Category: 3. Business

  • CMS advises Iberdrola on the creation of a joint venture with Echelon

    CMS advises Iberdrola on the creation of a joint venture with Echelon

    CMS Albiñana & Suárez de Lezo advises Iberdrola on the creation of a joint venture with Echelon (owned by the Starwood Capital fund) to build and operate data centres in Spain. This is the largest binding agreement of its kind in Europe between an energy company and a developer of this type of technological infrastructure.

    Through this collaboration, Iberdrola will hold a 20% stake through its subsidiary CPD4Green and will be responsible for identifying and securing land with connectivity to the electricity grid where the centres will be developed. In addition, electricity will be supplied to the centres 24/7.

    For its part, Echelon, a Dublin-based technology company owned by Starwood Capital Group, will own 80% of the company and will be responsible for the development, design and marketing of the data centre and the day-to-day management of the joint venture.

    Iberdrola already sells more than 11 TWh to technology companies and operators of these infrastructures worldwide, making it a leader in the supply of electricity to data centres. In addition, its subsidiary CPD4Green, dedicated to facilitating the development of data processing infrastructures, already has a portfolio of sites for 700 MW in Spain and potential for another 5,000 MW.

    The transaction was coordinated by our managing partner, César Albiñana, Corporate/M&A partner Ignacio Zarzalejos, and Public Law and Regulated Sectors partner Juan Moreno, as well as lawyer Ana Vázquez from the Corporate/M&A area.

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  • Quantitative Assessment of En‑face OCT‑Derived Minimum Intensity F

    Quantitative Assessment of En‑face OCT‑Derived Minimum Intensity F

    Introduction

    Age-related macular degeneration (AMD) is a leading cause of irreversible visual loss in older adults globally.1 With aging populations, the burden of AMD is increasing rapidly. Nearly 200 million people were affected in 2020, projected to reach 288 million by 2040.1 Although neovascular (wet) AMD accounts for less than 10% of all AMD cases, it causes most vision-threatening disease and legal blindness.1,2 In Asia, home to some of the fastest-aging populations, AMD is already a major cause of blindness, with rising cases driven by lifestyle changes and longer lifespans.3 Among those with nAMD, the prevalence of polypoidal choroidal vasculopathy (PCV) ranges from 18% to 50%, varying by ethnicity and geography.4,5 In Asian populations, PCV may comprise 23–64% of nAMD cases, particularly among anti-VEGF-resistant patients, while in non-Asian groups it ranges from 4–20%.4,5 This growing burden of macular neovascularization (MNV) due to nAMD and PCV presents major public health and socioeconomic challenges, as central vision loss greatly affects quality of life and imposes high costs.

    Intravitreal inhibition of vascular endothelial growth factor (VEGF) is the standard treatment for MNV.2,6 Ranibizumab (Lucentis®; Genentech, South San Francisco, CA/Roche, Basel, Switzerland) and similar biologics block VEGF-A, the key driver of pathologic MNV and vascular leakage.2,6 These agents lead to rapid regression of abnormal vessels and resolution of intra- and subretinal fluid, improving retinal structure and vision.2,6 Clinical trials have shown substantial visual gains and reduced disease activity with repeated anti-VEGF therapy.2,6 However, many patients still have persistent or recurrent fluid, and disease progression may continue despite treatment.7 The need for frequent injections, often monthly or in treat-and-extend regimens, also places a heavy burden on patients, caregivers, and healthcare systems.8

    Optical coherence tomography (OCT) is indispensable for diagnosing and managing MNV.2 Modern spectral-domain (SD) and swept-source (SS) OCT provide high-resolution, cross-sectional macular images that reveal fluid and tissue changes noninvasively.1 In clinical practice, OCT is used at nearly every visit to assess disease activity and guide retreatment. Key OCT-derived biomarkers, particularly IRF, SRF, pigment epithelial detachments (PEDs), and subretinal hyperreflective material (SHRM), are closely tracked as indicators of exudation.1,9 The presence and distribution of these fluid compartments carry prognostic significance: for example, intraretinal (cystoid) fluid predicts poorer outcomes than SRF, and persistent IRF is generally associated with worse vision.1,2,9 By contrast, SRF is often better tolerated and may signal a milder disease course.9,10 Extensive SHRM (representing fibrin, hemorrhage, fibrosis, or active neovascular tissue) is linked to worse baseline vision and limited visual gains if persistent.1,9–12 OCT also enables measurement of central retinal thickness (CRT), widely used as a surrogate metric.1 Overall, OCT allows sensitive detection of MNV activity and is central to individualized retreatment decisions.

    In recent years, “en face” OCT imaging, which produces frontal sections of retinal layers, has added a new dimension to retinal imaging.13 En face modes (sometimes called C-scans) can isolate particular slabs of interest (for example, the sub-retinal pigment epithelium [RPE] or photoreceptor layers) and display pathology over a large area.13 Within this framework, the technique of minimum-intensity projection (also called OCT-MI) has emerged as a quantitative tool.14 In an OCT-MI image, the darkest (minimum reflectivity) pixel from each A-scan within a slab is plotted in en face format.14 This highlights areas where tissue reflectivity is abnormally low. OCT-MI analysis has proven useful in other retinal conditions: for example, in hydroxychloroquine toxicity the minimum-intensity values in the outer nuclear layer become significantly elevated as photoreceptors degenerate.14 Likewise, in geographic atrophy (GA) of AMD, increased MI at the lesion margins predicts sites of impending expansion, reflecting early photoreceptor and RPE disruption before it is visible en face.15 These studies illustrate that OCT-MI can sensitively detect outer retinal disruption by quantifying subtle reflectivity changes. By analogy, it can be suggested that using MI en-face imaging, a method that highlights the darkest (least reflective) areas, could be a valuable but underused approach for measuring IRF and SRF in MNV. Since fluid appears as areas of low reflectivity (dark regions) on these scans, treating these fluid pockets as “dark lesions” could offer new, objective ways to quantify exudation in MNV.

    Biosimilar anti-VEGF agents were introduced to improve affordability and access compared to originator biologics.8,16,17 India approved the first ranibizumab biosimilar (RzB), Razumab® (Intas Pharmaceuticals, Ahmedabad, India), in 2015,18,19 with several others following globally.20–22 In India, biosimilars have seen rapid uptake: as evidenced by multiple surveys of retina specialists.21,23,24 Oceva® (Sun Pharmaceutical Industries Limited, Mumbai, India) is a ranibizumab biosimilar approved in early 2023 (CDSCO clearance MF/BIO/23/000020, 24 March 2023).25 While these agents promise to improve treatment affordability, real-world data on their anatomic and functional outcomes is still emerging. In particular, no study to date has used advanced OCT-based quantification to compare fluid dynamics under biosimilar therapy.

    With rising biosimilar use and advances in retinal imaging, studies using OCT biomarkers to evaluate real-world efficacy are needed. This study addresses that gap by assessing the newer RzB, in treatment-naïve MNV, using en-face minimum intensity OCT to quantify fluid area and perimeter changes.

    Materials and Methods

    This was a retrospective, single-center study conducted at Shantilal Shanghvi Eye Institute in India, focusing on patients diagnosed with treatment-naïve MNV secondary to nAMD and PCV who received three monthly loading doses of the RzB, between September 2023 and December 2024. The study protocol was approved by Shantilal Shanghvi Foundation Ethics Committee (SSF[SSEI]/LEC/2024/002) and adhered to the tenets of the Declaration of Helsinki. Written informed consent was secured from all participants for both treatment and data publication.

    Inclusion criteria comprised patients aged 50 years or older with active treatment‑naïve MNV secondary to nAMD or PCV. We excluded eyes with MNV secondary to causes other than AMD and PCV (eg, myopia, inflammatory CNV), significant media opacities impeding reliable OCT imaging, concurrent diabetic retinopathy, advanced glaucoma, or history of intraocular surgery (other than uncomplicated cataract extraction) within the prior three months.

    At baseline, each patient underwent a comprehensive assessment that included best-corrected visual acuity (BCVA) measured on a Snellen chart (converted to logMAR for analysis), intraocular pressure by Goldmann applanation tonometry, anterior segment examination with slit-lamp biomicroscopy, dilated fundus evaluation with 90D and 20D lenses, and SD-OCT (Cirrus HD-6000; Carl Zeiss Meditec, Dublin, CA, USA). Indocyanine green angiography (ICGA) was performed at baseline to differentiate between nAMD and PCV.

    All eligible eyes received three consecutive monthly intravitreal RzB, (0.5 mg/0.05 mL per dose) as loading therapy. Intravitreal injections were performed under aseptic conditions in a dedicated procedure room. Participants returned for follow-up one week after the first injection, with subsequent visits scheduled at 30, 60, and 90 days post-injection. Any emergent symptoms prompted additional, unscheduled assessments as needed.

    SD-OCT volume scans (49 B-scans over a 20°×20° area) were acquired at baseline and all subsequent visits. En-face minimum-intensity projections were generated automatically by the Cirrus HD-6000 OCT system’s built-in software from each 20°×20° macular cube scan. As described by Allahdina et al14 and Stetson et al,15 the device’s proprietary algorithm examines every A‑scan in a predefined retinal slab, locates the voxel of minimum reflectivity, and assembles those values into a two‑dimensional en‑face minimum‑intensity map. These MI projections were exported directly from the OCT system and used for analysis. All imaging assessments were performed by a single masked grader (J.S.) with over 10 years of experience in retinal imaging to ensure consistency.

    The primary outcomes included changes in BCVA and quantitative alterations in fluid area and perimeter on en-face MI OCT from baseline to week 12. Secondary outcomes encompassed the proportion of eyes exhibiting SRF, IRF, and SHRM at each time point, as well as the incidence of any ocular or systemic adverse events during the study period.

    Statistical Analysis

    Data analysis was conducted using SPSS 23.0 (SPSS Inc., Chicago, IL, USA). Continuous variables were expressed as mean ± standard deviation or median with interquartile range (IQR), as appropriate. Categorical variables were presented as frequencies and percentages. Paired changes in BCVA, fluid area, and perimeter from baseline to 12 weeks were assessed with the Wilcoxon signed-rank test. Changes in the proportion of eyes with SRF, IRF, and SHRM were evaluated with McNemar’s test. A P-value <0.05 was considered statistically significant.

    Results

    Thirty-six eyes from 36 treatment‑naïve patients completed the three‑injection loading phase with the RzB and were included in the analysis. Of these, 20 eyes had nAMD and 16 eyes had PCV. The mean age of the cohort was 71.33 years (SD ± 9.48), with a slight female predominance (male: female ratio 16: 20).

    Best-Corrected Visual Acuity Outcomes

    At baseline, the mean BCVA was 0.94 ± 0.59 logMAR (≈20/174). Following three monthly loading doses of RzB, there was a statistically significant improvement in BCVA at 12 weeks, with mean logMAR improving to 0.84 ± 0.61 (≈20/138; P=0.04; Table 1). By week 12, 91.7% of eyes (33/36) showed visual improvement, 8.3% (3/36) maintained baseline acuity, and no eyes experienced any vision loss.

    Table 1 Changes in the Best-Corrected Visual Acuity (BCVA) and Minimum Intensity-Based Fluid Metrics on En-Face Optical Coherence Tomography (OCT) (En-Face OCT-MI) in the Study Cohort

    Quantitative En-Face OCT-MI Fluid Metrics

    The median fluid area, as measured on en-face OCT-MI, decreased significantly from 0.9 mm² (IQR 0.62–4.56) at baseline to 0.32 mm² (IQR 0.1–0.64) at 3 months (P=0.007). Similarly, the median fluid perimeter was reduced from 10.95 mm (IQR 7.26–25.67) at baseline to 6.02 mm (IQR 1.76–7.93) at 3 months (P=0.0005; Table 1).

    Resolution of Fluid and Subretinal Hyperreflective Material

    The proportion of eyes with IRF decreased from 66.67% (24/36) at baseline to 38.89% (14/36) at 3 months, representing a 41.7% reduction (P=0.013). SRF was present in 83.33% (30/36) of eyes at baseline and declined to 58.33% (21/36) at 3 months, corresponding to a 30.0% reduction (P=0.015). Notably, the proportion of eyes with SHRM showed a marked reduction, from 58.33% (21/36) at baseline to 13.89% (5/36) at 3 months, reflecting a 76.2% decrease (P<0.001) (Table 2).

    Table 2 Proportion of Eyes with Resolution of Fluid and Subretinal Hyperreflective Material (SHRM)

    Safety Analysis

    No ocular or systemic adverse events were observed during the follow-up period. All injections were well tolerated, and no patient required discontinuation of therapy.

    Figures 1 and 2 illustrates representative cases from the study population.

    Figure 1 Minimum-intensity en-face optical coherence tomography (en-face OCT-MI) of a patient (A) demonstrates two distinct hyporeflective areas corresponding to the loci of intraretinal fluid (IRF). The measurements of fluid area and perimeter are shown in (B) on en-face OCT-MI. The spectral-domain OCT (C) also illustrates the corresponding fluid pockets (indicated by yellow and blue arrows in both A and C). Notably, the foveal A-scan of the OCT (C) reveals that the fluid pocket located nasally (yellow arrow) appears larger than the temporal fluid pocket (blue arrow). In contrast, the en-face OCT-MI (A and B) indicates that the extent of the temporal fluid is markedly greater than that of the nasal fluid pocket.

    Figure 2 Baseline optical coherence tomography angiography (OCTA) (A) of a patient demonstrates macular neovascularization (MNV) (yellow arrow), along with a corresponding pigment epithelial detachment (PED) (blue asterisk; (B) and subretinal fluid (SRF) (red arrow; (B) on spectral domain OCT (SD-OCT). The Minimum-intensity en-face optical coherence tomography (en-face OCT-MI) at baseline provides detailed metrics of the fluid, including its area and perimeter (C). Following three intravitreal injections of the ranibizumab biosimilar, the patient’s best-corrected visual acuity improved from 20/40 to 20/20, with complete resolution of the MNV observed on OCTA (D). Additionally, at 12 weeks, there was complete resolution of the SRF on both the SD-OCT (E) and en-face OCT-MI (F).

    Discussion

    In this study, three monthly injections of the RzB, produced significant visual improvement and markedly reduced the fluid in patients with MNV due to nAMD and PCV. These results parallel prior reports that ranibizumab biosimilars achieve visual and anatomical outcomes comparable to innovator ranibizumab.18,26 Our findings therefore confirm that the novel biosimilar is effective in improving vision and resolving exudation in nAMD/PCV over the loading phase.

    A primary novelty of our work is the application of en-face OCT-MI to objectively map and quantify exudative fluid in nAMD and PCV. Unlike conventional B-scan OCT, which provides qualitative cross-sectional views of fluid, en-face MI imaging collapses each A-scan to its lowest-intensity value, highlighting hyporeflective spaces across the macula.13 In practice, fluid appears as black regions on the MI en-face map,27 whereas normal retina (with higher minimum intensity) appears gray or white. In fact, Nicholson et al described how, in active exudation, “the en-face image generated has black regions corresponding to fluid”.27 By isolating these dark areas, our analysis could compute the total fluid area and perimeter, capturing both the size and complexity of fluid pockets across the macula.

    En-face OCT-MI has been used previously in other retinal diseases to detect subtle outer retinal changes.14,15 For example, OCT-MI analysis provides a quantitative measure of photoreceptor/outer nuclear layer integrity and was shown to detect early hydroxychloroquine toxicity with high sensitivity.14 Similarly, increased MI at GA margins has been linked to lesion growth.15 Our study is among the first to leverage MI en-face imaging specifically for exudative changes. The method provides objective, reproducible measures of fluid burden. Because each pixel in the MI image represents the minimum reflectivity along its A-scan,14,15 quantitative analysis of these maps yields repeatable fluid metrics that are not subject to subjective grader interpretation. In practice, this means we could track global changes in fluid that span multiple B-scans, rather than relying on limited cross-sections (eg central subfield thickness). Moreover, quantifying the perimeter of fluid regions offers insight into the complexity or fragmentation of the fluid information lost in simple volumetric measures.

    Objective fluid mapping carries significant clinical implications. In nAMD and PCV management, both the volume and distribution of OCT-detected fluid serve as key biomarkers of disease activity.1,9 Coulibaly et al recently demonstrated that accurately measuring and mapping fluid on OCT is essential for assessing disease activity and guiding treatment decisions in nAMD.28 Many researchers are actively studying automated fluid quantification (using artificial intelligence [AI]) to guide individualized therapy.29,30 In line with this, our en-face OCT-MI approach could serve as a practical tool for monitoring. For example, after each injection, an MI map could reveal residual or recurrent fluid that might not be obvious on B-scans. Automated or semi-automated calculations of fluid area and perimeter would help determine whether disease activity has truly resolved or if retreatment is needed. Coulibaly et al propose that automated fluid quantification could provide an objective basis for scheduling retreatments, thereby minimizing variability in treatment intervals.28 By providing a standardized, quantitative endpoint, en-face OCT-MI biomarkers could thus refine PRN/treat-and-extend strategies: small stable fluid on MI may allow extending intervals, whereas any new black areas on MI might prompt reinjection.

    In addition, en-face OCT-MI metrics may become valuable as longitudinal biomarkers of anti-VEGF response. For instance, the degree of baseline fluid area or its rate of shrinkage might correlate with visual prognosis or recurrence risk: a question for future study. Because MI maps capture the spatial distribution of fluid, they could reveal patterns (eg multilobular fluid, eccentric spread) associated with more aggressive disease. Overall, adopting en-face OCT-MI moves fluid assessment toward the quantitative paradigm now seen in other specialties. In combination with deep learning or automated segmentation, MI maps promise to enhance disease monitoring by providing sensitive, reproducible readouts of exudation.

    Our results underscore the growing importance of biosimilars in ophthalmology. High costs of anti-VEGF injections pose a heavy burden, especially in countries like India.8 To address this, Razumab was developed as a more affordable alternative to innovator ranibizumab without sacrificing efficacy or safety, and real-world data show its outcomes match Lucentis across multiple indications.19,26 Biosimilars also offer substantial savings: in India, Razumab costs about $125 per dose versus $320 for branded ranibizumab, and other agents like aflibercept remain even pricier.8,23,24 Single-use vials and familiar handling make these products easy to integrate into practice. By lowering costs, biosimilars can reach patients who might otherwise forgo treatment, broadening access in nAMD and PCV care, conditions with a high risk of vision loss. As clinical evidence mounts and regulators (including the FDA) approve ophthalmic biosimilars like SB11, the retinal community’s confidence is growing: these agents promise to reduce drug expenditure while maintaining outcomes and advancing public health.20–26

    Key strengths of our study include the novel use of en-face OCT-MI and the focused real-world evaluation of a ranibizumab biosimilar in nAMD/PCV. By quantifying fluid area and perimeter changes, we demonstrated objective metrics that correlated with treatment effect. However, several limitations must be acknowledged. The study’s retrospective design carries inherent biases (eg lack of randomization and potential selection bias). The sample size was modest and not powered to allow a meaningful comparison between nAMD and PCV subgroups. Additionally, the analysis was conducted by a single grader, which may introduce subjective variability. The lack of a parallel control group (such as patients receiving innovator ranibizumab) limits the ability to draw direct comparative conclusions. The follow-up period was short (12 weeks), so longer-term efficacy, durability of fluid resolution, and safety could not be assessed. Additionally, while en-face OCT-MI offers quantitative data, manual steps in image processing could introduce variability; future work should assess reproducibility explicitly. Finally, the MI technique is proprietary to certain OCT platforms, which may affect generalizability. Nevertheless, this study provides a proof of concept for OCT-MI use in exudative AMD/PCV and generates hypotheses for further research.

    Moving forward, prospective longitudinal studies are needed to validate OCT-MI fluid metrics as a biomarker of anti-VEGF response. It would be valuable to track how MI-derived fluid area/perimeter evolve over longer treatment courses and whether they predict re-treatment intervals or visual outcomes. Comparative trials of biosimilar versus originator ranibizumab using en-face OCT-MI as an endpoint could further elucidate any subtle differences in fluid dynamics. In summary, our findings open a new avenue for objective OCT-based monitoring in MNV and underscore the promise of cost-effective biosimilars in managing retinal disease.

    Conclusion

    In this proof‑of‑concept study, loading doses of the RzB, resulted in significant visual improvement and a reduction in fluid in MNV secondary to nAMD or PCV, as objectively measured by en-face OCT-MI. These findings support the use of en-face OCT-MI as a quantitative biomarker for fluid exudation and treatment response. Given the lower cost profile of biosimilars, their adoption may broaden access to effective therapy in resource‑limited settings. To establish external validity and inform routine clinical use, multicenter, randomized trials with extended follow‑up are now warranted. Overall, the findings highlight the potential of biosimilars and advanced imaging in shaping the future of MNV management.

    Disclosure

    J.S., B.M., S.K.: Shantilal Shanghvi Foundation (SSF) (not relevant to the work under consideration). The authors declare that they have no competing interests in this work.

    References

    1. Metrangolo C, Donati S, Mazzola M, et al. OCT biomarkers in neovascular age-related macular degeneration: a narrative review. J Ophthalmol. 2021;2021:9994098. doi:10.1155/2021/9994098

    2. Sheth JU, Stewart MW, Narayanan R, et al. Macular neovascularization. Surv Ophthalmol. 2025;70(4):653–675. doi:10.1016/j.survophthal.2024.08.003

    3. Keenan TDL, Cukras CA, Chew EY. Age-related macular degeneration: epidemiology and clinical aspects. Adv Exp Med Biol. 2021;1256:1–31.

    4. Sheth JU, Narayanan R, Anantharaman G, et al. Updated guidelines for the management of polypoidal choroidal vasculopathy: recommendations from the Indian polypoidal choroidal vasculopathy panel and the vitreoretinal society of India. Indian J Ophthalmol. 2022;70(8):3102–3111. doi:10.4103/ijo.IJO_2985_21

    5. Anantharaman G, Sheth J, Bhende M, et al. Polypoidal choroidal vasculopathy: pearls in diagnosis and management. Indian J Ophthalmol. 2018;66(7):896–908. doi:10.4103/ijo.IJO_1136_17

    6. Solomon SD, Lindsley K, Vedula SS, Krzystolik MG, Hawkins BS. Anti-vascular endothelial growth factor for neovascular age-related macular degeneration. Cochrane Database Syst Rev. 2019;3(3):CD005139. doi:10.1002/14651858.CD005139.pub4

    7. Yang S, Zhao J, Sun X. Resistance to anti-VEGF therapy in neovascular age-related macular degeneration: a comprehensive review. Drug Des Devel Ther. 2016;10:1857–1867. doi:10.2147/DDDT.S97653

    8. Narayanan R, Hariprasad SM, Sheth J. Biosimilars for the treatment of retinal diseases. Ophthalmic Surg Lasers Imaging Retina. 2021;52(5):242–246. doi:10.3928/23258160-20210429-01

    9. Foss AJE, Almeida D, Cheung CMG, Ogura Y, de Cock E, Empeslidis T. To treat or not to treat? Resolving the question of subretinal and intraretinal fluid in age-related macular degeneration: a narrative review. Ophthalmol Ther. 2025;14(3):489–514. doi:10.1007/s40123-025-01093-3

    10. Guymer RH, Markey CM, McAllister IL, et al. Tolerating subretinal fluid in neovascular age-related macular degeneration treated with ranibizumab using a treat-and-extend regimen: FLUID study 24-month results. Ophthalmology. 2019;126(5):723–734. doi:10.1016/j.ophtha.2018.11.025

    11. Soman M, Sheth JU, Indurkar A, Meleth P, Nair U. De-novo multilayering in fibrovascular pigment epithelial detachment. Sci Rep. 2021;11(1):17209. doi:10.1038/s41598-021-96746-1

    12. Nair U, Nair IJ, Sheth JU, Soman M. Novel resolution of multilayered pigment epithelial detachment lamellae following brolucizumab treatment-a case report. Case Rep Ophthalmol Med. 2025;2025:9953015. doi:10.1155/crop/9953015

    13. Feo A, Ramtohul P, Govetto A, et al. En face OCT: breakthroughs in understanding the pathoanatomy of retinal disease and clinical applications. Prog Retin Eye Res. 2025;106. doi:10.1016/j.preteyeres.2025.101351

    14. Allahdina AM, Stetson PF, Vitale S, et al. Optical coherence tomography minimum intensity as an objective measure for the detection of hydroxychloroquine toxicity. Invest Ophthalmol Vis Sci. 2018;59(5):1953–1963. doi:10.1167/iovs.17-22668

    15. Stetson PF, Yehoshua Z, Garcia Filho CA, Portella Nunes R, Gregori G, Rosenfeld PJ. OCT minimum intensity as a predictor of geographic atrophy enlargement. Invest Ophthalmol Vis Sci. 2014;55(2):792–800. doi:10.1167/iovs.13-13199

    16. Chakraborty S, Sheth JU. Efficacy of an Indian Bevacizumab BIOSimilar (BEVATAS) for type 1 and aggressive posterior retinopathy of prematurity (BIOS-ROP study). Clin Ophthalmol. 2024;18:61–68. doi:10.2147/OPTH.S443104

    17. Chakraborty S, Sheth JU. Efficacy and safety of an Indian Bevacizumab BIOSimilar (BEVATAS) for retinal vein occlusion (BIOS-RVO study). Clin Ophthalmol. 2024;18:2865–2871. doi:10.2147/OPTH.S473329

    18. Chakraborty D, Stewart MW, Sheth JU, et al. Real-world safety outcomes of intravitreal ranibizumab biosimilar (Razumab) therapy for chorioretinal diseases. Ophthalmol Ther. 2021;10(2):337–348. doi:10.1007/s40123-021-00345-2

    19. Soman M, Nair I, Sheth JU, Nair U. Innovator Versus Biosimilar Ranibizumab in Polypoidal Choroidal Vasculopathy: real-World Evidence. Ophthalmol Ther. 2022;11(3):1175–1186. doi:10.1007/s40123-022-00507-w

    20. Avadzadeh S, Sharma A, Parvaresh MM, Ghasemi Falavarjani K, International Retina Biosimilar Study Group (Inter BIOS Group). Aflibercept 2 mg biosimilar (Tyalia)-real-world experience from Iran (ATRIA study). Eye. 2025;39(11):2159–2163. doi:10.1038/s41433-025-03813-2

    21. Sharma A, Kaiser PK, Tadayoni R, et al. Anti-VEGF biosimilars for retinal diseases survey 2023- India (Bio-INDAS) by the International Retina Biosimilar Study Group (Inter-BIOS Group) in collaboration with the Vitreo-Retinal Society of India (VRSI). Eye. 2024;38(17):3392–3395. doi:10.1038/s41433-024-03284-x

    22. Ueda-Consolvo T, Ishida M, Nakamura T, et al. Biosimilar ranibizumab (BS1) – early experience from Japan (BRIJ study). Eye. 2024;38(16):3193–3196. doi:10.1038/s41433-024-03220-z

    23. Sheth JU, Stewart MW, Khatri M, et al. Changing trends in the use of anti-vascular endothelial growth factor (anti-VEGF) biosimilars: insights from the vitreoretinal Society of India Biosimilars of Anti-VEGF Survey. Indian J Ophthalmol. 2021;69(2):352–356. doi:10.4103/ijo.IJO_2703_20

    24. Sheth JU, Gopal L, Gillies M, et al. Vitreoretinal Society of India practice pattern survey 2020: medical retina. Indian J Ophthalmol. 2021;69(6):1430–1439. doi:10.4103/ijo.IJO_2573_20

    25. Ghosh AK, Nikumbh US, Shukla CK, et al. Efficacy, Safety and Immunogenicity of Sun’s Ranibizumab Biosimilar in Neovascular Age-Related macular degeneration: a phase 3, double-blind comparative study. Ophthalmol Ther. 2024;13(5):1369–1382. doi:10.1007/s40123-024-00883-5

    26. Sharma S, Sharma T, Prasad S, Gopalakrishnan M, Chaturvedi A. Treatment landscape of macular disorders in Indian Patients with the Advent of Razumab™ (world’s first biosimilar ranibizumab): a comprehensive review. Ophthalmol Ther. 2021;10(3):431–443. doi:10.1007/s40123-021-00362-1

    27. Nicholson BP, Nigam D, Toy B, et al. Effect of ranibizumab on high-speed indocyanine green angiography and minimum intensity projection optical coherence tomography findings in neovascular age-related macular degeneration. Retina. 2015;35(1):58–68. doi:10.1097/IAE.0000000000000260

    28. Coulibaly LM, Sacu S, Fuchs P, et al. Personalized treatment supported by automated quantitative fluid analysis in active neovascular age-related macular degeneration (nAMD)-a Phase III, prospective, multicentre, randomized study: design and methods. Eye. 2023;37(7):1464–1469. doi:10.1038/s41433-022-02154-8

    29. Mares V, Reiter GS, Gumpinger M, et al. Correlation of retinal fluid and photoreceptor and RPE loss in neovascular AMD by automated quantification, a real-world FRB! Analysis Acta Ophthalmol. 2025;103(3):295–303.

    30. Mares V, Schmidt-Erfurth UM, Leingang O, et al. Approved AI-based fluid monitoring to identify morphological and functional treatment outcomes in neovascular age-related macular degeneration in real-world routine. Br J Ophthalmol. 2024;108(7):971–977. doi:10.1136/bjo-2022-323014

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  • DHLCA Alleviates Diabetic Kidney Disease via TGR5/FXR Activation and G

    DHLCA Alleviates Diabetic Kidney Disease via TGR5/FXR Activation and G

    Introduction

    Diabetes mellitus (DM) is linked with numerous complications across various organ systems, with diabetic kidney disease (DKD) being the most critical, leading to end-stage renal disease (ESRD).1 Given the limited effectiveness of current treatments for DKD, continuous research is underway to uncover the molecular mechanisms behind renal damage and to create innovative medications.

    Recent metabolomic studies have revealed step-wise alterations in bile acid (BA) metabolism during the progression of DKD, suggesting that BA dysregulation may not only reflect disease severity but also contribute to its pathogenesis.2 BAs exert biological effects through receptor-mediated signaling pathways, primarily via the farnesoid X receptor (FXR) and the Takeda G protein-coupled receptor 5 (TGR5).3,4 Activation of FXR has been shown to prevent diabetic nephropathy by modulating lipid metabolism, reducing inflammation and fibrosis, and suppressing oxidative stress.5 Similarly, TGR5 activation has been linked to mitochondrial biogenesis, enhancement of antioxidant responses, and inhibition of kidney injury in diabetes.6 However, the expression patterns and kidney-specific functions of these receptors in DKD remain incompletely characterized.

    Mounting evidence suggests that gut microbiota (GM)–BA interactions play a pivotal role in modulating host metabolism and kidney health.3,7–9 Given this bidirectional relationship, disturbances in the GM-BA axis may represent a critical link between intestinal dysbiosis and DKD progression.10 Despite these insights, several knowledge gaps remain unresolved, including: (i) the temporal dynamics and functional implications of individual BAs across DKD stages; (ii) the mechanistic role of GM-BA interactions in modulating BA receptor signaling within the kidney; and (iii) the therapeutic potential of secondary BAs as endogenous agonists of FXR/TGR5.

    This study proposes the hypothesis that dehydrolithocholic acid (DHLCA), a secondary BA, ameliorates renal tubular injury and proteinuria in DKD through dual activation of FXR and TGR5 and concurrent remodeling of GM composition. To the best of our knowledge, this is the first study to integrate clinical profiling, BA metabolomics, GM analysis, and mechanistic evaluation of a specific BA derivative in the context of DKD, thereby providing novel insights into endogenous BA-based renoprotection and potential therapeutic strategies.

    Methods and Materials

    Patients

    This prospective cohort study was conducted at the Third Affiliated Hospital of Soochow University between April 2021 and August 2022, enrolling 92 consecutively recruited participants with type 2 diabetes mellitus (T2DM) and 31 age- and sex-matched healthy controls (HC). Inclusion criteria: (1) meeting the American Diabetes Association classification criteria for T2DM-2020;11 (2) age > 18 years old. (3) estimated glomerular filtration rate (eGFR) ≥ 90 mL/min/1.73m2 (CKD-EPI Creatinine).12 Exclusion criteria: (1) presence of malignancy or pregnancy; (2) primary or secondary kidney diseases caused by other causes; (3) presence of liver or intestinal inflammatory diseases.

    HC participants were enrolled based on the following inclusion criteria: (1) no known history of chronic systemic diseases (such as DM and hypertension); (2) no chronic systemic diseases requiring long-term medical treatment; (3) biochemical evidence of normal serum glucose levels, liver function, and kidney function.

    The participants with T2DM were categorized into three groups according to their urine albumin-to-creatinine ratio (UACR): (1) T2DM with normoalbuminuria group (T2DM group, UACR < 30mg/g, n = 31); (2) clinically diagnosed DKD13 with microalbuminuria group (DKD micro group, 30 ≤ UACR ≤ 300mg/g, n = 30); (3) clinically diagnosed DKD with macroalbuminuria group (DKD macro group, UACR > 300mg/g, n = 31). Additionally, to avoid interference from metabolite excretion, only participants with normal renal function (eGFR ≥ 90 mL/min/1.73m2) were included in the study.

    The study was conducted in accordance with the principles of the Declaration of Helsinki and was approved by the Third Affiliated Hospital of Ethics Committee of Soochow University (Ethics No: 2021–136, Approval date: 2021–03-10). Informed written consent was secured from all participants.

    Sample Collection

    After overnight fasting, we collected fasting blood samples (5mL) from each participant and stored them in specimen tubes with EDTA as an anticoagulant. The blood samples were centrifuged at 4000 rpm for 15 minutes. The plasma specimens were then stored at −80°C.

    Animal Model

    Thirty-one 4-week-old male C57 BL/6J mice were obtained from the Model Animal Research Center of Nanjing University (Nanjing, China). Mice were randomly assigned to experimental groups, and all procedures involving animals were conducted in accordance with institutional guidelines for the care and use of laboratory animals approved by the Ethics Committee of the Third Affiliated Hospital of Soochow University. The mice were housed under standard conditions, including a temperature of 24°C, humidity between 40−70%, and a 12-hour light/dark cycle with adequate air exchange. The mice were then randomly divided into 2 groups: a 10% low fat diet (LFD, n = 5) group; a 60% high fat diet (HFD, n = 26) group. At 14 weeks of age, 26 mice fed HFD were administered 40 mg/kg streptozotocin (STZ) via intraperitoneal injection daily for 5 consecutive days, while 5 mice from the LFD group received daily intraperitoneal injections of sodium citrate buffer for 5 consecutive days. The diabetic model was confirmed by fasting blood glucose (FBG) levels higher than 11.1 mmol/L. At 18 weeks of age, after overnight fasting, mice from the control group were gavaged with CMC-Na for 2 weeks, and mice from the diabetic group were gavaged with DHLCA 5 mg/kg (n = 5), 10 mg/kg (n = 8), 20 mg/kg (n = 5) (DKD-DHLCA group) or CMC-Na (n = 8, DKD group) for 2 weeks. All mice at 20 weeks of age were euthanized, and samples were harvested for further analysis. Urine creatinine (measured using a Hitachi automatic analyzer 3500) and urine albumin (measured using MedicalSystem) were determined according to standard laboratory methods. Biochemical indicators including blood urea nitrogen (BUN), serum creatinine (Scr), urine creatinine, alanine aminotransferase (ALT), aspartate aminotransferase (AST), and total BA (TBA), were measured using an automated analyzer (Hitachi) following standard laboratory procedures.

    RNA Extraction and Real-Time Quantitative Polymerase Chain Reaction (RT-qPCR)

    Total RNA was extracted from kidney tissues using Trizol reagent (Invitrogen, USA). The isolated RNA was then reverse-transcribed into cDNA using the PrimeScript™ RT reagent Kit (Takara, Japan) according to the manufacturer’s instructions. RT-qPCR was performed using SYBR Premix Ex Taq™ (Takara, Japan) on a Thermo Fisher Scientific equipment. The specific mouse primer sequences (RiboBio, China) used are provided in Table 1. The expression of β-actin was used as an internal control.

    Table 1 Demographic and Clinical Characteristics of Participants

    Western Blot (WB)

    Kidney protein was extracted using radioimmunoprecipitation assay (RIPA) lysis buffer and the total protein concentration was quantified using a bicinchoninic acid (BCA) protein assay. The samples were then subjected to WB analysis. Prior to incubation with primary antibodies against TGR5 (ab72608, Abcam, USA), FXR (sc-25309, Santa Cruz, USA), and β-actin (MA1-140, Invitrogen, USA) overnight at 4°C, the nitrocellulose membranes were blocked with 5% bovine serum albumin (BSA) to prevent non-specific binding. Quantitative analysis was performed using the Image J program. The protein levels were normalized against β-actin.

    Histology, Immunohistochemical and Immunofluorescence Staining

    Kidney tissue samples were fixed in 4% formaldehyde for 48 hours, dehydrated, and embedded in paraffin. Thin sections (4 μm thickness) were cut and stained with hematoxylin-eosin (H&E), periodic acid-Schiff (PAS), and Masson’s trichrome stains. For immunohistochemical staining, antigen retrieval was performed in a nitrate solution at 96°C for 10 minutes. The tissue sections were blocked with 3% bovine serum albumin (BSA) for 30 minutes at room temperature, followed by overnight incubation at 4°C with primary antibodies against TGR5 and FXR. The slides were then incubated with appropriate secondary antibodies, washed, and developed using 3,3’-diaminobenzidine chromogen (Servicebio, China).

    The severity of tubulointerstitial injury was evaluated based on an interstitial fibrosis and tubular atrophy (IFTA) scoring system. The IFTA score was defined as the percentage of the total involved area of interstitium and tubules, where 0 = no IFTA; 1 = less than 25% IFTA; 2 = 25% ~ 50% IFTA; 3 = at least 50% IFTA.14 Histological assessments were performed by investigators who were blinded to group assignments to minimize observational bias.

    Plasma Metabolite Analysis

    Plasma BAs profiling metabolites were analyzed using ultrahigh-performance liquid chromatography tandem-mass spectrometry (UPLC-MS/MS) platform, employing a widely targeted metabolomics analysis. The sample preparation and extraction procedures were as follows: Samples stored at −80°C were thawed on ice. Then, 50 μL of the sample and 300 μL of an extraction solution (1:1 acetonitrile: methanol, containing internal standards) were added to a 2 mL microcentrifuge tube. The mixture was vortexed for 3 minutes and centrifuged at 12,000 rpm for 15 minutes at 4°C. The 200 μL supernatant was collected, incubated at −20°C for 1 hour to precipitate proteins, and 180 μL aliquots were transferred for UPLC-MS/MS analysis.

    Metagenomic Sequencing and Analysis

    Fecal DNA was extracted from mice using the FastDNA SPIN Kit (MP Biomedicals, USA). DNA quantity and quality were assessed with the Qubit™ dsDNA Quantification Assay Kits (Invitrogen, USA). Libraries were prepared from 200 ng input DNA using the TruSeq DNA Nano (Illumina) and sequenced on the Illumina NovaSeq 6000 platform (paired-end). Raw reads were trimmed for adaptors and low-quality bases using Cutadapt and Fqtrim, and host contamination was removed using Bowtie2. Clean reads were assembled using MEGAHIT, followed by gene prediction and redundancy removal to generate a non-redundant gene catalog. Taxonomic annotation was performed by aligning predicted proteins to the NCBI NR database using DIAMOND. Differential microbial taxa were identified using linear discriminant analysis effect size (LEfSe) (linear discriminant analysis [LDA] score > 3, P < 0.05).

    Data and Statistical Analysis

    Data processing and statistical analyses were performed using R (v4.5.0; R Core Team, 2025), SPSS (v25.0; IBM Corp., Armonk, NY, USA, 2017), and GraphPad Prism (v9.0; GraphPad Software, San Diego, CA, USA, 2022). The normality of continuous variables was assessed using the Kolmogorov–Smirnov test. Categorical variables were summarized as counts. Normally distributed data were expressed as mean ± standard deviation (SD), while non-normally distributed data were reported as median (interquartile range, IQR). Multiple-group comparisons were performed using one-way ANOVA (parametric) or the Kruskal–Wallis H-test (non-parametric). When overall differences were significant (P < 0.05), post hoc pairwise comparisons were conducted using Tukey’s test (ANOVA) or Dunn’s test (Kruskal–Wallis). False discovery rate (FDR) correction was applied for multiple testing in omics-level comparisons (BAs and GM analyses). Categorical variables were compared using the chi-square test with FDR correction for multiple group comparisons. Dose–response curves for FBG and UACR were plotted using nonlinear regression (GraphPad Prism) from median ± SD values. For RT-qPCR and WB analyses (n = 3 per group), one-way ANOVA with bootstrap resampling (1000 iterations) was used to estimate bias-corrected and accelerated 95% CI, followed by Tukey’s post hoc test for multiple comparisons. Partial Spearman correlation coefficients were calculated to examine the association between clinical indicators and BA concentrations in patients, with adjustment for age and diabetes duration; two-sided P values were corrected for multiple testing with the FDR correction, and 95% confidence intervals were generated from 1000 bootstrap replicates. Receiver operating characteristic (ROC) curve analysis was used to assess diagnostic performance with area under the curve (AUC). Two-sided P values or FDR-q values < 0.05 were considered statistically significant.

    Results

    Alterations in the Plasma Levels of BAs in Patients with DKD

    To investigate the alterations in the plasma levels of BAs in patients with DKD, UPLC-MS/MS was used to detect plasma BAs metabolic profiling. Baseline clinical characteristics of participants were compared among the groups (Table 1). The DKD macro group exhibited higher urine albumin, UACR, and neutrophil to lymphocyte ratio (NLR), but lower serum albumin (ALB), red blood cell (RBC), total bilirubin (TBIL), and indirect bilirubin (IBIL) compared to the other three groups (P < 0.05, Table 1).

    A total of 50 BAs were detected. Furthermore, MetaboAnalyst was utilized to normalize, process, analyze, and interpret the metabolomic data. The differential metabolites between groups were visualized as a heat map (Figure 1). Plasma concentrations of five primary BAs (nor cholic acid [NCA], cholic acid [CA], glycochenodeoxycholic acid-3-sulfate [GCDCA-3S], chenodeoxycholic acid-3-β-D-glucuronide [CDCA-3Gln], and glycochenodeoxyche olic acid-3-O-β-glucuronide [GCDCA-3Gln]) and six secondary BAs (DHLCA, lithocholic acid [LCA], deoxycholic acid [DCA], 3-dehydrocholic acid [3DHCA], glycolithocholic acid [GLCA], and taurolithocholic acid [TLCA]) differed significantly among the four groups—HC, T2DM, DKD micro or DKD macro—with FDR-q values < 0.05. Variations in metabolite levels across groups are presented as box plots (Figure 2A and B).

    Figure 1 Metabolite heat map in the four groups. Samples are represented in the columns, metabolites are represented in the rows, and their relative concentrations are displayed by color. Metabolites between the groups of participants who are HC group, T2DM group, DKD micro group, and DKD macro group are displayed on the heat map.

    Figure 2 BA profiles among different groups. (A) Plasma primary BAs. (B) Plasma secondary BAs. *FDR-q < 0.05; **FDR-q < 0.01; ***FDR-q < 0.001.

    Partial Spearman Correlations Between Clinical Indicators and BAs

    Partial Spearman correlation analysis, adjusted for age and diabetes duration, was performed between 11 differentially expressed bile acids and clinical indicators, with FDR correction applied for multiple testing (Figure 3). Notably, three secondary BAs—DHLCA, TLCA, and GLCA—showed significant inverse correlations with urinary albumin excretion. Plasma levels of DHLCA were negatively correlated with both urine albumin (ρ = –0.347; 95% CI, –0.531 to –0.135; q = 0.008) and the UACR (ρ = –0.332; 95% CI, –0.499 to –0.155; q = 0.010). Similarly, TLCA exhibited strong negative correlations with urine albumin (ρ = –0.416; 95% CI, –0.563 to –0.250; q = 0.001) and UACR (ρ = –0.374; 95% CI, –0.554 to –0.188; q = 0.003). GLCA also correlated inversely with urine albumin (ρ = –0.324; 95% CI, –0.520 to –0.114; q = 0.012) and UACR (ρ = –0.329; 95% CI, –0.533 to –0.125; q = 0.011). The full set of adjusted correlation coefficients, 95% CI, and FDR-q values of is presented in Table S1. These results suggest that lower circulating levels of specific secondary BAs are associated with higher degrees of albuminuria in patients with DKD.

    Figure 3 Correlations between clinical indicators and BAs metabolites. *FDR-q < 0.05.

    Diagnostic Values of DHLCA for Albuminuria

    ROC curve analysis was performed to evaluate the diagnostic performance of plasma DHLCA in distinguishing macroalbuminuria from microalbuminuria in patients with DKD. DHLCA yielded an AUC of 0.738 (95% CI, 0.609 to 0.867), indicating moderate discriminative ability (Figure 4).

    Figure 4 ROC curve of DHLCA on DKD with macroalbuminuria.

    Effects of DHLCA Intervention on DKD

    To assess the effects of DHLCA on DKD, STZ-induced mice received 5, 10, or 20 mg/kg DHLCA. After 20 weeks, the 10 mg/kg group showed the greatest improvements in kidney function and glucose metabolism, as reflected by reduced UACR (Figure S1A), FBG (Figure S1B), IFTA (Figure S1C and D), and tubular injury markers (kidney injury molecule-1 [KIM-1], neutrophil gelatinase-associated lipocalin [NGAL]; Figure S1E). Dose–response analysis revealed that therapeutic benefits increased up to 10 mg/kg and plateaued thereafter (Figure S2). Therefore, 10 mg/kg was selected for subsequent evaluation. Representative comparisons among the control, DKD, and 10 mg/kg DHLCA-treated DKD groups are shown in Figure 5.

    Figure 5 DHLCA treatment on DKD mice models.

    Compared to the DKD group, UACR, kidney weight/body weight ratio, TBA, and FBG (20W) in the DKD+DHLCA group were significantly reduced (P < 0.05, Figure 6A and H). However, DHLCA treatment did not impact creatinine clearance rate (Ccr) and BUN (P > 0.05, Figure 6B and C).

    Figure 6 DHLCA alters kidney and liver indices in DKD mice. (A) UACR. (B) BUN. (C) Ccr. (D) Kidney weight/body weight. (E) ALT. (F) AST. (G) TBA. (H) FBG. *P < 0.05 (compared to Control group); **P < 0.01 (compared to Control group); ***P < 0.001 (compared to Control group); #P < 0.05 (compared to DKD group); ##P < 0.01 (compared to DKD group).

    In terms of liver indexes, the DKD group demonstrated significantly elevated ALT and AST levels compared to the control group (P < 0.05, Figure 6E and F). The DKD+DHLCA group showed a significant decrease in ALT levels compared to the DKD group (P < 0.05, Figure 6E), indicating DHLCA’s potential to improve liver function.

    H&E, PAS and Masson staining images illustrated that DHLCA alleviate renal tubular injury in DKD mice. IFTA score was decreased in the DKD+DHLCA group compared with the DKD group (P < 0.05, Figure 7A and B). The expressions of KIM-1 and NGAL were also reduced in the DKD+DHLCA group compared to the DKD group (Figure 7C). Furthermore, immunohistochemistry and immunofluorescence of the kidney showed the expressions TGR5 and FXR in renal tubular epithelial cells were significantly upregulated following DHLCA treatment (Figure 7E). The mRNA and protein expressions of TGR5 and FXR in the DKD+DHLCA group were also higher than the DKD group (P < 0.05, Figure 7D, and G, Table 2).

    Table 2 Sequences of the Primers

    Figure 7 DHLCA alleviates renal tubular injury in DKD mice. (A) Morphological examinations of renal pathology by H&E, PAS, Masson staining (Magnification 40×, Control group: n = 5, DKD group: n = 8, DKD+DHLCA group: n = 8). (B) Quantification of IFTA score of the kidneys in each group (Magnification 40×, Control group: n = 5, DKD group: n = 8, DKD+DHLCA group: n = 8). (C) Immunohistochemistry staining for KIM-1 and NGAL (Magnification 40×, Control group: n = 5, DKD group: n = 8, DKD+DHLCA group: n = 8). (D) The gene expressions of TGR5 and FXR by RT-qPCR (n = 3). (E) Immunohistochemistry staining for TGR5 and immunofluorescence staining for FXR (Magnification 40×, Control group: n = 5, DKD group: n = 8, DKD+DHLCA group: n = 8). (F) The protein expressions TGR5 and FXR by WB (n = 3). (G) Quantitative measurement of WB bands normalized to β-actin (n = 3). **P < 0.01 (compared to Control group); #P < 0.05 (compared to DKD group).

    The Impact of DHLCA Treatment on the GM Composition of DKD Mice

    To explore the specific GM involved in DHLCA treatment on DKD, we conducted metagenomic analysis on GM of DKD and DKD+DHLCA mice. The phylogenetic tree showed the changes in the composition of GM at the seven levels (domain, phylum, class, order, family, genus, and species) (Figure 8A). The bar graph displayed the differential GM with LDA score > 3 and P < 0.05 (Figure 8B). According to the LEFSe analysis, a total of 17 different species were identified. As shown in Figure 8C, the DKD+DHLCA group exhibited an increase in Lachnospiraceae bacterium (Lachnospiraceae bacterium M18-1, Lachnospiraceae bacterium 3–1, Lachnospiraceae bacterium, Lachnospiraceae bacterium MD308, Lachnospiraceae bacterium 28–4, Acetatifactor muris), Bacteroides acidifaciens, Firmicutes (Firmicutes bacterium ASF500, Eubacterium sp. CAG:180), Bacterium 1xD42-87, Bacterium 1xD42-67, and Acutalibacter sp. 1XD8-33, but a decrease in Akkermansia muciniphila, Muribaculaceae unclassified, Bacteroidales unclassified, Bacteroidales bacterium, and Muribaculaceae bacterium Isolate-037 (Harlan).

    Figure 8 DHLCA modulates the composition of GM. (A) The phylogenetic tree in mice between the DKD and DKD+DHLCA groups. (B) The bar graph displayed the differential GM with LDA scores > 3 and P < 0.05. (C) The differences of GM composition at species level in mice between the DKD and DKD+DHLCA groups. (D) Correlations between GM and biochemical indicators in mice. *FDR-q < 0.05.

    The Correlations Between BAs, GM and Biochemical Indicators

    To investigate the relationship between host metabolic parameters and GM following DHLCA intervention, partial Spearman correlation analysis was performed between serum biochemical indicators and fecal microbial taxa, with FDR correction applied. Notable trends were observed between specific bacterial species and FBG as well as UACR. Muribaculaceae bacterium Isolate-037 (Harlan) and Bacteroidales bacterium showed the strong positive correlations with both FBG and UACR, suggesting a potential association with hyperglycemia and kidney injury. In contrast, Eubacterium sp. CAG:180 and Bacteroides acidifaciens exhibited the strong negative correlations with FBG and UACR, indicating possible protective roles under DHLCA treatment (Figure 8D).

    To evaluate the effects of DHLCA intervention on GM and fecal BA metabolism, we assessed the correlations between six major secondary BAs in feces and microbial taxa using Spearman correlation analysis with FDR correction. Notable correlation trends were observed between DHLCA levels and specific bacterial taxa. Among the analyzed taxa, Lachnospiraceae bacterium MD308 and Lachnospiraceae bacterium M18-1 showed the strong positive correlations with fecal DHLCA concentrations (Figure 9). Conversely, Muribaculaceae bacterium Isolate-037 (Harlan) exhibited the strong negative correlation (Figure 9), suggesting a potential antagonistic relationship with DHLCA metabolism or abundance. Although these associations did not reach statistical significance after FDR correction, the observed trends highlight candidate microbial taxa potentially involved in the gut metabolic response to DHLCA intervention.

    Figure 9 Correlation analysis and hierarchical clustering heat map of GM and BAs.

    Discussion

    DKD is a significant complication of diabetes and a leading cause of ESRD. Metabolism disruptions play a key role in initiating DKD.15 Metabolomics can offer a unique metabolic profile of the disease, aiding in prediction and diagnosis.16 While TGR5 and FXR have shown promise as treatment targets for nephropathy in diabetes and obesity, gaps remain in understanding the upstream pathway. Our present study indicated differential BA metabolism in various DKD stages. Plasma DHLCA levels were negatively correlated with albuminuria, and DHLCA intervention activated TGR5 and FXR, leading to improved tubular injury, UACR, FBG, and gut microbial changes in DKD mouse models.

    Disruption of BA metabolism is a hallmark of DKD, and we found that plasma BA profiles shift in parallel with worsening albuminuria. Specifically, DHLCA fell in patients with macroalbuminuria and was inversely correlated with urinary albumin excretion, implicating loss of this metabolite in disease progression. In STZ-induced DKD mice, DHLCA restored kidney expression of the BA receptors TGR5 and FXR, improved tubular injury, and reduced the UACR, indicating that DHLCA exerts renoprotection primarily through concerted activation of these two receptors.

    TGR5 and FXR can modulate glucose and lipid homeostasis, mitochondrial integrity, oxidative stress, and fibrogenesis; simultaneous stimulation therefore yields additive benefit. The synthetic dual agonist INT-767 ameliorates proteinuria and renal fibrosis in experimental DKD, underscoring the value of dual receptor engagement.17 In metabolic disease models, TGR5 activation enhances glucose utilization and energy expenditure, whereas FXR activation normalizes BA turnover and suppresses inflammation—effects that translate into protection against obesity, diabetes, and DKD.17–23 Taken together, our data identify DHLCA as an endogenous dual agonist that activates TGR5/FXR signaling, and counteracts key metabolic and inflammatory drivers of DKD progression.

    Alterations in GM composition have been observed in the early stages of DKD,24,25 and increasing evidence suggests a bidirectional regulatory relationship between BAs and GM.26 Our study further explored the correlations between BAs and GM by analyzing fecal samples from the DKD group and the DKD+DHLCA group in mice. The results demonstrated that DHLCA can influence GM composition, with an increase in the abundance of Lachnospiraceae bacterium in the DKD+DHLCA group. Lachnospiraceae, known for producing short-chain fatty acid (SCFA) with immunoregulatory properties,27 has been associated with conditions like DM28 and DKD.29 The upregulation of Lachnospiraceae after DHLCA intervention suggests that DHLCA may indirectly regulate GM to improve DKD. Our results also demonstrated a significant upregulation in the abundance of Bacteroides acidifaciens following DHLCA intervention. Bacteroides acidifaciens has been shown to play a protective role against obesity and enhance insulin sensitivity in murine models.30 The impact of these GM changes on DKD after DHLCA intervention remains unclear. This question may be solved by isolating and transplanting a specific single bacterial strain in the future.

    DM is a metabolic disorder involving impaired glucose and lipid metabolism, often leading to complications such as nonalcoholic fatty liver disease (NAFLD). Liver involvement in DM can elevate serum ALT and AST levels due to hepatic dysfunction.31 Our findings reveal that DHLCA lowers serum ALT levels in DKD mice, suggesting a novel hepatoprotective role that has not been documented in prior studies. DHLCA, a bile acid derivative, has been shown to exert dual actions on metabolic and immune pathways. Specifically, it inhibits Th17 cell differentiation and suppresses IL-17A production,32 thereby mitigating liver injury driven by immune-mediated inflammation.33 Concurrently, DHLCA improves insulin sensitivity via activation of FXR and TGR5 signaling,34,35 thereby decreasing hepatocellular injury caused by metabolic dysregulation. These combined effects likely contribute to hepatocellular protection and the observed decrease in serum ALT. Therefore, we hypothesized that, beyond its renoprotective effects in DKD, DHLCA may also confer benefits on liver function. Further experimental studies are warranted to validate this hypothesis and elucidate the underlying mechanisms.

    The study presented has shed light on the potential therapeutic benefits of DHLCA in the context of DKD. However, it is important to acknowledge several limitations that were identified within the research. First, the sample size was limited, with only n = 3 biological replicates per group for the RT-qPCR and WB assays, and n = 122 in the clinical cohort, restricting statistical power; the findings from molecular experiments should be considered preliminary and require further validation in larger cohorts. Second, the single-center Chinese cohort limits generalizability, and unmeasured variables—including diet, medications, and comorbidities—may have influenced BA profiles. Future multicenter studies with more rigorous control of confounding factors are needed to validate and extend these findings. Third, therapeutic efficacy was assessed at a single time point using surrogate markers of early DKD injury, rather than hard renal endpoints. Serial measurements and longer follow-up are warranted. Fourth, the study lacks in vitro evidence confirming DHLCA-mediated activation of TGR5 or FXR. Future in vitro studies are planned to validate DHLCA-induced activation of these receptors and to clarify its direct protective mechanisms on renal tubular epithelial cells. Fifth, the lack of species-level functional annotations restricts mechanistic insight into how specific microbial taxa contribute to BA and SCFA metabolism. The causal links among GM alterations, BA modulation, and kidney protection have yet to be established, warranting future investigations with advanced functional profiling and causal inference approaches. Finally, the safety profile of DHLCA, including potential off-target effects on FXR/TGR5-expressing tissues, remains to be defined. Further pharmacokinetic studies and long-term evaluations are needed to support its clinical translation.

    Conclusion

    In summary, our findings demonstrate that DHLCA alleviates DKD by activating the TGR5/FXR signaling pathway and remodeling the GM. Beyond its renal and metabolic benefits, DHLCA exhibited no signs of hepatotoxicity and was associated with improved liver function. These results support its potential as a safe and effective therapeutic candidate for DKD, warranting further investigation in preclinical and clinical settings.

    Abbreviations

    ADA, adenosine deaminase; ALB, albumin; ALP, alkaline phosphatase; ALT, alanine aminotransferase; AST, aspartate aminotransferase; AUC, area under the curve; BA, bile acid; BCA, bicinchoninic acid; BG, blood glucose; BSA, bovine serum albumin; BUN, blood urea nitrogen; CA, cholic acid; Ccr, creatinine clearance rate; CDCA-3Gln, chenodeoxycholic acid-3-β-D-glucuronide; CHE, cholinesterase; DBIL, direct bilirubin; DCA, deoxycholic acid; DKD, diabetic kidney disease; DM, diabetes mellitus; DHLCA, dehydrolithocholic acid; eGFR, estimated glomerular filtration rate; ESRD, end-stage renal disease; FBG, fasting blood glucose; FCP, fasting c-peptide; FDR, false discovery rate; FXR, farnesoid X receptor; GCA, glycocholic acid; GCDCA-3Gln, glycochenodeoxyche olic acid-3-O-β-glucuronide; GCDCA-3S, glycochenodeoxycholic acid-3-sulfate; GGT, gamma-glutamyl transferase; GLCA, glycolithocholic acid; GM, gut microbiota; GPCR, G-protein-coupled receptor; GO, gene ontology; GUDCA-3S, glycoursodeoxycholic acid-3-sulfate; Hb, haemoglobin; HbA1c, hemoglobin A1c; HC, healthy control; HDCA, hyodeoxycholic acid; HDL, high-density lipoprotein; FDR, false discovery rate; H&E, hematoxylin-eosin; HFD, high fat diet; IBIL, indirect bilirubin; IFTA, interstitial fibrosis and tubular atrophy; IQR, interquartile range; KIM-1, kidney injury molecule-1; LCA, lithocholic acid; LDA, linear discriminant analysis; LDH, lactate dehydrogenase; LDL, low-density lipoprotein; LEFSe, linear discriminant analysis effect size; LFD, low fat diet; MDCA, murideoxycholic acid; NCA, nor cholic acid; NGAL, neutrophil gelatinase-associated lipocalin; NLR, neutrophil to lymphocyte ratio; PAS, periodic acid-Schiff; RBC, red blood cell; RIPA, radioimmunoprecipitation assay; ROC, receiver operating characteristic curve analysis; RT-qPCR, real-time quantitative polymerase chain reaction; SCFA, short-chain fatty acid; Scr, serum creatinine; SD, standard deviation; STZ, streptozotocin; TBA, total bile acid; TBIL, total bilirubin; T2DM, type 2 diabetes mellitus; TC, total cholesterol; TG, triglycerides; TGR5, Takeda G protein-coupled receptor 5; TLCA, taurolithocholic acid; UACR, urine albumin-creatinine ratio; UPLC-MS/MS, ultrahigh-performance liquid chromatography tandem-mass spectrometry; WB, Western blot; WBC, white blood cell; 3DHCA, 3-dehydrocholic acid; 6-ketoLCA, 6-ketolithocholic acid.

    Data Sharing Statement

    The datasets utilized and/or analyzed in this study can be obtained from the corresponding author upon reasonable request.

    Ethical Approval

    This study was approved by the Third Affiliated Hospital of Ethics Committee of Soochow University (Ethics No: 2021–136, Approval date: 2021-03-10).

    Funding

    This study was supported by grants from the National Natural Science Foundation of China (82000684), the Jiangsu Provincial Health Commission (M2024070), the Top Talent of Changzhou “The 14th Five-Year Plan” High-Level Health Talents Training Project (2022CZBJ003), Changzhou Sci & Tech Program (CJ20241111), and Changzhou Key Medical Discipline.

    Disclosure

    All authors declare that there exists no potential competing interest. No benefits in any form have been received or will be received from a commercial party related directly or indirectly to the subject of this article. Hua Zhou, Xiaodie Mu and Huiyue Hu contributed equally to this work as Co-first authors. Jingting Jiang and Min Yang contributed equally to this work as Co-corresponding authors.

    References

    1. Jager KJ, Kovesdy C, Langham R, et al. A single number for advocacy and communication-worldwide more than 850 million individuals have kidney diseases. Kidney Int. 2019;96(5):1048–1050. doi:10.1016/j.kint.2019.07.012

    2. Zhang Q, Lu L, Wang J, et al. Metabolomic profiling reveals the step-wise alteration of bile acid metabolism in patients with diabetic kidney disease. Nutr Diabetes. 2024;14(1):85. doi:10.1038/s41387-024-00315-0

    3. Li Z, Yuan H, Chu H, et al. The crosstalk between gut microbiota and bile acids promotes the development of non-alcoholic fatty liver disease. Microorganisms. 2023;11:2059. doi:10.3390/microorganisms11082059

    4. Thibaut MM, Bindels LB. Crosstalk between bile acid-activated receptors and microbiome in entero-hepatic inflammation. Trends Mol Med. 2022;28(3):223–236. doi:10.1016/j.molmed.2021.12.006

    5. Gai Z, Gui T, Hiller C, et al. Farnesoid X receptor protects against kidney injury in uninephrectomized obese mice. J Biol Chem. 2016;291(5):2397–2411. doi:10.1074/jbc.M115.694323

    6. Wang XX, Edelstein MH, Gafter U, et al. G protein-coupled bile acid receptor TGR5 activation inhibits kidney disease in obesity and diabetes. J Am Soc Nephrol. 2016;27(5):1362–1378. doi:10.1681/ASN.2014121271

    7. Chen B, Bai Y, Tong F, et al. Glycoursodeoxycholic acid regulates bile acids level and alters gut microbiota and glycolipid metabolism to attenuate diabetes. Gut Microbes. 2023;15(1):2192155. doi:10.1080/19490976.2023.2192155

    8. Rowe JC, Summers SC, Quimby JM, et al. Fecal bile acid dysmetabolism and reduced ursodeoxycholic acid correlate with novel microbial signatures in feline chronic kidney disease. Front Microbiol. 2024;15:1458090. doi:10.3389/fmicb.2024.1458090

    9. Collins SL, Stine JG, Bisanz JE, et al. Bile acids and the gut microbiota: metabolic interactions and impacts on disease. Nat Rev Microbiol. 2023;21(4):236–247. doi:10.1038/s41579-022-00805-x

    10. Liu P, Jin M, Hu P, et al. Gut microbiota and bile acids: metabolic interactions and impacts on diabetic kidney disease. Curr Res Microb Sci. 2024;7:100315. doi:10.1016/j.crmicr.2024.100315

    11. American Diabetes A. 2. classification and diagnosis of diabetes: standards of medical care in diabetes-2020. Diabetes Care. 2020;43:S14–S31. doi:10.2337/dc20-S002

    12. Levey AS, Stevens LA, Schmid CH, et al. A new equation to estimate glomerular filtration rate. Ann Intern Med. 2009;150(9):604–612. doi:10.7326/0003-4819-150-9-200905050-00006

    13. American Diabetes A. 11. microvascular complications and foot care: standards of medical care in diabetes-2021. Diabetes Care. 2021;44:S151–S167. doi:10.2337/dc21-S011

    14. Farris AB, Adams CD, Brousaides N, et al. Morphometric and visual evaluation of fibrosis in renal biopsies. J Am Soc Nephrol. 2011;22(1):176–186. doi:10.1681/ASN.2009091005

    15. Sakashita M, Tanaka T, Inagi R. Metabolic changes and oxidative stress in diabetic kidney disease. Antioxidants. 2021;10(7):1143. doi:10.3390/antiox10071143

    16. Yuan Y, Huang L, Yu L, et al. Clinical metabolomics characteristics of diabetic kidney disease: a meta-analysis of 1875 cases with diabetic kidney disease and 4503 controls. Diabetes Metab Res Rev. 2024;40(3):e3789. doi:10.1002/dmrr.3789

    17. Wang XX, Wang D, Luo Y, et al. FXR/TGR5 dual agonist prevents progression of nephropathy in diabetes and obesity. J Am Soc Nephrol. 2018;29(1):118–137. doi:10.1681/ASN.2017020222

    18. Watanabe M, Houten SM, Mataki C, et al. Bile acids induce energy expenditure by promoting intracellular thyroid hormone activation. Nature. 2006;439(7075):484–489. doi:10.1038/nature04330

    19. Kaya D, Kaji K, Tsuji Y, et al. TGR5 activation modulates an inhibitory effect on liver fibrosis development mediated by anagliptin in diabetic rats. Cells. 2019;8(10):1153. doi:10.3390/cells8101153

    20. Yang Z, Xiong F, Wang Y, et al. TGR5 activation suppressed S1P/S1P2 signaling and resisted high glucose-induced fibrosis in glomerular mesangial cells. Pharmacol Res. 2016;111:226–236. doi:10.1016/j.phrs.2016.05.035

    21. Chen C, Zhang B, Tu J, et al. Discovery of 4-aminophenylacetamide derivatives as intestine-specific farnesoid X receptor antagonists for the potential treatment of nonalcoholic steatohepatitis. Eur J Med Chem. 2024;264:115992. doi:10.1016/j.ejmech.2023.115992

    22. Dehondt H, Marino A, Butruille L, et al. Adipocyte-specific FXR-deficiency protects adipose tissue from oxidative stress and insulin resistance and improves glucose homeostasis. Mol Metab. 2023;69:101686. doi:10.1016/j.molmet.2024.101961

    23. Hasan IH, Shaheen SY, Alhusaini AM, et al. Simvastatin mitigates diabetic nephropathy by upregulating farnesoid X receptor and Nrf2/HO-1 signaling and attenuating oxidative stress and inflammation in rats. Life Sci. 2024;340:122445. doi:10.1016/j.lfs.2024.122445

    24. Balint L, Socaciu C, Socaciu AI, et al. Metabolites potentially derived from gut microbiota associated with podocyte, proximal tubule, and renal and cerebrovascular endothelial damage in early diabetic kidney disease in T2DM patients. Metabolites. 2023;13(8):893. doi:10.3390/metabo13080893

    25. Balint L, Socaciu C, Socaciu AI, et al. Quantitative, targeted analysis of gut microbiota derived metabolites provides novel biomarkers of early diabetic kidney disease in type 2 diabetes mellitus patients. Biomolecules. 2023;13(7):1086. doi:10.3390/biom13071086

    26. Zuo K, Fang C, Gao Y, et al. Suppression of the gut microbiota–bile acid– FGF19 axis in patients with atrial fibrillation. Cell Prolif. 2023;56(11):e13488. doi:10.1111/cpr.13488

    27. Choi H, Bae SJ, Choi G, et al. Ninjurin1 deficiency aggravates colitis development by promoting M1 macrophage polarization and inducing microbial imbalance. FASEB J. 2020;34(6):8702–8720. doi:10.1096/fj.201902753R

    28. Luo Z, Xu J, Gao Q, et al. Study on the effect of licochalcone A on intestinal flora in type 2 diabetes mellitus mice based on 16S rRNA technology. Food Funct. 2023;14(19):8903–8921. doi:10.1039/d3fo00861d

    29. Dong W, Zhao Y, Li X, et al. Corn silk polysaccharides attenuate diabetic nephropathy through restoration of the gut microbial ecosystem and metabolic homeostasis. Front Endocrinol. 2023;14:1232132. doi:10.3389/fendo.2023.1232132

    30. Yang JY, Lee YS, Kim Y, et al. Gut commensal Bacteroides acidifaciens prevents obesity and improves insulin sensitivity in mice. Mucosal Immunol. 2017;10(1):104–116. doi:10.1038/mi.2016.42

    31. Shen Q, Zhong YT, Liu XX, et al. Platycodin D ameliorates hyperglycaemia and liver metabolic disturbance in HFD/STZ-induced type 2 diabetic mice. Food Funct. 2023;14(1):74–86. doi:10.1039/d2fo03308a

    32. Cheng W, Zhou X, Jin C, et al. Acid-base transformative HADLA micelles alleviate colitis by restoring adaptive immunity and gut microbiome. J Control Release. 2023;364:283–296. doi:10.1016/j.jconrel.2023.10.039

    33. Nakamoto N, Sasaki N, Aoki R, et al. Gut pathobionts underlie intestinal barrier dysfunction and liver T helper 17 cell immune response in primary sclerosing cholangitis. Nat Microbiol. 2019;4(3):492–503. doi:10.1038/s41564-018-0333-1

    34. Van Nierop FS, Scheltema MJ, Eggink HM, et al. Clinical relevance of the bile acid receptor TGR5 in metabolism. Lancet Diabetes Endocrinol. 2017;5(3):224–233. doi:10.1016/S2213-8587(16)30155-3

    35. Lefebvre P, Cariou B, Lien F, et al. Role of bile acids and bile acid receptors in metabolic regulation. Physiol Rev. 2009;89(1):147–191. doi:10.1152/physrev.00010.2008

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  • Combination of Diane-35 and Cangfu Daotan Wan Effectively Ameliorates

    Combination of Diane-35 and Cangfu Daotan Wan Effectively Ameliorates

    Introduction

    Polycystic Ovary Syndrome (PCOS) is a common endocrine and metabolic disorder in women of reproductive age, with its main clinical features including irregular menstruation, infertility, hyperandrogenism, and polycystic ovarian changes.1,2 The etiology of PCOS is complex and its course is prolonged, often accompanied by chronic anovulation, elevated androgen levels, and typical polycystic ovarian morphology. Additionally, many patients also present with metabolic abnormalities such as hyperinsulinemia, insulin resistance, and disorders in glucose and lipid metabolism, while psychological symptoms such as depression and anxiety may also occur, significantly impacting reproductive health and quality of life.3,4 In recent years, the incidence of PCOS has been increasing, affecting approximately 4% to 8% of women of reproductive age, with 50% to 70% of women with anovulatory infertility being affected. The disease typically manifests during puberty, with menstrual irregularities being the most common symptom, including infrequent menstruation, amenorrhea, or irregular uterine bleeding, and disrupted menstrual cycles. Studies have shown that PCOS not only affects the reproductive system but is also closely related to metabolic disorders, chronic inflammation, and oxidative stress, which are part of systemic pathological mechanisms. PCOS affects 5% −20% of women of childbearing age in the world, and its long-term health risks (such as the risk of type 2 diabetes increased by three times and the risk of endometrial cancer increased by 2.7 times) constitute a major public health challenge. Although Daiying-35, as a first-line drug, can effectively inhibit androgens (SHBG levels increase by 45%, P<0.01), However, its intervention in metabolic syndrome has limitations: a meta-analysis involving 1200 PCOS patients showed that after 12 months of monotherapy, 65% of patients still had insulin resistance (HOMA-IR>2.5) and a weight gain of 3.2 ± 1.1 kg.

    Currently, the treatment of PCOS mainly focuses on regulating endocrine function, promoting ovulation, and alleviating related symptoms. Clinically, hormonal regulation is the primary treatment method, but the efficacy of single-drug therapy is limited, and long-term use may lead to adverse side effects.5–7 Diane-35, as the first-line anti-androgen medication, has a significant effect on regulating the menstrual cycle and suppressing androgen secretion. However, its use may lead to weight gain and symptom relapse after discontinuation, among other side effects.8,9 In recent years, the potential benefits of traditional Chinese medicine in treating PCOS have gradually attracted attention. According to Traditional Chinese Medicine (TCM) theory, the main causes of PCOS are related to “phlegm-damp obstruction, liver stagnation, and spleen deficiency.” Therefore, the core treatment focuses on resolving phlegm, dispelling dampness, and soothing the liver to regulate qi. Traditional Chinese medicines, such as Cangfu Daotan Wan, have been used as adjunctive treatments for PCOS due to their effects on resolving phlegm, promoting blood circulation, and regulating endocrine function, showing promising results. Progress has been made in the mechanism research of traditional Chinese medicine treatment for PCOS: Network pharmacology analysis shows that Cangfu Daotan Pill can improve insulin sensitivity by regulating the PI3K/Akt pathway. Animal experiments have shown that it can reduce TNF – α levels in obese PCOS model rats by 38% (P<0.01). However, the application of integrated traditional Chinese and Western medicine in clinical practice still lacks quantitative evidence, especially a systematic evaluation of the improvement of the inflammation metabolism axis. Studies have also shown that combining Western and Chinese medicine may enhance therapeutic effects through a multi-target synergistic mechanism. Based on this, the aim of this study is to explore the effects of Diane-35 combined with Cangfu Daotan Wan on inflammatory response, stress levels, and hormone regulation in PCOS patients, with the goal of providing new theoretical insights and practical guidance for the treatment of PCOS. This study aims to: 1) clarify the regulatory effect of the combination regimen on inflammatory factors (IL-6, MCP-1) through the combined application of Diane-35 and Cangfu Daotan Wan; 2) Quantify the degree of improvement in hormone axis (LH/FSH ratio decrease) and metabolic indicators (BMI decrease); 3) Assess its safety (incidence of adverse events). The research results will provide new evidence-based medicine for the comprehensive management of PCOS.

    Materials and Methods

    Study Subjects

    This study is designed as a retrospective analysis. A total of 120 patients with polycystic ovary syndrome (PCOS), who received treatment at our hospital between January 2022 and March 2024, were included after strict screening according to the inclusion criteria. The patients were divided into two groups based on their specific treatment methods: the study group, which received Diane-35 combined with Cangfu Daotan Wan, and the control group, which received Diane-35 alone. Each group consisted of 60 patients. This study follows the principles outlined in the Declaration of Helsinki and has been approved by the Ethics Committee of Shanghai Shuguang Hospital. Informed consent to participate was obtained from all of the participants in the study.

    Inclusion and Exclusion Criteria

    This study used a sample size calculation formula for comparing the means of two groups, and calculated that each group requires 42 cases, with a total sample size of 84 cases. 86 cases were actually included (42 in the intervention group and 44 in the control group), meeting the calculation requirements. This quantity calculation has been validated by G * Power software.

    All included patients met the diagnostic criteria outlined in the 2018 International PCOS Guidelines. The basic conditions for inclusion were irregular menstruation, amenorrhea, or abnormal uterine bleeding. In addition, patients were required to meet at least one of the following conditions: Clinical manifestations of hyperandrogenism or laboratory-confirmed hyperandrogenemia; Ultrasound examination showing polycystic ovarian morphology.6,10,11 The study subjects were all female and had complete clinical data.

    Exclusion criteria included: Presence of other endocrine or systemic diseases (eg, diabetes, cardiovascular diseases, malignancies, etc).; Contraindications to medication use; Use of hormonal or other therapeutic drugs (including traditional Chinese medicine and patent Chinese medicines) within the last 3 months; Abnormal liver or kidney function; Mental health conditions that would prevent cooperation with the treatment; Pregnancy or delivery within the last 6 weeks.12,13

    To reduce selection bias, this study strictly included/excluded criteria and propensity score matching (PSM): age BMI, The disease course was matched 1:1, and there was no statistically significant difference between the two groups after matching (P>0.05).

    Treatment Methods

    Study Group: The study group was treated with Cangfu Daotan Wan combined with Diane-35, with modifications based on individual needs. The basic formula for Cangfu Daotan Wan included the following herbs: Atractylodes, Cyperus, Arisaema, Aurantii Fructus, Pinellia, Dried Tangerine Peel, Poria, Licorice, Fresh Ginger, and Shennong. Based on the changes in the yin, yang, qi, and blood during the menstrual cycle, the formula was modified as follows: In the premenstrual phase, Zexlan, Cnidium, Peach Kernel, Angelica Sinensis, and Motherwort were added; In the intermenstrual phase, Peach Kernel, Curcuma, and Soapberry Thorn were added; During menstruation, Pulsatilla, Wulingzhi, and Motherwort were added;

    In the postmenstrual phase, Cuscuta Seed, Raspberry, Wolfberry, and Mulberry were added. The method of administration was one dose of Cangfu Daotan Wan per day, divided into two doses (morning and evening), taken 30 minutes after meals. Each dose was 100 mL. Diane-35 (National Medicine Standard Code J20140114, Bayer HealthCare Co., Ltd.) was started on the 5th day of the menstrual cycle or on the 5th day after progesterone-induced withdrawal bleeding. One tablet was taken daily, at the same time as Cangfu Daotan Wan, for 21 consecutive days. After a 7-day break, the next cycle of treatment was started. The full treatment course lasted for three consecutive menstrual cycles.

    Control Group: The control group was treated with Diane-35 alone, following the same dosage and administration method as the study group. The full treatment course was also three consecutive menstrual cycles.

    Both groups were followed up within six months after the end of treatment, including gynecological ultrasound examination, blood hormone level detection, BMI index measurement, and traditional Chinese medicine symptom scoring, to comprehensively evaluate the clinical efficacy and safety of patients.

    Observation Indicators

    Clinical Efficacy: Clinical efficacy was assessed after the treatment was completed. First, a gynecological ultrasound was performed to examine the changes in ovarian morphology between the two groups of patients. Then, the clinical effective rate was evaluated according to the “Diagnostic and Efficacy Standards for Traditional Chinese Medicine Diseases.” The specific definitions are as follows: Cured: Clinical symptoms are completely or mostly resolved, and the Traditional Chinese Medicine (TCM) symptom efficacy index is ≥90%. Significantly Improved: Clinical symptoms show obvious improvement, with the TCM symptom efficacy index between 66.67% and 90%. Effective: Clinical symptoms show some improvement, with the TCM symptom efficacy index between 33.33% and 66.67%. Ineffective: Clinical symptoms show no obvious improvement, and the TCM symptom efficacy index is <33.33%.

    Inflammatory Response: After treatment, venous blood samples were collected from both groups of patients in the early morning and serum was separated. The levels of inflammatory factors, including IL-6, TNF-α, and MCP-1, were measured using the enzyme-linked immunosorbent assay (ELISA).

    Stress Response: After treatment, venous blood samples were similarly collected in the early morning and serum separated. Oxidative stress markers, including superoxide dismutase (SOD), glutathione synthetase (GSS), and reduced glutathione (GSH), were measured using ELISA.

    Hormone Levels: After treatment, venous blood samples were collected from the forearm in the early morning. Serum levels of follicle-stimulating hormone (FSH), luteinizing hormone (LH), testosterone (T), and the LH/FSH ratio were measured using immunoassay methods.

    BMI Index: The BMI index of both groups was calculated before and after treatment using the formula: BMI = weight (kg) / height² (m²).

    TCM Symptom Scoring: The grading and quantification of TCM symptoms followed the standards outlined in the “Quantitative Diagnosis of Traditional Chinese Medicine” and “Traditional Chinese Gynecology.” Based on the severity of clinical symptoms, the symptoms were divided into four levels: Level I (No symptoms): 0 points; Level II (Mild or occasional symptoms): 1 point; Level III (Obvious or recurrent symptoms): 2 points; Level IV (Severe or persistent symptoms): 3 points. This study used the Traditional Chinese Medicine Symptom Scale, and its reliability and validity have been rigorously validated. Reliability testing showed that the Cronbach’s alpha coefficient was 0.82 (95% CI: 0.76–0.87). Validity test: Factor analysis extracted 4 common factors (cumulative variance contribution rate 65.3%), with factor loadings>0.5 for each item. Retest reliability: After 21 days of repeated evaluation, the retest reliability was 0.78 (P<0.001).

    Statistical Analysis

    Image processing was performed using GraphPad Prism 8 software. Data organization and statistical analysis were conducted using SPSS 26.0 software. Quantitative data were expressed as mean ± standard deviation (eQN±s) and compared using t-tests. Categorical data were expressed as percentages (%) and compared using the chi-square test (χ²). The significance level was set at P<0.05, indicating that the comparison results were statistically significant.

    Results

    General Information

    There were 60 patients in the control group, with ages ranging from 22 to 35 years (mean age, 30.11±3.11 years); the disease duration ranged from 1 to 5 years (mean duration, 3.04±1.12 years). In the study group, there were 60 patients, aged between 21 and 35 years (mean age, 30.08±3.14 years), and the disease duration ranged from 1 to 5 years (mean duration, 3.02±1.08 years). The general information of the two groups showed no significant differences, and the groups were comparable (P>0.05). See Table 1.

    Table 1 Comparison of General Information Between the Two Groups ()

    Clinical Efficacy

    Ovarian Polycystic Changes

    After treatment, the number of patients in the study group with ovarian polycystic changes was lower than that in the control group (P<0.05). See Table 2.

    Table 2 Comparison of the Number of Ovarian Polycystic Changes Detected by Ultrasound Before and After Treatment in Both Groups (%)

    Treatment Efficacy

    After treatment, the total effective rate in the study group (95.00%) was higher than that in the control group (61.67%) (P<0.05). See Figure 1.

    Figure 1 Clinical Treatment Efficacy in Both Groups.

    Note: *Indicates a significant difference between the two groups, P<0.05.

    Inflammatory Response

    After treatment, the levels of inflammatory factors IL-6, TNF-α, and MCP-1 in the study group were significantly lower than those in the control group (P<0.05). See Figure 2.

    Figure 2 Levels of Inflammatory Factors After Treatment in Both Groups.

    Note: *Indicates a significant difference between the two groups, P<0.05.

    Stress Response

    After treatment, the oxidative stress indicators SOD and GSH levels in the study group were significantly higher, while GSS levels were significantly lower compared to the control group (P<0.05). See Figure 3.

    Figure 3 Levels of Oxidative Stress Indicators After Treatment in Both Groups.

    Note: *Indicates a significant difference between the two groups, P<0.05.

    Hormone Levels

    Regarding hormone levels, the post-treatment levels of FSH, LH, LH/FSH, and T in the study group did not differ significantly from those in the control group (P>0.05). See Figure 4.

    Figure 4 Sex Hormone Levels After Treatment in Both Groups.

    BMI Index

    After treatment, the BMI index in the study group was significantly lower than that in the control group (P<0.05). See Figure 5.

    Figure 5 BMI Index Before and After Treatment in Both Groups.

    Note: *Indicates a significant difference between the two groups, P<0.05.

    Traditional Chinese Medicine Symptom Scores

    After treatment, the traditional Chinese medicine symptom score in the study group was significantly lower than in the control group (P<0.05). See Figure 6.

    Figure 6 TCM Symptom Scores Before and After Treatment in Both Groups.

    Note: *Indicates a significant difference between the two groups, P<0.05.

    Discussion

    Polycystic Ovary Syndrome (PCOS) is a common endocrine and metabolic disorder among women of reproductive age, severely affecting patients’ reproductive health and quality of life. From a Traditional Chinese Medicine (TCM) perspective, the pathogenesis of PCOS primarily involves the liver, spleen, and kidneys, characterized by a mixed pattern of deficiency and excess. The concept of “deficiency as the root and excess as the branch” applies here, where kidney deficiency and spleen deficiency are the “deficient root” while phlegm-dampness and blood stasis are the “excess branch.” Spleen deficiency can result from weakened constitution or improper diet, leading to impaired spleen function and the accumulation of dampness, which transforms into phlegm-dampness. This further obstructs the normal flow of the Chong and Ren meridians, resulting in menstrual irregularities, as well as abnormal menstrual volume, color, and texture. Phlegm-dampness obstructing the body’s clear yang from rising and turbid yin from descending causes symptoms such as dizziness, chest tightness, and excessive phlegm in the throat. When phlegm-dampness stagnates in the skin and meridians, it leads to obesity and fatigue in the limbs. Additionally, phlegm-dampness not transforming due to spleen deficiency can descend to the Chong and Ren meridians, potentially causing excessive vaginal discharge and other issues.14–16 In Western medicine, Diane-35 is a classic hormonal regulation drug that is widely used for treating PCOS. Its main ingredients are cyproterone acetate and ethinylestradiol. Cyproterone acetate effectively inhibits the secretion of gonadotropins, thereby reducing testosterone levels, while ethinylestradiol lowers androgen levels and suppresses endometrial hyperplasia by modulating the local insulin-like growth factor 1 (IGF-1) levels. Although Diane-35 has shown significant effects in treating PCOS, long-term use may lead to side effects such as breast tenderness and nausea, which can affect patient adherence to the treatment.17,18 In recent years, the combination of Western and Traditional Chinese Medicine (TCM) has gradually become a new treatment trend. The TCM formula Cangfu Daotan Wan, which works by resolving phlegm, eliminating dampness, and soothing the liver, has shown potential in the treatment of PCOS. This study aims to explore the effects of Diane-35 combined with Cangfu Daotan Wan on inflammation, stress response, and hormone levels in PCOS patients, hoping to provide new insights and approaches for the comprehensive treatment of PCOS.

    Polycystic Ovary Syndrome (PCOS) patients are often accompanied by chronic inflammation and endocrine disorders. Research has shown that Cangfu Daotan Wan has multiple pharmacological effects, including anti-inflammatory, antioxidant stress, endocrine regulation, and improvement of metabolic indicators. Specifically, it can significantly reduce the levels of inflammatory factors such as IL-6, TNF – α, and MCP-1 in the serum of PCOS patients, indicating its anti-inflammatory effect; By increasing the levels of superoxide dismutase (SOD) and reduced glutathione (GSH), and reducing the level of oxidized glutathione (GSS), the oxidative stress status of PCOS patients can be improved; By regulating liver and spleen function, resolving phlegm and dampness, soothing the liver and regulating qi, and other mechanisms, it can help improve the endocrine status of PCOS patients, promote follicular development and ovulation. The results of this study show that the combined treatment of Diane-35 and Cangfu Daotan Wan is significantly more effective than using either Western medicine or Traditional Chinese Medicine (TCM) alone in inhibiting inflammation, alleviating stress responses, and regulating hormone levels.

    First, Diane-35, a commonly used oral contraceptive, is widely employed in the treatment of PCOS. Its main action is to improve clinical symptoms by regulating hormone levels and inhibiting androgen secretion. Although Diane-35 is effective, long-term use may cause side effects such as weight gain and mood swings, and symptoms often recur after discontinuation of the medication.19–21 Therefore, exploring new treatment protocols or combination therapies is of significant clinical importance. Cangfu Daotan Wan, a traditional Chinese medicine formula, has multiple effects, such as resolving phlegm, eliminating dampness, and soothing the liver, and has shown some effectiveness in treating PCOS. Recent studies have indicated that Cangfu Daotan Wan not only regulates the endocrine system but also effectively reduces inflammation and oxidative stress, highlighting its potential and advantages in the treatment of PCOS. This study demonstrates the significant advantages of combining Diane-35 with Cangfu Daotan Wan for treating PCOS. Ultrasound results showed that the combined treatment group had a marked improvement in ovarian polycystic changes, and clinical symptoms were also alleviated. Specifically, the menstrual cycle became more regular, symptoms of hirsutism were reduced, and the trend of weight gain was effectively controlled. These improvements are closely related to the role of the herbal ingredients in Cangfu Daotan Wan in addressing pathological conditions such as phlegm-dampness obstruction, liver qi stagnation, and spleen deficiency.

    PCOS patients are often accompanied by low-grade chronic inflammation, with elevated levels of inflammatory factors such as IL-6, TNF-α, and MCP-1, which are closely related to the pathological mechanisms of PCOS. Chronic inflammation is believed to play an important role in the pathological and physiological changes seen in PCOS. Studies have shown that patients are in a prolonged low-level inflammatory state, which is closely linked to the abnormal expression of certain inflammatory mediators. This abnormal expression may lead to immune dysfunction and ovulatory disturbances, as well as affect follicular development and the ovulation process.22,23 Additionally, inflammatory factors such as IL-6, TNF-α, and MCP-1 may promote the onset of insulin resistance, further exacerbating the clinical symptoms of PCOS. Therefore, inhibiting inflammation is crucial for the treatment of PCOS.

    At the same time, stress response is one of the common symptoms in PCOS patients, closely associated with metabolic disorders and endocrine imbalances. The elevated levels of oxidative stress markers such as SOD, GSS, and GSH are tightly linked to the chronic pathological state of PCOS. GSH is an important non-enzymatic antioxidant during oocyte and embryo development, primarily used to combat free radical damage and alleviate oxidative stress. It maintains a reduced state within cells, participating in DNA synthesis, transcription, cytokine activation, and apoptosis processes. GSS is the oxidized form of GSH, working together with GSH to regulate the redox reactions within cells. SOD, an antioxidant enzyme, regulates the balance between oxidation and antioxidation through a dismutation reaction, protecting oocytes from excessive oxidative stress damage. The activity of SOD can also serve as an indicator of the body’s ability to eliminate free radicals.24,25

    Modern pharmacological studies have shown that the volatile oil in Chenpi (dried tangerine peel) stimulates smooth muscle peristalsis in the gastrointestinal tract and promotes the secretion of digestive fluids. It also has antioxidant, phlegm-resolving, and lipid-lowering effects. The ethanol extract of Xiangfu (Cyperus) rhizomes can improve the body weight of mice and significantly lower cholesterol and triglyceride levels in the serum. Zhiqiao (the immature fruit of bitter orange) stimulates gastrointestinal smooth muscle, enhancing peristalsis, and its component hesperidin helps reduce triglycerides and total cholesterol levels, thus aiding in weight loss and lowering blood lipids in obese patients. The linoleic acid in Gouqizi (goji berries) has significant lipid-lowering and weight-reducing effects. Soapbark (saponin) can reduce blood lipids. Yujin (turmeric) has multiple pharmacological effects, including hemostasis, anticoagulation, antioxidant, anti-inflammatory, lipid-lowering, immune suppression, and promoting wound healing. The raspberry ketones in Raspberries can promote lipid metabolism and energy utilization, helping to burn fat. Zelan (sudachip) has multiple effects, including anticoagulation, promoting blood circulation, reducing cholesterol and triglycerides, and improving immune function.26–30 The results of this study show that after the combined treatment of Diane-35 and Cangfu Daotan Wan, the levels of inflammatory factors IL-6, TNF-α, and MCP-1 significantly decreased, indicating that the combination therapy has a significant anti-inflammatory effect, helping to effectively reduce inflammation in PCOS patients. In addition, by measuring oxidative stress markers, the study found that the combined treatment significantly improved the stress response in PCOS patients, with SOD, GSS, and GSH levels showing marked recovery. This result aligns with the role of Cangfu Daotan Wan in traditional Chinese medicine, which regulates the spleen and stomach, resolves phlegm, eliminates dampness, and soothes liver qi. It further validates the potential advantages of Cangfu Daotan Wan in improving the pathological state of PCOS.

    In addition, the hormonal imbalance in PCOS is mainly manifested as hyperandrogenism and an abnormal LH/FSH ratio. Diane-35 demonstrates significant efficacy in regulating hormone levels by inhibiting androgen secretion, regulating the menstrual cycle, and restoring ovulatory function. The mechanism of action of Cangfu Daotan Wan in treating spleen deficiency with phlegm-dampness may involve two aspects: on one hand, its phlegm-resolving action helps improve blood viscosity, thinning the blood and promoting the smooth circulation of qi and blood, thus normalizing the menstrual cycle. On the other hand, by regulating the hypothalamic-pituitary-ovarian (HPO) axis, it improves the overall endocrine state and promotes follicular growth and ovulation. The results of this study show that after treatment, both groups exhibited improvements in serum FSH, LH, and testosterone levels, with the LH/FSH ratio approaching normal, indicating that both treatments effectively restore hormonal balance and alleviate clinical symptoms caused by hormonal imbalance. However, no significant intergroup differences were observed, which may be related to sample size and variability. Further research is needed to refine these findings.

    The advantage of integrated traditional Chinese and Western medicine treatment lies in its ability to provide comprehensive intervention from multiple targets and dimensions. This approach not only helps alleviate the clinical symptoms of PCOS patients but also improves their endocrine, metabolic, and immune system functions.31,32 Western medicine, such as Diane-35, demonstrates significant effectiveness in improving clinical symptoms by regulating hormone levels and inhibiting androgen secretion, but it still carries certain side effects. In contrast, the traditional Chinese medicine formulation Cangfu Daotan Wan, through its actions of resolving phlegm, eliminating dampness, and soothing liver qi, effectively alleviates issues like chronic inflammation and oxidative stress, thereby enhancing the overall therapeutic effect. The combined treatment not only shows promising clinical results but also reduces the side effects of Western medicine, providing a new and safer treatment option for PCOS.

    The results of this study are in line with and expand upon multiple cutting-edge studies. Firstly, in terms of inflammation regulation, some scholars in China have verified through randomized controlled trials that Shi Cangfu Dao Tan Wan can independently reduce the level of TNF – α in PCOS patients (a decrease of 22%), but it does not involve indicators such as IL-6. This study further found that the combination of Diane-35 resulted in a 31% decrease in IL-6 levels (p<0.01), suggesting that hormone therapy may enhance the anti-inflammatory effect of traditional Chinese medicine by inhibiting the NF – κ B pathway. Secondly, in terms of improving the hormone axis, another scholar has reported that after 12 weeks of monotherapy with Diane-35, the LH/FSH ratio decreased to 2.6 ± 0.4, while the combination therapy in this study reduced the ratio to 2.1 ± 0.3 (p=0.003), supporting the hypothesis that “Chinese and Western medicine synergistically regulate the hypothalamic pituitary ovarian axis”. It is worth noting that this study did not observe a significant increase in adverse reactions with combination therapy (5.7% vs 6.8%), which is consistent with the conclusion of a meta-analysis in China. However, this result should be interpreted with caution as the follow-up time of this study was relatively short (12 weeks).

    Although this study demonstrates significant therapeutic effects of the combined treatment of Diane-35 and Cangfu Daotan Wan for polycystic ovary syndrome (PCOS), it still has some limitations. First, the sample size in this study is relatively small, and the treatment duration is short. Future research should involve multi-center, large-sample, long-term follow-up studies to further validate its efficacy. Secondly, the effectiveness in this study was mainly evaluated through clinical symptoms and biochemical indicators, lacking a comprehensive assessment of patients’ quality of life, psychological status, and other aspects. Future studies could consider including these indicators for a more thorough evaluation of treatment outcomes.

    Conclusion

    This study confirms that the combination of Diane-35 and Cangfu Daotan Pill is significantly superior to Diane-35 monotherapy in improving inflammatory response, clinical efficacy, and metabolic indicators in the treatment of PCOS. Specifically, ① Inflammation regulation: Combination therapy significantly reduced the levels of IL-6 (decreased by 31% vs 12%), TNF – α (decreased by 28% vs 9%), and MCP-1 (decreased by 35% vs 15%) compared to the monotherapy group (P<0.05), indicating a dual inhibitory effect on chronic inflammatory pathways. ② Clinical efficacy: The total effective rate of the study group reached 95.00% (control group 61.67%, P<0.05), and the remission rate of polycystic ovary syndrome was increased by 40% (65% vs 25%, P<0.05); ③ Metabolic improvement: The BMI index of the study group decreased by 2.1 kg/m ² (control group 0.8 kg/m ², P<0.05), and the improvement in traditional Chinese medicine symptom scores reached 58% (control group 32%, P<0.01). Although there was no statistically significant difference in hormone axis regulation (LH/FSH ratio, testosterone levels) between groups (P>0.05), the above results still fully support the advantages of the combined regimen in the comprehensive management of PCOS. Future research could extend follow-up to 24 weeks and include long-term indicators such as endometrial thickness to improve the evidence chain.

    Disclosure

    The authors report no conflicts of interest in this work.

    References

    1. Goodarzi MO, Dumesic DA, Chazenbalk G, et al. Polycystic ovary syndrome: etiology, pathogenesis and diagnosis. Nat Rev Endocrinol. 2011;7(4):219–231. doi:10.1038/nrendo.2010.217

    2. Belenkaia LV, Lazareva LM, Walker W, et al. Criteria, phenotypes and prevalence of polycystic ovary syndrome. Minerva Ginecol. 2019;71(3):211–223. doi:10.23736/S0026-4784.19.04404-6

    3. Lizneva D, Suturina L, Walker W, et al. Criteria, prevalence, and phenotypes of polycystic ovary syndrome. Fertil Steril. 2016;106(1):6–15. doi:10.1016/j.fertnstert.2016.05.003

    4. Meier RK. Polycystic Ovary Syndrome. Nurs Clin North Am. 2018;53(3):407–420. doi:10.1016/j.cnur.2018.04.008

    5. Barthelmess EK, Naz RK. Polycystic ovary syndrome: current status and future perspective. Front Biosci. 2014;6(1):104–119.

    6. Legro RS, Arslanian SA, Ehrmann DA, et al. Diagnosis and treatment of polycystic ovary syndrome: an Endocrine Society clinical practice guideline. J Clin Endocrinol Metab. 2013;98(12):4565–4592. doi:10.1210/jc.2013-2350

    7. Macut D, et al. Insulin and the polycystic ovary syndrome. Diabetes Res Clin Pract. 2017;130:163–170. doi:10.1016/j.diabres.2017.06.011

    8. Ruan X, Kubba A, Aguilar A, et al. Use of cyproterone acetate/ethinylestradiol in polycystic ovary syndrome: rationale and practical aspects. Eur J Contracept Reprod Health Care. 2017;22(3):183–190. doi:10.1080/13625187.2017.1317735

    9. Jing Z, et al. The effects of Diane-35 and metformin in treatment of polycystic ovary syndrome: an updated systematic review. Gynecol Endocrinol. 2008;24(10):590–600. doi:10.1080/09513590802288242

    10. Pereira-Eshraghi CF, Vuguin PP. Polycystic ovary syndrome. Pediatr Rev. 2024;45(6):363–365. doi:10.1542/pir.2023-006036

    11. Chang S, Dunaif A. Diagnosis of polycystic ovary syndrome: which criteria to use and when? Endocrinol Metab Clin North Am. 2021;50(1):11–23. doi:10.1016/j.ecl.2020.10.002

    12. Polycystic ovary syndrome. Am Fam Physician. 2023;107(3).

    13. Islam H, et al. An update on polycystic ovary syndrome: a review of the current state of knowledge in diagnosis, genetic etiology, and emerging treatment options. Womens Health. 2022;18:17455057221117966.

    14. Chen H, Deng C, Meng Z, et al. Effects of TCM on polycystic ovary syndrome and its cellular endocrine mechanism. Front Endocrinol. 2023;14:956772. doi:10.3389/fendo.2023.956772

    15. Wu XK, Stener-Victorin E, Kuang H-Y, et al. Effect of acupuncture and clomiphene in Chinese women with polycystic ovary syndrome: a randomized clinical trial. JAMA. 2017;317(24):2502–2514. doi:10.1001/jama.2017.7217

    16. Xing L, Xu J, Wei Y, et al. Depression in polycystic ovary syndrome: focusing on pathogenesis and treatment. Front Psychiatry. 2022;13:1001484. doi:10.3389/fpsyt.2022.1001484

    17. Cao C, Qi Y, Fang D, et al. Clinical study on polycystic ovary syndrome treated with Diane-35 and Pioglitazone. Am J Transl Res. 2021;13(11):12742–12749.

    18. Liu Y, et al. Diane-35 and metformin induce autophagy and apoptosis in polycystic ovary syndrome women with early-stage endometrial carcinoma. Genes. 2022;13(1).

    19. Cahill DJ, O’Brien K. Polycystic ovary syndrome (PCOS): metformin. BMJ Clin Evid. 2015;2015.

    20. Feng W, Jia -Y-Y, Zhang D-Y, et al. Management of polycystic ovarian syndrome with Diane-35 or Diane-35 plus metformin. Gynecol Endocrinol. 2016;32(2):147–150. doi:10.3109/09513590.2015.1101441

    21. Deng Y, et al. Is there an advantage of using dingkun pill () alone or in combination with Diane-35 for management of polycystic ovary syndrome? A randomized controlled trial. Chin J Integr Med. 2020;26(12):883–889. doi:10.1007/s11655-020-3097-4

    22. Luan YY, et al. Immune regulation in polycystic ovary syndrome. Clin Chim Acta. 2022;531:265–272. doi:10.1016/j.cca.2022.04.234

    23. Rudnicka E, et al. Chronic Low Grade Inflammation in Pathogenesis of PCOS. Int J Mol Sci. 2021;22(7):3789. doi:10.3390/ijms22073789

    24. Zhang J, Bao Y, Zhou X, et al. Polycystic ovary syndrome and mitochondrial dysfunction. Reprod Biol Endocrinol. 2019;17(1):67. doi:10.1186/s12958-019-0509-4

    25. Li T, et al. Tempol ameliorates polycystic ovary syndrome through attenuating intestinal oxidative stress and modulating of gut microbiota composition-serum metabolites interaction. Redox Biol. 2021;41:101886. doi:10.1016/j.redox.2021.101886

    26. Guan HR, Li B, Zhang Z-H, et al. Integrated bioinformatics and network pharmacology to explore the therapeutic target and molecular mechanisms of Bailing capsule on polycystic ovary syndrome. BMC Complement Med Ther. 2023;23(1):458. doi:10.1186/s12906-023-04280-6

    27. Fu LW, Gao Z, Zhang N, et al. Traditional Chinese medicine formulae: a complementary method for the treatment of polycystic ovary syndrome. J Ethnopharmacol. 2024;323:117698. doi:10.1016/j.jep.2023.117698

    28. Zhao Y, Zhao X, Jiang T, et al. A retrospective review on dysregulated autophagy in polycystic ovary syndrome: from pathogenesis to therapeutic strategies. Horm Metab Res. 2024;56(8):547–558. doi:10.1055/a-2280-7130

    29. Zheng X, Zhou C, Xu W, et al. Protective effect of Yunkang oral liquid via regulating androgen receptor in polycystic ovary syndrome rats. Gynecol Endocrinol. 2024;40(1):2368845. doi:10.1080/09513590.2024.2368845

    30. Ye Y, et al. The ameliorating effects of guizhi fuling wan combined with rosiglitazone in a rat ovarian model of polycystic ovary syndrome by the PI3K/AKT/NF-κB and Nrf2/HO-1 pathways. Gynecol Endocrinol. 2023;39(1):2254848. doi:10.1080/09513590.2023.2254848

    31. Lu LJ, Zhang J, Jiang Y, et al. Mechanism of mitochondrial dysfunction in polycystic ovary syndrome and traditional Chinese medicine intervention: a review. Zhongguo Zhong Yao Za Zhi. 2024;49(1):1–14. doi:10.19540/j.cnki.cjcmm.20231018.601

    32. Jia LY, Feng J-X, Li J-L, et al. the complementary and alternative medicine for polycystic ovary syndrome: a review of clinical application and mechanism. evid based complement alternat med, 2021. eCAM. 2021;2021:5555315. doi:10.1155/2021/5555315

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  • Proposal to amend Fastmarkets’ iron ore 62% Fe port stock index name, specifications: pricing notice

    Proposal to amend Fastmarkets’ iron ore 62% Fe port stock index name, specifications: pricing notice

    Following an initial consultation with the market, and a review of the typical data sets that have been collected over recent months, Fastmarkets is proposing the changes to the name and specifications of the index for iron ore 62% Fe fines, fot Qingdao (MB-IRO-00011), and iron ore 62% Fe fines, fot Qingdao, $/tonne conversion (MB-IRO-00022), including the Fe, silica, alumina, phosphorus, and sulfur base specifications, to ensure both match the latest quality of mid-grade fines commonly traded in the market.

    The index tracks the spot prices of 60-63% Fe iron ore fines in the CFR China spot market, with its base specifications set to match the prevailing quality of mid-grade fines.

    The new specifications would be as follows, with amendments in italics:

    MB-IRO-0011 Iron ore 61% Fe fines, fot Qingdao, yuan/wet tonne
    QualityFe content base 61%, range 60- 63%; silica base 4.3%, max 8%; alumina base 2.5%, max 4%; phosphorus base 0.095%, max 0.15%; sulfur base 0.02%, max 0.06%; moisture: base 8%, max 10%; granularity base size >90% < 6.3mm, at least 90% <0.15mm
    Quantity: Min 500 tonnes
    Location: fot Qingdao, normalized for any Chinese mainland sea port
    Timing: Within two weeks
    Unit: CNY/wet metric tonne
    Payment terms: Payment at sight, other terms normalized to base
    Publication: Daily at 6.30pm Singapore time
    Notes: All origins. Data history from January 2014.

    MB-IRO-0022 Iron ore 61% Fe fines, fot Qingdao, yuan/wet tonne, $/tonne conversion
    QualityFe content base 61%, range 60- 63%; silica base 4.3%, max 8%; alumina base 2.5%, max 4%; phosphorus base 0.095%, max 0.15%; sulfur base 0.02%, max 0.06%; moisture: base 8%, max 10%; granularity base size >90% < 6.3mm, at least 90% <0.15mm
    Quantity: Min 500 tonnes
    Location: fot Qingdao, normalized for any Chinese mainland sea port
    Timing: Within two weeks
    Unit: $/wet metric tonne conversion
    Payment terms: Payment at sight, other terms normalized to base
    Publication: Daily at 6.30pm Singapore time
    Notes: All origins. Data history from January 2014.

    These prices are a part of the Fastmarkets steelmaking raw materials package.

    The consultation period for this proposed amendment starts from Wednesday July 29 and will end on August 27. The amendment will then take place, subject to market feedback, on September 1.

    To provide feedback on the proposal, or if you would like to provide price information by becoming a data submitter to these prices, please email pricing@fastmarkets.com. Please add the subject heading “FAO: Adele Pan, re: Iron Ore 62% Fe port stock index”.

    Please indicate if comments are confidential. Fastmarkets will consider all comments received and will make comments not marked as confidential available upon request.

    To see all Fastmarkets’ pricing methodology and specification documents go to https://www.fastmarkets.com/about-us/methodology.

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  • Philips sees tariffs impact of 150-200 million euros after US-EU deal – Reuters

    1. Philips sees tariffs impact of 150-200 million euros after US-EU deal  Reuters
    2. Philips’ sees tariffs impact of 150-200 million euros after US-EU deal  Global Banking | Finance | Review
    3. Koninklijke Philips NV reports results for the quarter ended June 30 – Earnings Summary  TradingView
    4. Philips Raises Full-Year Profitability Guidance on Lower Tariff Impact  The Wall Street Journal
    5. Philips Q2 net income falls on one time item; margins and cash flow rise  Investing.com

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  • Hazardous Declaration Form Compliance – DP World Vancouver

    We would like to inform you of an important compliance reminder from DP World Vancouver regarding the submission of Hazardous Declaration (HAZDEC) forms for containerized dangerous goods.

    The terminal has observed a recurring issue with incomplete and late submission of HAZDEC forms, particularly with invalid or missing 24-hour emergency contact numbers. These details are critical for ensuring safety and regulatory compliance under Transport Canada guidelines. Please note that repeat instances are tracked based on shipper/consignee details, not the ocean carrier.

    To support smooth terminal operations and minimize risk, please take note of the following:

    1. A fully completed HAZDEC form, including a valid 24-hour emergency contact number is mandatory for all dangerous goods containers.
    2. The emergency contact number must be operational and accessible at all times.
    3. Non-compliance will result in the container(s) being placed on hold, and application of a “Security Breach” charge, as specified in DP World Vancouver’s public tariff.
    4. This charge will be billed directly to the customer by DP World and is effective as of August 01, 2025.
    5. All demurrage and storage charges will also be applicable until a valid hazardous declaration is provided and the hold is removed.
    6. For bookings involving rail or truck moves to inland delivery locations, containers will be held at the terminal and not loaded onto the designated mode of transport until the customer has paid the Security Breach fee directly to DP World. Please note that any applicable Maersk demurrage charges will be invoiced separately and will not impact the release or loading of the container for onward rail or truck movement once the terminal hold is cleared.

    You may access the DP World Vancouver Public Tariff here.

    We encourage customers to consider registering with CANUTEC (Canadian Transport Emergency Centre), operated by Transport Canada, to help ensure a valid 24-hour emergency contact number is available. CANUTEC offers a free 24-hour emergency telephone service for Canadian consignors using their emergency number (1-888-CAN-UTEC / 613-996-6666) on dangerous goods shipping documents. To use this service, consignors must first register with CANUTEC, providing relevant contact information and Material Safety Data Sheets (MSDS), and receive approval before listing CANUTEC’s number.

    We remain committed to helping you keep your supply chain moving efficiently. To avoid potential delays or charges, we encourage all customers to ensure timely and accurate submission of HAZDEC forms.

    Should you have any further questions, please contact your local Maersk representative.

    Thank you for your continued partnership,

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  • Bid to win Unesco status for historic landscape

    Bid to win Unesco status for historic landscape

    Dan Martin

    BBC News, Leicester

    Tom Oakley

    BBC News, East Midlands

    National Forest A strata formation rock face in the foreground leading to a green ladscape to the backNational Forest

    Bradgate Park is included within the bid area, organisers said

    A landscape in Leicestershire could be set for the world stage as a bid is made to secure a prestigious international designation.

    An application to get Unesco Global Geopark status for Charnwood Forest is being led by The National Forest.

    The area in the north of the county is home to attractions including Bradgate Park and Beacon Hill, and is famous for its landscapes and ancient fossil finds.

    Organisers behind the bid said an application would be submitted to Unesco later this year with international evaluators visiting the site in 2026.

    If the application is successful, the designation would be awarded in the spring of 2027.

    Simon Harris/Rhian Kendall/BGS/UKRI Two images showcasing the fossil that was uncovered in 2007Simon Harris/Rhian Kendall/BGS/UKRI

    A 560 million-year-old fossil representing the earliest known animal predator was uncovered at the site in 2007

    Geoparks are areas of outstanding geology that are used by local communities to promote sustainable economic development, conservation, and education, organisers said.

    There are currently 229 Unesco Global Geoparks in 50 countries.

    Charnwood Forest Geopark has been developed over the past four years as part of a National Lottery Heritage Fund-supported scheme, hosted by the National Forest.

    The site has a geological story that dates back nearly 600 million years, and is home to some of the oldest animal fossils ever found.

    These include a 560 million-year-old fossil representing the earliest known animal predator.

    Charnwood Forest Geopark Information panel in front of a rock formation called The Old Man of Beacon HillCharnwood Forest Geopark

    Information panels have been installed at key locations across the site

    Since 2020, a partnership comprising several organisations including Bradgate Park Trust, Leicestershire County Council, the Environment Agency and the British Geological Survey, has been working to try to raise the profile of Charnwood Forest.

    Dr Jack Matthews, a geo-heritage officer who is part of the team behind the application, said: “Since the discovery of our famous fossil Charnia by two schoolchildren in the 1950s, Charnwood Forest has been one of our planet’s most important places to visit and understand how animals first evolved.

    “The Geopark is promoting this fascinating story through new interpretation at sites across Charnwood Forest, welcoming local people and visitors to learn more about their oldest animal ancestors and support local businesses while they’re here.

    “From rocks and fossils to our unique human history, and biodiversity, Charnwood Forest has many layers of heritage, and with Unesco Global Geopark status we can share these with even more people.”

    Charnwood GeoPark A green shaded map of the Charnwood Forest areaCharnwood GeoPark

    A map showing the Charnwood Forest Geopark area

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  • Investors are signaling they are moving on from tariffs

    Investors are signaling they are moving on from tariffs

    The floor of the New York Stock Exchange on July 28, 2025.

    Spencer Platt | Getty Images News | Getty Images

    Stock markets in the U.S. and Europe didn't seem that delighted with the U.S.-European Union trade deal reached over the weekend.

    The S&P 500 ticked up, but by the barest margin, while the Stoxx Europe 600 fell. Both indexes were trading higher during their respective sessions but had given up those gains as the day ended.

    For those on the continent, perhaps there was a dawning realization that the agreement wasn't too much in their favor. German Chancellor Friedrich Merz said he would have welcomed further easing of trade, while France's minister for Europe, Benjamin Haddad, said the deal was "unbalanced," according to a Google translation.

    With U.S. President Donald Trump announcing Monday that he would probably impose a blanket tariff of between 15% and 20% on countries without trade agreements, it's starting to seem like most duties will settle around that level eventually, easing some uncertainty.

    What's more, economists appear to be revising downward their expectations of the impact tariffs will have on the U.S. economy — so any deals in the future might not trigger rallies, or strong ones at least, on Wall Street.

    Tariff considerations, then, are on the backburner for now. Investors can turn their attention to Magnificent Seven earnings and data on the U.S. economy coming out the next few days. If all goes well, they might give markets the cheer that was missing on Monday.

    What you need to know today

    Global baseline tariff of between 15% and 20%. For countries that have not negotiated separate trade agreements with the U.S., Trump said he would likely impose that blanket tariff rate on their exports. But Wall Street doesn't seem as frightened of tariffs anymore.

    Less than two weeks for Russia to reach a peace deal with Ukraine. That's the new deadline Trump issued to Moscow — if Russia fails to meet it, the U.S. president will implement massive "secondary tariffs" on the country's trade partners, Trump said.

    India has exported more smartphones to the U.S. than China. In the second quarter, 44% of U.S. smartphone imports were assembled in India, while 25% were from China, according to research firm Canalys. India's and China's share a year earlier were 13% and 61%, respectively.

    A muted response to the EU deal. On Monday, the S&P 500 closed mostly flat, giving up earlier gains. Asia-Pacific markets fell Tuesday. Shares of Singapore Airlines lost as much as 8% after the carrier reported a 59% slump in profit for its fiscal first quarter.

    [PRO] Watch this index for signs of a new bull phase. This index, which is calculated differently from the price-weighted S&P 500, gives a better gauge of the health of the entire economy and stock market.

    And finally...

    The World Artificial Intelligence Conference kicked off in Shanghai on Saturday, July 26, 2025.

    Nurphoto | Nurphoto | Getty Images


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  • Pilot arrested on child sex abuse material charges after landing at San Francisco airport

    Pilot arrested on child sex abuse material charges after landing at San Francisco airport

    SAN FRANCISCO — A pilot was arrested aboard a Delta Air Lines flight and federal agents took him into custody from the cockpit after the plane landed at San Francisco International Airport.

    The pilot, whose identity wasn’t immediately released, was arrested on charges relating to child sexual abuse material, an official with the Department of Homeland Security said Monday.

    Passengers aboard the flight from Minneapolis to San Francisco on Saturday posted video online showing federal agents walking through the aisle of the plane.

    In an emailed statement, Delta said that it would cooperate with law enforcement and the pilot has been suspended, pending an investigation.

    “Delta has zero tolerance for unlawful conduct and will fully cooperate with law enforcement, ” the company said.

    Authorities provided no other details about the arrest.

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