was preserved in China General Microbiological Culture Collection Center on February 24, 2025, and the address is No. 1, Xibaixili, Chaoyang District, Beijing, and the preservation number is CGMCC NO. 33627. The Lactobacillus reuteri -NCU-37 of the present application can relieve perimenstrual syndrome induced by leuprolide in assisted reproductive treatment, specifically relieve perimenstrual syndrome and related emotional disorders (including anxiety, depression and insomnia), improve the hormone level of infertile women with perimenstrual syndrome, and relieve the symptoms of infertile women with perimenstrual syndrome by regulating intestinal flora diversity.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, specifically to a strain of *Lactobacillus reuteri* and its application in alleviating perimenopausal syndrome. Background Technology

[0002] Leuprorelin, a commonly used gonadotropin-releasing hormone (GnRH) analog, binds to GnRH receptors in the anterior pituitary gland, leading to a decrease in the secretion of pituitary gonadotropins (FSH and LH), thereby inhibiting the secretion of estrogen and progesterone from the ovaries. It has significant efficacy in the treatment of gynecological diseases, such as controlling the menstrual cycle, reducing the size of uterine fibroids, and improving the symptoms of endometriosis. However, its use may also induce a series of side effects, the most significant being perimenopausal syndrome. Perimenopausal syndrome is a series of physiological and psychological symptoms caused by the gradual decline in ovarian function and estrogen levels, such as hot flashes, night sweats, mood swings (e.g., irritability, anger, depression, emotional instability), insomnia, decreased libido, osteoporosis, and lower back and leg pain. These symptoms mainly arise because the decline in estrogen levels affects the normal function of multiple systems. Generally, after discontinuing leuprorelin, as ovarian function gradually recovers and estrogen levels rise, perimenopausal syndrome-like side effects will gradually lessen or disappear. However, current treatment options for menopausal symptoms after using leuprorelin are limited. Currently, for patients experiencing significant perimenopausal syndrome symptoms, doctors can provide appropriate symptomatic treatments, such as estrogen supplementation, dosage adjustment, or switching to other treatment regimens. Therefore, finding safer and more effective treatments to alleviate perimenopausal syndrome induced by leuprorelin is urgently needed.

[0003] In recent years, an increasing number of studies have focused on the application of probiotics in perimenopausal syndrome. Probiotics are a class of live microorganisms that are beneficial to the host's health. They maintain gut health by regulating the balance of gut microbiota, inhibiting the growth of harmful bacteria, and promoting the absorption and metabolism of nutrients. In patients with perimenopausal syndrome, probiotic supplementation can help restore the balance of gut microbiota and alleviate various discomfort symptoms caused by dysbiosis. Some studies have found that supplementation with specific types of probiotics (such as Bifidobacterium lactis and Lactobacillus plantarum) can alleviate certain symptoms of perimenopausal syndrome, such as hot flashes and mood swings. This may be related to probiotics' ability to regulate hormone levels and improve nervous system function. Meanwhile, studies have also shown that combined intervention with Lactobacillus plantarum 30M5 and soy isoflavones (SIF) can effectively alleviate metabolic disorder symptoms in ovariectomized mice, including lowering blood glucose, total cholesterol, and high-density lipoprotein cholesterol levels. This reveals the important role of probiotics in regulating gut microbiota and provides new insights into their application in perimenopausal syndrome. Summary of the Invention

[0004] The purpose of this invention is to at least solve one of the technical problems existing in the prior art, and to provide a strain of *Lactobacillus reuteri* and its application in alleviating perimenopausal syndrome.

[0005] The technical solution of the present invention is as follows:

[0006] In a first aspect, the present invention provides a strain of *Lactobacillus reuteri*-NCU-37, wherein *Lactobacillus reuteri* ( Limosilactobacillus reuteri NCU-37 was deposited on February 24, 2025, at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, with accession number CGMCCNO.33627.

[0007] Optionally, the gene sequence of the 16S rDNA of *Lactobacillus reuteri*-NCU-37 is shown in SEQ ID No. 1.

[0008] Secondly, the present invention provides the use of the aforementioned *Lactobacillus reuteri*-NCU-37 in the preparation of products that alleviate and / or improve perimenopausal syndrome.

[0009] Optionally, the perimenopausal syndrome is perimenopausal syndrome induced by leuprorelin during assisted reproductive treatment.

[0010] Optionally, the product may function to relieve and / or improve symptoms of perimenopausal syndrome, including at least one of hot flashes, sweating, mood swings, fatigue, and joint pain.

[0011] Optionally, the product may function to alleviate and / or improve mood disorders caused by perimenopausal syndrome, including at least one of anxiety, depression, and insomnia.

[0012] Optionally, the product may function to increase hormone levels in infertile women with perimenopausal syndrome, the hormones including anti-Müllerian hormone, estradiol, follicle-stimulating hormone, and luteinizing hormone.

[0013] Optionally, the product may function to regulate the gut microbiota and reduce the proliferation and abundance of pathogenic bacteria of the genus Bacteroides.

[0014] Optionally, the product may include a drug or a microbial agent.

[0015] Thirdly, the present invention provides a product for alleviating and / or improving perimenopausal syndrome, the product comprising the aforementioned *Lactobacillus reuteri*-NCU-37 or its ferments or metabolites.

[0016] This invention has at least one of the following beneficial effects:

[0017] This invention obtained a strain of *Lactobacillus reuteri*-NCU-37 through screening. Research revealed that this bacterium can alleviate perimenopausal syndrome induced by leuprorelin during assisted reproductive technology (ART). Studies have shown that *Lactobacillus reuteri*-NCU-37 of this invention can significantly alleviate perimenopausal syndrome and related mood disorders (including anxiety, depression, and insomnia), and can significantly increase hormone levels in infertile women with perimenopausal syndrome (LIPS). Furthermore, it may alleviate symptoms in infertile women with perimenopausal syndrome (LIPS) by regulating gut microbiota diversity, particularly by reducing the proliferation of potentially pathogenic bacteria such as *Bacteroides* and reducing the relative abundance of *Bacteroides* in the gut microbiota. Attached Figure Description

[0018] Figure 1 The figures show the results of acid and bile salt tolerance tests and microscopic morphology of *Lactobacillus reuteri* NCU-37; where A in the figure represents the results of the acid tolerance test; B represents the results of the bile salt tolerance test; and C represents the microscopic morphology.

[0019] Figure 2 The severity of perimenopausal symptoms and mood was assessed in all participants. In the figure, A represents the Modified Cooperman Index (MKI) score; B represents the Hamilton Anxiety Rating Scale (HAMA) score; C represents the Hamilton Depression Rating Scale (HAMD) score; and D represents the Athens Insomnia Scale (AIS) score. C: Healthy group (n=50); MP: Placebo group (n=46); ML: Probiotic group (n=47). Data are expressed as median and interquartile range. Normality of the dataset was analyzed using the Shapiro-Wilk test. Continuous variables were compared using one-way repeated measures ANOVA, followed by multiple comparisons using the Dunn test. Significance is expressed as *p<0.05.

[0020] Figure 3The levels of hormones associated with perimenopausal symptoms in the blood of all participants are shown in the figure; where A: P (progesterone, ng / mL); B: AMH (anti-Müllerian hormone, ng / mL); C: E2 (estradiol, pg / mL); D: FSH (follicle-stimulating hormone, mIU / mL); E: LH (luteinizing hormone, mIU / mL). C: healthy group (n=50); MP: placebo group (n=46); ML: probiotic group (n=47). Data are expressed as median and interquartile range. Normality of the dataset was analyzed using the Shapiro-Wilk test. Continuous variables were compared using one-way repeated measures ANOVA, followed by multiple comparisons using the Dunn test. Significance is expressed as *p<0.05.

[0021] Figure 4 The figures show the differences between groups of participants; where A: α diversity index (Chao1 index, Faith_pd index, Shannon index, and number of observed species); B: Principal coordinate analysis (PCoA); C: Venn diagram; D: Principal coordinate analysis (PCoA); E: Dilution curve. C: Healthy group (n=35); MP: Placebo group (n=35); ML: Probiotic group (n=35). Data are expressed as median and interquartile range. The normality of the dataset was analyzed using the Shapiro-Wilk test. Continuous variables were compared using one-way repeated measures ANOVA, followed by multiple comparisons using the Dunn test. Significance is expressed as *p<0.05.

[0022] Figure 5 Analysis of fecal microbiota at the phylum and genus levels for each group; where, A in the figure: taxonomic composition at the phylum level; B: relative abundance of Firmicutes; C: relative abundance of Bacteroidetes; D: taxonomic composition at the genus level; E: relative abundance of Blautia; F: relative abundance of Bacteroides; G: relative abundance of Parabacteroides; H: relative abundance of Coprococcus; I: relative abundance of Prevotella; J: relative abundance of *Lactobacillus reuteri* in fecal microbiota detected by RT-qPCR. Limosilactobacillus reuteriThe relative levels of the probiotics group (n=35) were calculated. C: healthy group (n=35); MP: placebo group (n=35); ML: probiotic group (n=35). Data are expressed as mean ± standard deviation (SD) or median and interquartile range. Normality of the datasets was analyzed using the Shapiro-Wilk test. Continuous variables were compared using one-way repeated measures ANOVA, followed by multiple comparisons using the Tukey test or Dunn test. Significance is expressed as *p<0.05.

[0023] Figure 6 Differential species analysis of fecal microbiota was performed for each group. In the figure, A: LEfSe analysis; B: random forest analysis at the genus level. C: healthy group (n=35); MP: placebo group (n=35); ML: probiotic group (n=35).

[0024] Figure 7 This study analyzed the correlation between gut microbiota, hormones, and perimenopausal symptoms using Spearman correlation analysis. Red indicates a positive correlation, and blue indicates a negative correlation. Detailed Implementation

[0025] To make the technical problems solved, the technical solutions, and the beneficial effects of this invention clearer, the invention will be further described in detail below with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention.

[0026] Example 1: Screening and identification of Lactobacillus reuteri-NCU-37

[0027] In this invention, *Lactobacillus reuteri* NCU-37 was isolated from a normal healthy human fecal sample (26-year-old female). The specific isolation method is as follows:

[0028] Under anaerobic conditions, take an appropriate amount of fresh human fecal sample and perform serial dilutions using sterile phosphate-buffered saline (PBS). Select an appropriate dilution (e.g., 10⁻⁶). -1 Up to 10 -7 The sample dilution was spread onto MRS agar plates containing 0.2% L-cysteine ​​hydrochloride to promote the growth of lactic acid bacteria and maintain an anaerobic environment. The inoculated plates were then placed in an anaerobic incubator at 37°C for 72 hours. Single colonies that resembled lactobacilli (e.g., small, milky-white, round, smooth-surfaced colonies with regular edges) were picked from the plates and examined under a Gram stain. Gram-positive, slender rod-shaped bacteria were identified as the initial screening targets. The initially screened colonies were repeatedly streaked onto fresh MRS plates for purification to obtain pure cultures.

[0029] Through comprehensive analysis of the strain's morphology, physiological and biochemical characteristics, 16S rRNA gene sequence (as shown in SEQ ID No. 1), and dnaK gene sequence, the target strain was finally identified as *Lactobacillus reuteri*. Limosilactobacillus reuteri NCU-37 was deposited on February 24, 2025, at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, with accession number CGMCCNO. 33627. Its microscopic morphology (oil immersion) is shown below. Figure 1 As shown in C.

[0030] SEQ ID No. 1:

[0031] GGGGGGCAGGGGGGGGGGCTAATGATGGCGGGTCGTACGCACTGGCCCAACTGATTGATGGTGCTTGCACCTGATTGACGATGGATCACCAGTGAGTGGCGGACGGGTGAGTAACACGTAGGTAACCTGCCCCGGAGCGGGGGATAACATTTGGAAACAGATGCTAATACCGCATAACAACAAAAGCCGCATGGCTTTTGTTTGAAAGATGGCTTTGGCTATCACTCTGGGATGGACCTGCGGTGCATTAGCTAGTTGGTAAGGTAACGGCTTACCAAGGCGATGATGCATAGCCGAGTTGAGAGACTGATCGGCCACAATGGAACTGAGACACGGTCCATACTCCTACGGGAGGCAGCAGTAGGGAATCTTCCACAATGGGCGCAAGCCTGATGGAGCAACACCGCGTGAGTGAAGAAGGGTTTCGGCTCGTAAAGCTCTGTTGTTGGAGAAGAACGTGCGTGAGAGTAACTGTTCACGCAGTGACGGTATCCAACCAGAAAGTCACGGCTAACTACGTGCCAGCAGCCGCGGTAATACGTAGGTGGCAAGCGTTATCCGGATTTATTGGGCGTAAAGCGAGCGCAGGCGGTTGCTTAGGTCTGATGTGAAAGCCTTCGGCTTAACCGAAGAAGTGCATCGGAAACCGGGCGACTTGAGTGCAGAAGAGGACAGTGGAACTCCATGTGTAGCGGTGGAATGCGTAGATATATGGAAGAACACCAGTGGCGAAGGCGGCTGTCTGGTCTGCAACTGACGCTGAGGCTCGAAAGCATGGGTAGCGAACAGGATTAGATACCCTGGTAGTCCATGCCGTAAACGATGAGTGCTAGTGTTGGAGGGTTTCCGCCCTTCAGTGCCGGAGCTAACGCATTAGCACTCCTCCTGGGGAGTACGACCGCAGGGTTGGAGACTCAATGGATTTGACTGGGCCCGGCACAAGCGCTGGCACCATGTGGA。

[0032] Example 2: Determination of acid and bile salt tolerance of Lactobacillus reuteri-NCU-37

[0033] (1) Acid resistance test

[0034] Lactobacillus reuteri NCU-37 was cultured overnight in MRS liquid medium at 37°C in an aerobic incubator. 100 µL of the culture was then diluted with PBS buffer. 1 times, 10 3 times, 10 5 Centrifuge at 4000 rpm for 3 min and discard the supernatant. Add PBS buffer (pH 2, 3, 4, 5, 7), incubate for 4 h, mix well, and spread 10 μL onto a plate. Incubate in an anaerobic incubator at 37℃ for 48 h and count the viable bacteria.

[0035] (2) Bile salt tolerance test

[0036] Lactobacillus reuteri-NCU-37 was cultured in MRS liquid medium containing 0.3% and 0.5% ox bile salts and incubated at 37°C for 24 h. After mixing, 10 μL was plated and incubated at 37°C for 48 h. Viable bacteria were counted and experimental records were made.

[0037] The results are as follows Figure 1 A and Figure 1 As shown in B, by Figure 1 As shown in A, the viable count of *Lactobacillus reuteri*-NCU-37 is approximately 10 in a strongly acidic environment at pH 2. 6 CFU / mL; by Figure 1 As shown in B, the viable count of *Lactobacillus reuteri*-NCU-37 in a 0.5% bovine bile salt environment is approximately 10. 8 CFU / mL. The results showed that this strain had good tolerance to both strong acids and bile salts.

[0038] Example 3: Study on the role of *Lactobacillus reuteri*-NCU-37 in alleviating leuprorelin-induced perimenopausal syndrome during assisted reproductive treatment.

[0039] I. Research Subjects

[0040] The study was conducted from October 1, 2024 to August 31, 2025, collecting data on perimenopausal syndrome induced by leuprorelin in healthy women and infertile women during assisted reproductive technology. The subjects were from the Reproductive Hospital of Nanchang City, Jiangxi Province.

[0041] II. Inclusion and Exclusion Criteria

[0042] (1) Normal healthy women

[0043] Inclusion criteria: 1. Healthy women with regular menstrual cycles (menstrual cycle of 21-35 days, menstrual period of 2-7 days). 2. Age 20-40 years. 3. Voluntary participation and signing of informed consent.

[0044] Exclusion criteria: 1. Menstrual irregularities within the past two months. 2. Planning pregnancy or currently pregnant, breastfeeding, or lactating. 3. History of miscarriage (medical or surgical) within the past six months. 4. Use of antibiotics or birth control pills within the past month. 5. Female-factor infertility and / or female infertility due to male factors. 6. No significant organ dysfunction or other serious illnesses. 7. Currently participating in other clinical trials.

[0045] (2) Menopausal syndrome patients

[0046] Inclusion criteria: ① Infertile women aged 20-40 years (female-factor infertility and / or female-cause infertility due to male factors), with baseline hormone levels (before leuprorelin intervention): FSH: 3mIU / mL-10mIU / mL, LH: 3mIU / mL-10mIU / mL, E2: 10pg / mL-70pg / mL, AMH ≥1.2ng / mL, and a total of >7 antral follicles. ② Meeting IVF indications. ③ No unexplained abnormal uterine and vaginal bleeding. ④ Developing perimenopausal syndrome symptoms after leuprorelin use, such as hot flashes, sweating, mood swings, insomnia, fatigue, and joint pain, with one or more symptoms present, and these symptoms must reach a certain severity and duration to meet the diagnostic criteria for perimenopausal syndrome. ⑤ Treatment history: No recent (e.g., within the last 6 months) use of other hormonal drugs or treatments that may affect the study results. ⑥ Informed consent: Patients must fully understand the research content, purpose, risks and benefits, and sign an informed consent form to indicate their willingness to participate in the research.

[0047] Exclusion Criteria: ① Pregnant or lactating women are not suitable for this type of study. ② Unexplained abnormal uterine and vaginal bleeding. ③ Premature ovarian failure not caused by organic factors or surgery, or ovarian insufficiency caused by chromosomal abnormalities. ④ Dysfunction of vital organs, such as severe dysfunction of the heart, liver, kidneys, etc. ⑤ Patients with other endocrine disorders. ⑥ Those who have used antibiotics or oral probiotics within the past month. ⑦ Those allergic to gonadotropin-releasing hormone agonists (such as leuprorelin) and excipients. ⑧ Other serious diseases: Those with serious genetic, physical, or mental illnesses that make pregnancy unsuitable, as well as those with acute infectious diseases of the reproductive and urinary systems or sexually transmitted diseases, are not suitable for inclusion in the study. ⑨ Recent exposure to harmful substances: Such as recent exposure to teratogenic doses of radiation, toxic substances, or taking teratogenic drugs or narcotics and being within the period of their effects. ⑩ Other non-compliance conditions: Such as other specific exclusion criteria specified in the study design, such as specific types of endometriosis, specific degrees of menopausal syndrome symptoms, etc.

[0048] III. Experimental grouping and treatment: A randomized, controlled, double-blind trial was conducted.

[0049] Grouping and Treatment:

[0050] (1) Normal healthy group (Group C, n=50 people): Collect normal healthy people without intervention.

[0051] (2) Placebo group (MP group, n=50 people): Infertile women who experienced perimenopausal syndrome due to taking leuprorelin during assisted reproduction were treated with oral placebo (maltodextrin) for 4 weeks.

[0052] (3) Probiotic group (MY group, n=50 people): Infertile women who experienced perimenopausal syndrome due to taking leuprorelin during assisted reproductive technology were treated with oral probiotics for 4 weeks. Probiotic selected: Lactobacillus reuteri-NCU-37.

[0053] IV. Indicator Testing:

[0054] 1. General Clinical Indicator Testing: ① Basic Information Statistics: Patient age, gender, BMI, etc. were collected. ② Blood Sex Hormone Indicator Testing (Before and After Intervention): Venous blood was drawn in the morning after fasting for 12 hours to test serum estradiol (E2), follicle-stimulating hormone (FSH), luteinizing hormone (LH), and anti-Müllerian hormone (AMH). ③ Blood Biochemical Indicator Testing: Venous blood was collected after leuprorelin administration to test liver function indicators. ④ Antral Follicle Recovery: The number and size of antral follicles and the recovery of ovarian function were detected by transvaginal ultrasound in both groups of patients.

[0055] 2. Perimenopausal Syndrome Scale Scores (Before and After Intervention)

[0056] Perimenopausal syndrome scale: Modified Kupperman score (KMI), see Table 1 for details.

[0057] Anxiety Scale: Hamilton Anxiety Scale (HAMA), see Table 2 for details.

[0058] Depression scale: Hamilton Depression Rating Scale (HAMD), see Table 3 for details.

[0059] Insomnia scale: Athens Insomnia Scale (AIS), see Chart 4 for details.

[0060] Table 1

[0061]

[0062] Table 2

[0063]

[0064] Table 3

[0065]

[0066] Table 4

[0067]

[0068] 3. High-throughput analysis of the characteristics of the two groups of microbial communities

[0069] ① Sample Collection: Fecal samples were collected from each group. ② DNA Extraction and Sequencing: Gut microbiota DNA was extracted according to standard operating procedures and sent to a professional company for high-throughput sequencing. The V4 region of the 16S rDNA gene was analyzed, and the PCR products were sequenced. ③ Data Analysis: Based on the effective sequencing data, the α-diversity and β-diversity of the gut microbiota were analyzed using QIIME software. SIMCA-P software was used for the identification, cluster analysis, and partial least squares discriminant analysis of different rich taxa.

[0070] 4. Statistical Analysis

[0071] Data analysis was performed using SPSS 25.0. Quantitative data conforming to a normal distribution were expressed as mean ± standard deviation, and independent samples t-tests were used for comparisons between groups. Data not conforming to a normal distribution were expressed as median (interquartile range), and Wilcoxon rank-sum tests were used for comparisons between groups. Categorical data were expressed as n (%), and chi-square tests were used for comparisons between groups. 2 The test or Fisher's exact test is used. Indicators that are significant in univariate analysis are included in the multivariate binary logistic regression model. P < 0.05 is considered statistically significant.

[0072] The experimental results are as follows:

[0073] (1) Baseline characteristics of the subjects

[0074] This study initially recruited 243 women. After rigorous screening based on inclusion and exclusion criteria, 45 did not meet the inclusion criteria, 11 refused to participate, and 37 withdrew for other reasons. Ultimately, 150 participants were enrolled and randomly assigned to either the healthy control group (Group C, n = 50), the placebo group (Group MP, n = 50), or the probiotic group (Group ML, n = 50). During follow-up, due to loss to follow-up and intervention interruption, a total of 143 participants completed the clinical trial: 50 in Group C, 46 in Group MP, and 47 in Group ML.

[0075] Baseline characteristic analysis showed that the ages of the three groups were 33.50 years, 34.00 years, and 35.00 years, respectively, with no significant difference between groups (p = 0.7926) (see Table 5). The mean BMI values ​​of the three groups were 22.55 ± 2.62, 22.46 ± 2.88, and 22.48 ± 3.14, respectively, with no statistically significant difference (p = 0.9880). The MKI scores of the three groups were 3.00, 13.50, and 13.00, respectively, with a significant difference (p < 0.0001). Compared with group C, the HAMA (anxiety), HAMD (depression), and AIS (insomnia) scores of the MP and ML groups were significantly higher. These results indicate that, compared with healthy individuals, infertile women with leuprorelin-induced perimenopausal symptoms (LIPS) experience more severe perimenopausal symptoms and tend to have mild anxiety, depression, and insomnia, while there were no significant differences in these indicators between the MP and ML groups. Furthermore, hormone testing results showed that the levels of E2, P, and AMH in the MP and ML groups were significantly lower than those in the C group, but there was no significant difference between the MP and ML groups. In summary, there were no significant differences between the placebo and probiotic groups on all baseline indicators, indicating that the two groups were highly homogeneous at the start of the study.

[0076] Table 5. Basic characteristics of all subjects

[0077]

[0078] Note: BMI: Body Mass Index; MKI: Modified Kupperman Index; HAMA: Hamilton Anxiety Rating Scale; HAMD: Hamilton Depression Rating Scale; AIS: Athens Insomnia Scale. FSH: Follicle-stimulating hormone; LH: Luteinizing hormone; E2: Estradiol; P: Progesterone; AMH: Anti-Müllerian hormone. C: Healthy group (n=50); MP: Placebo group (n=46); ML: Probiotic group (n=47). Data are expressed as mean ± standard deviation (SD) and median (interquartile range). " / " indicates no relevant data. The Shapiro-Wilk test was used to analyze the normality of the datasets. Continuous variables were compared using one-way repeated measures ANOVA, followed by multiple comparisons using the Tukey test or Dunn test. Significance is expressed as *p<0.05. There was no significant difference between the MP and ML groups.

[0079] (2) Lactobacillus reuteri-NCU-37 significantly improved perimenopausal symptoms and mood disorders in infertile women with perimenopausal syndrome (LIPS).

[0080] To assess these symptoms, infertile women with perimenopausal syndrome (LIPS patients) underwent a 4-week intervention with *Lactobacillus reuteri*-NCU-37. Figure 2 As shown, compared with the placebo group (MP group), all assessment scores in the probiotic group (ML group) were significantly lower: the modified Kupperman Index (MKI) score decreased from 15.00 to 8.00 (p<0.01). Figure 2 (A in the original text); Hamilton Anxiety Rating Scale (HAMA) score decreased from 11.50 to 7.00 (p<0.01, ...). Figure 2 (B in the text); Hamilton Depression Rating Scale (HAMD) score decreased from 9.00 to 7.00 (p<0.01, Figure 2 (C in the text); the Athens Insomnia Scale (AIS) score decreased from 7.50 to 5.00 (p<0.05, Figure 2 (D in the text). These results indicate that *Lactobacillus reuteri*-NCU-37 can significantly alleviate perimenopausal syndrome and related mood disorders (including anxiety, depression, and insomnia).

[0081] (3) Lactobacillus reuteri-NCU-37 significantly increased hormone levels in infertile women with perimenopausal syndrome (LIPS patients).

[0082] The levels of key hormones associated with perimenopause, including E2, progesterone (P), and anti-Müllerian hormone (AMH), were measured. Results showed that the probiotic group (ML group) had higher P levels compared to the placebo group (MP group) (MP group and ML group = 0.39 and 0.45, respectively), but the difference was not statistically significant. Figure 3 (A in the text). Meanwhile, the AMH level in the ML group was significantly higher than that in the MP group (MP group and ML group = 2.76 and 3.24, respectively, p < 0.05); Figure 3 (B in the text). E2 levels were also significantly elevated (MP group and ML group = 20.46 and 35.01, respectively, p < 0.05; Figure 3 In addition, follicle-stimulating hormone (FSH) levels were significantly elevated (MP group = ML group = 2.60 = 3.60, p < 0.05). Figure 3 The levels of luteinizing hormone (LH) were also significantly elevated in the MP group and ML group (p < 0.05, 0.64 and 1.15, respectively). Figure 3 (E in the text). These findings suggest that Lactobacillus reuteri-NCU-37 can significantly increase hormone levels in infertile women with perimenopausal syndrome (LIPS patients).

[0083] (4) Lactobacillus reuteri-NCU-37 significantly reduced gut microbiota diversity in infertile women with perimenopausal syndrome (LIPS patients).

[0084] To further investigate the role of gut microbiota in infertile women with perimenopausal syndrome (LIPS), high-throughput sequencing was performed on fecal samples from groups C, MP, and ML after intervention. Alpha diversity analysis showed that compared with group C, the Chao1 index, Faith_pd index, Shannon index, and Observed_species index were significantly increased in the MP group (p<0.01). After probiotic intervention, all these indices in the ML group were significantly lower than those in the MP group (p<0.05), indicating that *Lactobacillus reuteri*-NCU-37 can effectively reduce the alpha diversity of gut microbiota. Figure 4 In A). Principal coordinate analysis (PCoA) results showed that samples from different groups partially overlapped, but some inter-group differences could still be observed (in A). Figure 4 (B in the diagram). The Venn diagram shows that a total of 5713 operational taxonomic units (OTUs) were generated across all samples, of which 582 OTUs were common to all three groups. Groups C, MP, and ML had 953, 2183, and 1459 unique OTUs, respectively. Figure 4 (C) The comparison of distances between groups showed significant differences among all groups (p<0.001). Figure 4 (D in the original text). Furthermore, the dilution curves showed that, at the same sequencing depth, the plateau of the MP group was higher than that of the C and ML groups, further confirming the higher species richness of the MP group (D in the original text). Figure 4These results suggest that *Lactobacillus reuteri*-NCU-37 may alleviate symptoms in infertile women with perimenopausal syndrome (LIPS) by modulating gut microbiota diversity.

[0085] (5) Lactobacillus reuteri-NCU-37 modulates the gut microbiota composition of infertile women with perimenopausal syndrome (LIPS patients).

[0086] The taxonomic composition of the gut microbiota in each group after intervention was then analyzed. At the phylum level, the fecal microbiota of all subjects mainly consisted of Firmicutes, Bacteroidetes, Actinobacteria, and Proteobacteria. Figure 5 (A) Compared with group C, the relative abundance of Firmicutes in group ML was significantly decreased (p<0.001), while the relative abundance of Bacteroidetes was significantly increased (p<0.001). Figure 5 B- Figure 5 (C in the original text). These trends were reversed after probiotic intervention. At the genus level, the top 30 genera in terms of relative abundance were further analyzed. The fecal microbiota was mainly dominated by *Blautia*, *Collinsella*, *Bacteroides*, *Faecalibacterium*, and *Coprococcus*. Figure 5 (D) Compared with group C, the relative abundance of *Bacteroides* and *Parabacteroides* was significantly increased in group MP (p<0.001). After probiotic intervention, the relative abundance of these genera decreased in group ML (no significant difference). Furthermore, the relative abundance of *Prevotella* was significantly decreased after probiotic intervention (p<0.05). Notably, there was no significant difference in the relative abundance of *Blautia* and *Coprococcus* among the groups (p<0.05). Figure 5 E- Figure 5 (I) In addition, we used RT-qPCR to quantitatively detect the relative abundance of Lactobacillus in fecal samples. The results showed that compared with group C, the relative abundance of Lactobacillus in group MP was significantly reduced (p<0.05), while after probiotic intervention, the relative abundance of Lactobacillus in group ML was significantly increased compared with group MP (p<0.001). Figure 5 (J in the article). These findings suggest that *Lactobacillus reuteri*-NCU-37 may alleviate symptoms in infertile women with perimenopausal syndrome (LIPS) by modulating the gut microbiota, particularly by reducing the proliferation of potentially pathogenic bacteria such as *Bacteroides*.

[0087] In addition, differential species analysis was performed. LEfSe analysis showed that group C was significantly enriched with beneficial bacteria such as Bifidobacterium, while group MP was enriched with potentially harmful bacteria, including Bacteroides. Figure 6(A in the original text). Notably, after probiotic intervention, the ML group showed a significant enrichment of the beneficial bacteria *Akkermansia*. Random forest analysis further supported these findings, indicating that *Bacteroides* played an important role in the inter-group differences, and its relative abundance decreased after intervention (…). Figure 6 (B in the text). These results suggest that *Lactobacillus reuteri*-NCU-37 may improve symptoms in infertile women with perimenopausal syndrome (LIPS) by reducing the relative abundance of *Bacteroides* in the gut microbiota.

[0088] (6) Correlation analysis between gut microbiota, clinical symptoms and hormone levels

[0089] To investigate the relationship between gut microbiota, clinical symptoms, and hormone levels in infertile women with perimenopausal syndrome (LIPS patients), Pearson correlation analysis was performed. Results showed that the Modified Kupperman Index (MKI) score was positively correlated with Bacteroides (p<0.001), Bacteroides (p<0.05), Parabacteroides (p<0.01), and Prevotella (p<0.01), and negatively correlated with Firmicutes (p<0.001) and Blautia (p<0.05). The Hamilton Anxiety Rating Scale (HAMA) score was positively correlated with Bacteroides (p<0.01) and Prevotella (p<0.001), and negatively correlated with Firmicutes (p<0.05). The Athens Insomnia Scale (AIS) score was positively correlated with Bacteroides (p<0.01) and Firmicutes (p<0.01), and negatively correlated with Firmicutes (p<0.01). Furthermore, follicle-stimulating hormone (FSH) and luteinizing hormone (LH) were both negatively correlated with Bacteroides and Firmicutes, and positively correlated with Blautia. Notably, estradiol (E2) was negatively correlated with Bacteroides. Figure 7 These results indicate a close relationship between the severity of perimenopausal symptoms, hormonal changes, and gut microbiota imbalance in infertile women with perimenopausal syndrome (LIPS patients).

[0090] 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 reuteri* ( Limosilactobacillus reuteri The use of NCU-37 in the preparation of products for relieving and / or improving perimenopausal syndrome, characterized in that, The Lactobacillus reuteri-NCU-37 was deposited on February 24, 2025, at the China General Microbiological Culture Collection Center, located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, with accession number CGMCCNO.33627. The perimenopausal syndrome mentioned refers to the perimenopausal syndrome induced by leuprorelin during assisted reproductive treatment; The product's functions include: regulating the intestinal flora and reducing the proliferation and abundance of pathogenic bacteria of the genus Bacteroides.

2. The application according to claim 1, characterized in that, The product's functions include relieving and / or improving symptoms of perimenopausal syndrome, including at least one of hot flashes, sweating, mood swings, fatigue, and bone and joint pain.

3. The application according to claim 1, characterized in that, The product's functions include: relieving and / or improving mood disorders caused by perimenopausal syndrome, including at least one of anxiety, depression, and insomnia.

4. The application according to claim 1, characterized in that, The product's functions include increasing hormone levels in infertile women with perimenopausal syndrome, including anti-Müllerian hormone, estradiol, follicle-stimulating hormone, and luteinizing hormone.

5. The application according to claim 1, characterized in that, The products include pharmaceuticals or microbial agents.

6. A product for relieving and / or improving perimenopausal syndrome, characterized in that, The product includes Lactobacillus reuteri-NCU-37; The Lactobacillus reuteri-NCU-37 was deposited on February 24, 2025, at the China General Microbiological Culture Collection Center, located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, with accession number CGMCCNO.33627.

Images