A cohort study on inflammatory response and coagulation function changes in women with silver clip or silk ligation sterilisation.

The rate of contraception use worldwide is extremely high, exceeding 86.1% in China [ 1 ]. At present, multiple contraception methods are available, including permanent contraception, long-term reversible contraception (e.g., intrauterine devices and subdermal contraceptive implants), contraceptive drugs, and condoms [ 2–5 ]. Tubal sterilisation is a permanent method of contraception that prevents the ovum from being fertilised by sperm by blocking, clipping, or removing the fallopian tubes [ 6 , 7 ]. Over the last century, it has been widely accepted by women of childbearing age owing to its advantages of minimal invasiveness, low cost, and simplicity. The silver clip technique is minimally invasive, causes less damage to the ovaries, and is associated with few postoperative complications; therefore, it is commonly used for tubal sterilisation [ 8 ]. A silver clip, shaped like the letter ‘Π’, contains 99% silver. Two silver clips are surgically placed across the isthmus of both fallopian tubes, thereby mechanically occluding the passage of sperm and ova. Owing to changes in family planning policies in China, the application rate of silver clips for tubal sterilisation peaked at 49.5% between 1987 and 1993 and has declined since 2000 [ 9 , 10 ]. It can be inferred that most women who used silver clips have now entered middle age or older adulthood. However, the interaction between the metal material of silver clips and the surrounding tissue remains a matter of considerable concern. Hulka et al. [ 11 ] reported that 30-day placement of silver clips resulted in the formation of a thin fibrous membrane on their surface. After years of sterilisation, both the silver clips and the surrounding tissue may darken in colour [ 12 ]. Xiao et al. [ 13 ] described the deposition of silver particles in local fallopian tube tissue and small blood vessel walls after 6–7 years of sterilisation using the silver clip technique. Despite the recognised biocompatibility of metal implants, under conditions of long-term, chronic, and low-concentration exposure to silver, no systematic studies have evaluated the inflammatory status of the human body. Silver nanoparticles (AgNPs) can penetrate the circulatory system via phagocytosis, potentially leading to organ toxicity and lifelong effects on human health [ 14 , 15 ]. A case report described inflammation-associated microscopic haematuria in a 54-year-old postmenopausal woman with a 32-year history of tubal sterilisation using a silver clip [ 16 ]. He et al. found that AgNPs induce excessive production of mitochondrial reactive oxygen species (ROS) in vascular endothelial cells, leading to the aggregation of inflammatory cells [ 17 ]. Conversely, another study demonstrated that AgNPs downregulate the expression of inflammatory mediators such as interleukin-6 (IL-6) and tumour necrosis factor-α (TNF-α), exhibiting antioxidant and anti-inflammatory effects [ 18 ]. However, no consensus has been reached. Several studies have confirmed that vascular endothelial cells are activated during inflammation and release tissue factor (TF) and tissue factor pathway inhibitor (TFPI), thereby initiating coagulation [ 19 ]. In a randomised, controlled, double-blind clinical trial involving patients at high risk of thrombosis, plasma pro-inflammatory cytokines, such as IL-6, C-reactive protein (CRP), and TF, showed a downward trend after intervention. It has been speculated that pro-inflammatory cytokines enhance the procoagulant state predominantly by increasing TF expression [ 20 ]. In vivo , silver particles significantly affect activated partial thromboplastin time (APTT), thrombin time (TT), and prothrombin time (PT) [ 21 ]. The potential synergistic relationship between inflammation and coagulation provides a compelling rationale for studying these two systems simultaneously. Considering the interaction between silver clips and the human body, existing evidence from earlier reports and in vivo or animal studies lacks support from modern clinical investigations in humans, particularly rigorous cohort studies involving the large ageing population of women who received these clips. To date, no literature has reported systemic silver levels in these women or provided long-term follow-up data. We therefore designed a multicentre ambispective cohort study to analyse silver concentrations in body fluid samples, coagulation function, and inflammatory responses in women who underwent tubal sterilisation using silver clips several decades ago. This study aims to provide evidence to inform health monitoring and contraceptive decision-making among women worldwide.

Sponsored by the Second Hospital of Hebei Medical University in Hebei Province, China, this multicentre, ambispective cohort study will recruit patients between April 2025 and April 2026, with follow-up for one year until April 2027. Participants will be randomly recruited from five tertiary and five secondary hospitals in Hebei Province ( Table 1 ). Using a combination of stratified and cluster sampling, five tertiary hospitals will be randomly selected from five cities out of 11 cities, and five secondary hospitals will be selected from five counties out of 91 counties, with one hospital in each region. All procedures will be standardised and performed by pre-trained investigators. Clinical samples will be tested at the Hebei Food Inspection and Research Institute. Light and electron microscopy will be performed at the core facilities and centres of Hebei Medical University. All assessments will be conducted in a blinded manner. Laboratory personnel performing inductively coupled plasma mass spectrometry (ICP-MS), enzyme-linked immunosorbent assay (ELISA), and coagulation tests will be blinded to patient group assignment. Pathologists conducting stereological analyses of tissue samples will also be blinded. Study sites. The inclusion criteria were as follows: (i) women aged 40–70 years; (ii) long-term residence at the originally registered site; (iii) a history of contraception, including tubal sterilisation using a silver clip, silk ligation or modified Pomeroy ligation, or condom contraception; (iv) benign gynaecological diseases to be managed by elective surgery involving removal of the bilateral fallopian tubes and uterus; and (v) consumption of drinking water meeting the Standards for Drinking Water Quality of China [ 23 ]. The exclusion criteria were as follows: (i) a history of contraception using other metal clips or metal sutures; (ii) a history of metal implantation; (iii) exposure to heavy metal pollution in the living or working environment; (iv) long-term use of makeup, silver ornaments, silver tableware, or hair dye; and (v) severe pelvic or abdominal infectious diseases. In the retrospective cohort, silver concentrations in fluid samples, cytokine levels, and coagulation function were assessed preoperatively. The extent of pelvic adhesions was evaluated intraoperatively, and tissue surrounding the silver clip or ligation site, or the isthmus of the fallopian tube, was collected for postoperative assessment. In the prospective cohort, all patients will be followed up for 12 months postoperatively, with assessments at 1 day, 1 month, 6 months, and 12 months after surgery. Postoperative silver concentrations in fluid samples, cytokine levels, and coagulation function will be re-examined. The study procedure and workflow are shown in Figures 1 and 2 . Study procedure. Study workflow. Clinical data will be collected before surgery and at 1 day, 1 month, 6 months, and 12 months postoperatively, as well as during routine follow-up visits. Based on clinical records and experimental results, data will be recorded in Microsoft Excel and uploaded to a secure database. All personal and sensitive patient data will be de-identified, and patient identities encrypted. The workflow for recruitment, sample collection and analysis, and follow-up is detailed in Figure 3 , Table 2 , and Figure 4 . Workflow of participant recruitment. Follow-up timeline. Workflow for sample collection. Exposure factors include tubal sterilisation using a silver clip, silk ligation, and condom contraception. The silver clip technique involves placement of a metal clip containing 99% silver to clamp the isthmus. Silk ligation provides contraception by blocking the isthmus. Condom contraception is a physical method to prevent sperm from entering the uterus [ 8 ]. Primary variables include cytokine levels and coagulation function. Levels of TNF-α, IL-1, IL-6, IL-8, IL-10, TF, and TFPI in peritoneal fluid and venous blood will be measured using ELISA [ 24–27 ]. Venous blood samples will be collected after overnight fasting and subjected to coagulation tests. Coagulation-associated indicators include APTT, PT, TT, and fibrinogen level (FIB) [ 28–31 ]. Secondary variables include stereological parameters of the fallopian tube and pelvic adhesions. Stereology is a statistical methodology using random sampling to interpret three-dimensional structures from two-dimensional tissue sections [ 32–34 ]. Fallopian tubes will be examined using transmission and scanning electron microscopy. Stereological parameters, including number, volume, and surface density, will be quantified using Image Pro [ 35 , 36 ]. Pelvic adhesions will be assessed using a comprehensive scoring method proposed by the American Fertility Society. The pelvic cavity is divided into 23 anatomical parts, and adhesions are graded based on severity (0 = none; 1 = filmy/avascular; 2 = some vascularity or dense; 3 = cohesive adhesion) and extent (0 = none; 1 ≤ 25%; 2 = 26–50%; 3 ≥ 51%). Pelvic adhesions in each part will be graded 0–6, yielding a total score of 0–138. Overall adhesion severity will be classified as no adhesion (0–35), mild (36–69), moderate (70–105), and severe (106–138) [ 37–39 ]. Silver concentration is the predominant covariate and may be influenced by age and sterilisation duration [ 40 , 41 ]. It will be measured in preoperative serum, intraoperative peritoneal fluid, and postoperative serum samples at 1 day, 1 month, 6 months, and 12 months using ICP-MS [ 42–44 ]. Other covariates include age, menstrual cycle regularity, history of pregnancy and childbirth, date of last pregnancy, duration of lactation, sterilisation time, interval between last pregnancy and sterilisation, current sterilisation duration, history of sexually transmitted infections or pelvic inflammatory diseases, complications, and drug use. Adverse events occurring from the time of informed consent to study completion will be reported to the Research Ethics Committee within 24 h. Adverse reactions related to sample collection (e.g., bleeding at the puncture site or infection following blood draw) will be managed promptly, with financial compensation where appropriate. Potential adverse reactions related to silver clip or ligation sterilisation in the retrospective cohort will be observed, including new-onset thrombosis, autoimmune conditions, chronic pain, long-term oedema, haematuria, gastrointestinal bleeding, pruritus, and other allergic reactions. These events will also be monitored in the prospective cohort. Based on pre-experimental data for IL-6 levels with normal distribution (27.94 ± 8.16 ρg/mL in the silver clip group and 31.51 ± 8.87 ρg/mL in the silk ligation group), a sample size of 113 participants per group is required. Accounting for 20% loss to follow-up, 142 participants will be recruited per group, yielding a total of 426 participants. The primary comparison will be between the silver clip and silk ligation groups, as both involve surgical intervention. The condom contraception group will serve as a reference for ‘non-sterilised ‘ population norms. Statistical analyses will be conducted using SPSS version 25.0. Normality and homogeneity of variance will be assessed. Normally distributed data will be expressed as mean ± standard deviation and analysed using one-way ANOVA for intragroup comparisons and independent samples t test for intergroup comparisons. Non-normally distributed data will be expressed as median (interquartile range) and analysed using Wilcoxon rank-sum and Friedman tests for intragroup comparisons, and Mann–Whitney tests for intergroup comparisons. Multivariable regression models (e.g. linear regression for continuous outcomes, logistic regression for binary outcomes) will adjust for key covariates, including age, body mass index (BMI), sterilisation time, and comorbidities. Subgroup analyses based on age and sterilisation duration will also be performed. Per-protocol (PP) and best–worst-case analyses will address missing data. A two-tailed p value <0.05 will be considered statistically significant. This study was conducted in accordance with the principles of the Declaration of Helsinki. It was approved by the Institutional Review Board (IRB) of The Second Hospital of Hebei Medical University (2025-R193). Written informed consent will be obtained from all participants prior to enrolment.

This is a multicentre ambispective cohort study involving both retrospective and prospective components. In the retrospective cohort study, clinical data from patients with benign gynaecological diseases—including atypical/complex endometrial hyperplasia, high-grade squamous intraepithelial lesion of the cervix, uterine fibroids [haemoglobin >9 g/dl], uterine adenomyosis, and benign ovarian cysts—who were surgically treated with bilateral salpingectomy and hysterectomy and had received contraception many years earlier, were analysed. Clinical records were reviewed for patients who underwent surgery between January 1980 and December 2015. Patients were grouped according to their contraception method into silver clip sterilisation, silk ligation sterilisation, and condom contraception groups. The condom group did not undergo surgical sterilisation and served as a reference for ‘background levels’ in the population rather than for the effects of sterilisation itself. This clinical trial was designed in accordance with the SPIRIT 2025 Statement [ 22 ] and registered in the Chinese Clinical Trial Register (ChiCTR2500099975).

With advances in economic and cultural development, awareness of contraception has increased. An optimal contraceptive method should be selected based on individualised conditions to minimise long-term adverse effects. The silver clip technique is commonly used for tubal sterilisation [ 45 ]. During surgery, the fallopian tubes are initially isolated, followed by placement of a hinged clip around a small section of each tube. The clips compress the tubes, causing scarring and eventual separation [ 16 ], which is the likely mechanism of contraception. Tubal sterilisation using silver clips is generally considered safe and effective, with a low risk of failure and major complications [ 46 ]. Although complications from silver clip sterilisation are rare, long-term adverse effects such as inflammatory responses, systemic toxicity, and coagulation abnormalities have been reported. In vivo experiments in female New Zealand rabbits showed that surgical adhesives in the tubal lumen were associated with severe inflammation [ 47 ]. Animal studies have also demonstrated that AgNPs can be absorbed into the bloodstream, increasing the risk of organ toxicity as silver from clips corrodes over time [ 48–51 ]. Systemic silver poisoning can occur when the cumulative concentration of ionic silver or AgNPs in the human body reaches 70–1,500 mg/kg [ 41 , 52 , 53 ]. AgNPs have been reported to increase the procoagulant activity of red blood cells, potentially promoting thrombus formation [ 54 ]. Recent studies also suggest that silver may modulate vascular endothelial cell damage induced by inflammation, triggering the release of TF, formation of the factor VII–antithrombin complex, and activation of coagulation [ 7 , 55–58 ]. The long-term and chronic effects of these processes may lead to systemic changes in the human body. These findings provide a new perspective for research on silver clips. Vascular endothelial cells are likely central to this study, as their activation integrates the previously independent systems of inflammation and coagulation, functioning together to exert long-term effects in women undergoing silver clip sterilisation. This represents an innovative aspect of the present study. To date, clinical evidence on the long-term effects of silver clips on inflammatory and coagulation parameters remains limited. Therefore, we designed a multicentre ambispective cohort study to comprehensively examine the impact of tubal sterilisation using silver clips on cytokine levels and coagulation function. This study has some limitations. In the retrospective component, although strict criteria were applied to limit exposure to metallic silver in the environment, the observation period spans several decades, and some details may be difficult for participants to recall accurately. Additionally, as participants age and physiological functions decline, some complications may be attributed to age-related changes and overlooked. In the prospective component, surgical procedures among participants are not entirely uniform, which may introduce variability in postoperative inflammatory responses. The study is ongoing and is expected to be completed in 2027.

This study will provide long-term data for monitoring silver levels in women’s bodies. It will emphasise the importance of careful health surveillance for women who have undergone silver clip sterilisation, particularly regarding inflammation and coagulation function. The findings may contribute to the development of updated guidelines for contraception in women of childbearing age and inform broader public strategies.

This study was registered in the Chinese Clinical Trial Registry (CHiCTR2500099975).