Effect of hydroxychloroquine on pregnancy outcomes in women with unexplained recurrent pregnancy loss complicated with antinuclear antibody positivity: a multicentre prospective cohort study protocol

Abstract

Introduction

Unexplained recurrent pregnancy loss (URPL), defined as two or more pregnancy losses without identifiable cause, affects approximately 2% of reproductive-aged women. Antinuclear antibodies (ANA) may disrupt maternal-fetal immune tolerance in URPL, but the prognostic value of ANA titres and effectiveness of immunomodulatory therapies like hydroxychloroquine (HCQ) remain uncertain. This multicentre prospective cohort study aims to establish a multicentre clinical cohort of ANA-positive women with URPL to investigate whether HCQ improves pregnancy outcomes and to examine the relationship between changes in ANA titres during pregnancy and live birth rates. Additionally, mechanistic exploration will use a large-scale autoantibody microarray with bioinformatics analysis.

Method

This multicentre prospective cohort study across three Chinese tertiary hospitals will recruit 720 ANA-positive URPL patients. Participants will receive either low-dose aspirin alone or low-dose aspirin in combination with HCQ, starting two months before conception. Treatment duration depends on ANA titres: discontinued if negative or < 1:80 at 20 weeks; continued if ≥ 1:160 until 38 weeks. Primary outcome is live birth rate. Autoantibody microarrays will compare profiles between 30 ANA-positive and 30 ANA-negative patients and assess HCQ effects.

Discussion

This study will provide prospective evidence on the effectiveness of HCQ in improving live birth rates among ANA-positive women with URPL. Findings may inform future therapeutic strategies and contribute to personalized management for this challenging condition. If HCQ demonstrates benefit, it could offer a readily applicable treatment option in clinical settings. Furthermore, autoantibody profiling may uncover mechanistic pathways and biomarker signatures associated with treatment response and pregnancy success.

Trial registration

ChiCTR2500105183 (registration date 2025-06-30).

Background

Recurrent pregnancy loss (RPL), typically defined as the occurrence of two or more pregnancy losses, affects approximately 1%–2% of women of reproductive age [1, 2]. Aetiological factors contributing to RPL include chromosomal abnormalities, uterine structural anomalies, autoimmune diseases, and endometrial dysfunction, etc [3]. However, in nearly half of the cases, no identifiable clinical cause can be established, referred to as unexplained recurrent pregnancy loss (URPL) [2]. The pathophysiology of URPL remains elusive. Emerging evidence suggests that immunological dysregulation at the maternal–fetal interface, particularly related to impaired alloimmune tolerance, may play a significant role in a subset of URPL cases [4, 5]. Nevertheless, the underlying mechanisms remain poorly understood, and there is currently no consensus on effective therapeutic strategies. This underscores the need for continued investigation into the immunological basis of URPL and the development of targeted treatment approaches.

Growing evidence highlights a potential role for autoantibodies in the pathogenesis of RPL, particularly through their impact on early placental development. These autoantibodies may contribute to pregnancy failure by disrupting key trophoblast functions, including cellular invasion and angiogenesis [6]. Among them, antinuclear antibodies (ANA), which target nuclear components, have been extensively studied. While ANA are commonly detected in the serum of patients with autoimmune and rheumatic diseases, their presence is also observed in the general population (reported prevalence of 4%–13%), where their clinical significance remains uncertain [7]. Recent observational data suggest a positive association between ANA positivity and increased risk of RPL [8–10]. A comprehensive meta-analysis involving 21 case–control studies across 13 countries in Asia, Europe, North America, and Oceania found a significantly higher prevalence of ANA positivity in women with RPL compared with controls (OR 2.97, 95% CI 1.91 to 4.64; p < 0.00001; I²=75%) [8]. Furthermore, this association remained significant when analyses were restricted to cases of unexplained RPL (OR 3.27, 95% CI 2.01 to 5.31; p < 0.00001; I²=70%) [8]. Subgroup analysis revealed that low ANA titres (1:40–1:80) did not correlate with RPL (OR 2.44, 95% CI 0.42 to 14.06; p = 0.32), whereas high titres (≥ 1:160) were strongly associated with increased RPL risk (OR 45.89, 95% CI 8.44 to 249.45; p < 0.00001) [8]. Although the precise pathogenic mechanisms remain to be elucidated, it has been proposed that ANA may compromise embryo quality and interfere with implantation, ultimately reducing pregnancy success rates [11]. These findings have prompted increased interest in exploring ANA as a potential biomarker and therapeutic target in the management of unexplained RPL.

The association between ANA titres and pregnancy outcomes remains insufficiently characterised, with existing evidence yielding inconsistent results. A prospective study by Ticconi et al. (2016) involving 86 women with unexplained URPL found that the disappearance of ANA during early gestation was associated with improved pregnancy outcomes, suggesting that ANA negativity may serve as a favourable prognostic marker [10]. Conversely, a large retrospective cohort study of 1021 women with URPL demonstrated that the likelihood of live birth following a confirmed pregnancy exceeded 70%, with comparable outcomes observed irrespective of ANA status or titre thresholds [12]. Given the limited and conflicting nature of current data, further investigation is warranted to clarify the potential prognostic value of ANA titres in predicting live birth rates (LBR) among women with unexplained RPL.

Immunomodulatory therapies are currently under investigation as potential interventions for RPL. Acting as an antithrombotic agent, low-dose aspirin, either alone or in combination, has been widely administered in clinical practice for women with RPL. However, despite the presumed similarities in pathogenesis between URPL and the autoimmune diseases, clinical trials evaluating the efficacy of antithrombotic therapy in preventing recurrent pregnancy loss have not demonstrated consistent benefit [13–15]. So far, there is still a lack of consistent clinical data supporting effective therapies for URPL patients combined with ANA positivity. Prednisolone, a glucocorticoid with potent anti-inflammatory and immunosuppressive properties, is commonly used in the management of inflammatory and autoimmune conditions. First published in 1997, prednisolone was used to treat RPL women with the presence of the lupus anticoagulant [16]. A meta-analysis evaluating its role in recurrent miscarriage reported a significant association between prednisolone treatment and improved pregnancy outcomes (OR 7.99, 95% CI 5.01 to 12.73). However, substantial heterogeneity was observed (I²=71%, p = 0.02), likely attributable to variations in inclusion criteria, steroid dosage, and timing of administration [17]. Similarly, another meta-analysis examining oral immunosuppressive agents (including cyclosporine A and prednisolone) demonstrated that such therapies were associated with increased live birth rates (OR 3.6, 95% CI 2.1 to 6.15; p < 0.00001) and ongoing pregnancy rates (OR 8.82, 95% CI 2.91 to 26.75; p = 0.0001), as well as reduced miscarriage rates (OR 0.21, 95% CI 0.08 to 0.52; p = 0.0007) [18]. Despite these potential benefits, the use of corticosteroids such as prednisolone is not without risk. High dose or prolonged exposure may lead to adverse maternal and fetal outcomes, including gestational hypertension, hyperglycaemia, preterm birth, and low birth weight, thereby limiting its widespread clinical application [19].

Originally developed as an antimalarial agent, hydroxychloroquine (HCQ) is now widely employed as a disease-modifying antirheumatic drug, particularly in the management of autoimmune disorders such as systemic lupus erythematosus [20]. In addition to its antithrombotic properties, HCQ exhibits immunomodulatory effects, including inhibition of pro-inflammatory cytokine production [21]. Within the context of recurrent miscarriage, HCQ may modulate excessive immune activation at the maternal–fetal interface and facilitate vascular remodelling of the spiral arteries, a process essential for optimal placentation. Despite its theoretical benefits, clinical data on the use of HCQ in URPL remain limited. A small retrospective cohort study involving patients with refractory antiphospholipid syndrome (APS) reported a live birth rate of 78% following the introduction of HCQ [22].

Taken all together and given the current paucity of prospective and large-sample well-homogenous studies on pregnancy outcomes and therapeutic strategies in patients with URPL combined with ANA positivity, this study aims to address a critical gap in the literature. Specifically, we propose the establishment of a multicentre prospective cohort of ANA-positive URPL patients to evaluate whether treatment with HCQ is associated with improved pregnancy outcomes. The study will further investigate the association between changes in ANA titres during pregnancy and live birth outcomes, thereby exploring both the predictive value of ANA dynamics and the efficacy and safety profile of HCQ in this population. In addition, mechanistic exploration will be conducted using a microarray containing over 1,000 non-redundant autoantibodies, combined with bioinformatics analysis.

Method/design

Study objectives

This study aims to evaluate the efficacy and safety of HCQ in improving pregnancy outcomes among patients with URPL in the presence of ANA antibodies. In addition, the study seeks to investigate the relationship between changes in ANA titres before and during pregnancy and subsequent pregnancy outcomes. It will further explore whether ANA titre thresholds (e.g. low vs. high titre) are associated with live birth or miscarriage rates, and identify clinical or immunological predictors of treatment response. Findings from this research may inform future clinical strategies for the management of URPL with immunological involvement.

Study design and setting

This is a multicentre, prospective cohort study sponsored by Peking University Third Hospital. Recruitment and data collection will be conducted across three sites in China: Peking University Third Hospital, Beijing Obstetrics and Gynecology Hospital affiliated with Capital Medical University, and the Second Hospital of Hebei Medical University.

Eligible participants include 720 women diagnosed with URPL in the presence of ANA antibodies. Participants will receive either low-dose aspirin(75 mg per day) alone or low-dose aspirin(75 mg per day) in combination with HCQ(0.2 g twice a day). Pregnancy outcomes and longitudinal changes in ANA titres will be monitored from preconception through pregnancy and postpartum.

The study flow diagram is provided in Fig. 1.

Fig. 1.

Study flow diagram

This study will also investigate the etiology of URPL with ANA positivity and the underlying mechanisms of HCQ, using peripheral blood autoantibody microarray analysis combined with bioinformatics approaches. A microarray containing over 1,000 non-redundant autoantibodies will be applied to compare autoantibody expression profiles between 30 URPL patients with ANA positive and 30 URPL with ANA negative. In addition, autoantibody profiles of URPL patients with ANA positivity will be assessed before and after HCQ treatment and correlated with pregnancy outcomes.

Intervention

Eligible participants will be assigned to receive either a combination of oral low-dose aspirin (75 mg once daily) and HCQ 0.2 g twice daily), or aspirin alone (75 mg once daily). All participants will initiate treatment 2 months prior to planned conception and continue therapy into pregnancy. The duration of treatment will be determined based on changes in ANA titres during gestation: if ANA becomes negative or decreases to below 1:80, treatment will be discontinued at 20 weeks of gestation; however, if ANA remains positive or increases to 1:160 or higher, treatment will be continued until 38 weeks of gestation. Participants who do not achieve pregnancy within 48 weeks (1 year) of starting the preconception phase will be withdrawn from the study.

Trial population

Inclusion criteria

Participants must meet all of the following criteria to be eligible for inclusion:

1. RPL: History of two or more spontaneous abortions occurring before 24 weeks of gestation, with no previous full-term live births.

2. Age: Women aged between 20 and 40 years.

3. Genetic normalcy: Normal peripheral blood karyotypes in both partners and no known family history of hereditary disorders.

4. Anatomical integrity: No structural abnormalities of the reproductive tract, as confirmed by pelvic examination, transvaginal ultrasound, hysteroscopy, and hysterosalpingography.

5. Normal endocrine function: Regular menstrual cycles with biphasic basal body temperature, confirmed ovulation on ultrasound, and no history of endocrine disorders including diabetes or hyperthyroidism disease.

6. Exclusion of autoimmune disease: Negative for antiphospholipid antibody syndrome (APS) and other autoimmune diseases. Negative plasma anticardiolipin antibodies and β2-glycoprotein I antibodies (IgG and IgM) on two separate occasions, 12 weeks apart. No prior history of thrombosis, fetal growth restriction, or preeclampsia.

7. Normal metabolic and coagulation profile: Normal plasma homocysteine levels and coagulation function.

8. Compliance: Willingness and ability to provide written informed consent and comply with study procedures.

   • ANA positivity is defined as a titre of ≥1:80 on two separate tests.

Exclusion criteria

Participants will be excluded if they meet any of the following criteria:

1. Known causes of RPL: Presence of a confirmed aetiology for recurrent miscarriage.

2. Autoimmune disorders: Diagnosis of APS or any other autoimmune diseases.

3. Genetic abnormalities: Abnormal karyotypes in either partner or identified chromosomal abnormalities in all abortion embryos.

4. Endocrine disorders: Diagnosed conditions including luteal phase defect, polycystic ovary syndrome, premature ovarian insufficiency (FSH ≥ 20 mIU/ml in the follicular phase), hyperprolactinemia, thyroid dysfunction, diabetes mellitus, or other hypothalamic–pituitary–adrenal axis disorders.

5. Structural reproductive tract abnormalities: Presence of congenital uterine anomalies, cervical insufficiency, or uterine fibroids > 5 cm.

6. Active infection: Evidence of genital tract infection.

7. Hyperhomocysteinaemia: Elevated plasma homocysteine levels above 15µmol/L.

8. Significant systemic disease: Severe cardiovascular, hepatic, renal, hematological or endocrine disease. Specific laboratory exclusion thresholds include:

   • ○ ALT or AST ≥2× upper limit of normal
   • ○ Creatinine clearance <30 ml/min
   • ○ WBC <2.5 × 10⁹/L
   • ○ Hemoglobin <85 g/L
   • ○ Platelet count <50 × 10⁹/L

9. Thrombotic or adverse obstetric history: History of thromboembolism or fetal loss in mid-to-late gestation.

10. Placental disorders: Prior history of severe preeclampsia or preterm delivery due to placental insufficiency.

11. Drug allergies: Known hypersensitivity to HCQ or aspirin.

12. Gastrointestinal history: History of peptic ulcer disease or gastrointestinal bleeding.

13. Malignancy: History of any malignant neoplasm.

14. Psychiatric illness: Diagnosed mental health disorder.

15. Neurological disorders: Epilepsy or other neurologic conditions.

16. Ophthalmologic contraindications: Presence of retinal macular disease or visual field abnormalities precluding HCQ use.

Observation index

Outcome measures

1. Primary outcome

   • Live birth rate: Defined as the delivery of at least one live-born infant ≥ 28 weeks of gestation.
2. Secondary outcomes

   • Miscarriage: Pregnancy loss occurring before 28 weeks of gestation.
   • Fetal growth restriction (FGR): Defined as a birth weight below the 10th percentile for gestational age or more than two standard deviations below the mean for gestational age.
   • Preterm birth: Defined as delivery between 28 and 37 weeks of gestation.
   • Hypertensive disorders of pregnancy: Includes gestational hypertension and preeclampsia, defined as new-onset hypertension after 20 weeks’ gestation, or hypertension accompanied by proteinuria (>300 mg/day) and/or clinical signs of organ dysfunction (e.g., seizures suggestive of eclampsia).
   • Congenital anomalies: Structural or functional fetal anomalies detected during pregnancy or at birth, including congenital heart block and major fetal malformations.
   • Postpartum haemorrhage: Defined as blood loss exceeding 500 ml within 24 hours after fetal delivery.

Assessment of safety

Safety will be monitored throughout the study using the following assessments:

1. Vital signs: Routine monitoring of blood pressure, heart rate, respiratory rate, and body temperature.

2. Clinical evaluation: Documentation of clinical symptoms and any adverse reactions potentially related to the disease or treatment.

3. Treatment-related adverse events: Monitoring for known adverse effects of LDA and HCQ, including:

   • Thrombocytopenia
   • Gastrointestinal ulceration or bleeding
   • Mucocutaneous or systemic allergic reactions
   • Hepatic enzyme abnormalities
   • Retinal toxicity or visual disturbances
4. Laboratory monitoring: Regular testing will include:

   • Complete blood count
   • Liver and renal function tests
   • Coagulation profile
   • Autoimmune markers including ANA and extractable nuclear antigens (ENAs)

5. Neonatal outcomes: Congenital anomalies

6. Retinal abnormalities

7. Bleeding events

8. Concomitant medications: Recording of any medications used during the study period, excluding supportive therapies such as vitamins, luteal phase support, or treatments for anemia.

Baseline data collection

Baseline data are collected at enrollment, with all items recorded only if such information is available or has been previously obtained. This encompasses demographic characteristics such as name, age, height, and weight, along with detailed medical, obstetric, allergy, and medication histories, including any pregnancy-related contraindications. Physical and gynecological examination findings are documented, and chromosomal karyotyping is performed for both partners. Laboratory assessments include a complete blood count, biochemical profile (liver and renal function, fasting blood glucose), urinalysis, and coagulation parameters. Imaging evaluations, such as transvaginal ultrasound and hysteroscopy, are conducted to assess reproductive anatomy. In addition, Specialized immunological and coagulation-related testing relevant to recurrent pregnancy loss is carried out, including ANA panel, lupus anticoagulant, anticardiolipin antibodies, β2-glycoprotein I antibodies, homocysteine levels, and platelet aggregation rate. Thrombophilia screening assesses protein C and protein S levels, and baseline reproductive endocrine hormone profiles are also obtained.

Biological sample collection

Serum samples (3 ml) will be collected from URPL patients at 3 time points, cohort entry a, two months after treatment initiation (prior to pregnancy attempts), and 4–6 weeks of gestation. All samples will be clearly labelled with non-fading pens or tags and freezed at − 80 °C in sample bank. Sample information will be documented in a secure, specialist-managed electronic database with detailed storage and retrieval logs to ensure traceability.

Follow-up plan

The preconception period will span up to 48 weeks, during which participants will attend follow-up visits at baseline (start of the preconception phase), week 24, and week 48. In addition, telephone follow-ups will be conducted at weeks 12 and 36. Participants who do not achieve pregnancy within 48 weeks will be withdrawn from the study.

Upon a positive pregnancy test, participants will enter the pregnancy follow-up phase. The trial will be terminated upon either miscarriage or delivery. During pregnancy, participants will be in-person followed up in week 7,12,20, and phone follow up in week 38,42. The final follow-up visit will take place at 6 weeks postpartum to assess safety, medication usage, and pregnancy outcomes.

Sample size estimation

The sample size calculation is based on prior research evaluating the effect of prednisone combined with aspirin in women with URPL and positive ANA [23]. In that study, the live birth rate was reported as 93.1% in the prednisone plus aspirin group and 83.7% in the aspirin-only group [23]. Drawing on these data, we assume a live birth rate of 93% (p₁ = 0.93) for the HCQ plus aspirin group, and 83% (p₂ = 0.83) for the aspirin-only group. Assuming a power of 80%, a two-tailed alpha level of 0.05, and an allocation ratio (k) of 1:1 between the two groups, approximately 162 participants per group are required. To account for a potential 10% dropout rate, the total sample size should be increased to 360 participants (180 in each group). Furthermore, given the design effect of a prospective cohort study, we plan to double the calculated sample size, resulting in an estimated target recruitment of 720 participants across multiple study centres.

Statistical analysis

Data will be analysed using SPSS Statistics version 26.0 (SPSS Inc., Chicago, IL, USA). Continuous variables with a normal distribution will be expressed as mean ± standard deviation (SD), and intergroup differences will be assessed using the Student’s t-test. Non-normally distributed continuous variables will be presented as median and range, and compared using the Wilcoxon rank-sum test. Categorical variables will be summarised as frequencies and percentages, with comparisons between groups performed using the chi-square test or Fisher’s exact test, as appropriate.

Given the absence of a negative control group, multivariable logistic regression analysis will be used to adjust for potential confounding factors and centre effects, and to identify independent predictors of live birth outcomes. All statistical tests will be two-sided, and a p-value < 0.05 will be considered statistically significant.

Data collection and management

Data will be collected using a standardised Case Report Form (CRF) to record medication use, treatment details, and clinical observations. All source documents, including medical records and CRFs, will remain unaltered. Any corrections will be made separately, clearly signed and dated by the responsible clinical investigator. Laboratory test results obtained during the study will be recorded, and original laboratory reports will be retained. Any clinically significant abnormalities will be reviewed and confirmed by the study physician. All entries in paper CRFs will be handwritten in pen to ensure traceability.

Electronic data capture will be conducted using the REDCap system, maintained by the Clinical Epidemiology Research Center of Peking University Third Hospital. Electronic CRFs (eCRFs) will serve as the primary tool for data entry. To ensure completeness and data validation, paper CRFs will also be maintained as a backup. All supporting laboratory findings and imaging or examination results will be archived in parallel to the CRFs. Access to the eCRF will be limited to authorised personnel including the sponsor, principal investigators, and clinical trial assistants. Rights such as reading, writing, and access to specific CRF pages will be allocated based on the individual’s role in the study and their centre. User authentication will be achieved through a unique username and password customised for each user, with all connections to the eCRF logged in an audit trail.

Designated follow-up personnel will be responsible for conducting scheduled patient visits and for completing CRFs in a timely, accurate, and consistent manner. Data management staff will oversee the electronic data entry process, perform double data entry verification where necessary, and carry out routine data backups. They will also ensure the secure storage, confidentiality, and integrity of all study data throughout the duration of the trial.

Discussion

This is one of the first large-scale, multicentre prospective cohort studies specifically targeting women with URPL and ANA positivity. It aims to provide critical evidence regarding the efficacy and safety of HCQ as an immunomodulatory treatment for women with URPL and ANA positivity. By investigating both clinical outcomes and underlying immunological mechanisms, this research addresses a significant gap in the management of immunologically-mediated pregnancy loss.

The study’s design incorporates several innovative aspects: first, the predefined ANA titre-based treatment duration protocol represents a novel approach to personalized therapy in reproductive immunology. Second, the integration of large-scale autoantibody profiling with clinical outcomes represents a comprehensive approach to understanding URPL pathogenesis. The prospective multicentre design enhances the generalizability of findings, while the preconception initiation of treatment allows for assessment of its impact on early implantation events.

If HCQ demonstrates a significant improvement in live birth rates, it could offer a readily applicable and relatively safe treatment option for this challenging patient population. The correlation between dynamic ANA titre changes and pregnancy outcomes may establish ANA as a dynamic biomarker for treatment monitoring, potentially guiding individualized therapy duration. Furthermore, the identification of clinical and immunological predictors of treatment response could help stratify patients most likely to benefit from HCQ therapy.

However, several methodological considerations warrant attention. As an observational cohort study, the findings may be subject to confounding factors, despite efforts to control for known variables through multivariable analysis. Additionally, the open-label design, while practical for this initial investigation, introduces potential for bias in outcome assessment.

Future research directions emerging from this work may include randomized controlled trials to validate HCQ’s efficacy, studies exploring optimal dosing regimens, and investigations into combination therapies targeting different pathways of immune dysregulation. Long-term follow-up studies would also be valuable to assess the safety of HCQ exposure on offspring development.

In conclusion, this study represents a significant step toward personalized management of immunologically-mediated recurrent pregnancy loss. By integrating clinical outcomes with sophisticated immunological profiling, it may not only establish HCQ’s therapeutic role but also advance our understanding of the complex immunological mechanisms underlying pregnancy loss. The results have the potential to directly inform clinical practice guidelines and improve reproductive outcomes for affected women worldwide.

Abbreviations

URPL

Unexplained recurrent pregnancy loss

ANA

Antinuclear antibody

HCQ

Hydroxychloroquine

CRF

Case Report Form

Authors’ contributions

BM: Manuscript writing, methodology, and conception. Y-JH: Manuscript writing, conception and supervise. LL: Resources, methods, project management. H-YW: Concepts, methods, and supervise. LZ and TT: methodology and data analysis. L-JC, Z-LY, G-MH and YL: provide resources for project data collection. All authors reviewed the manuscript. All authors contributed to the article and approved the submitted version.

Funding

This work was supported by Beijing Research Ward Excellence Program, BRWEP2024W094090106 and Capital Health Development Research Special Project (2024-1-4093).

Data availability

The datasets used and/or analysed during the current study are available from the corresponding author on reasonable request.

Declarations

Ethics approval and consent to participate

This study will be conducted in accordance with the principles of the Declaration of Helsinki and relevant Good Clinical Practice (GCP) guidelines. Ethical approval has been obtained from Peking University Third Hospital Medical Science Research Ethics Committee (M20250133), as well as from the ethics committees of all participating centres. The study was registered with the Chinese Clinical Trial Register (ChiCTR2500105183), the registration date is 2025-06-30.

Written informed consent will be obtained from all participants prior to any study-related procedures. Participants will be informed of their right to withdraw from the study at any time without any impact on their standard medical care. To ensure participant confidentiality, all collected data will be de-identified and stored securely on password-protected servers, accessible only to authorised research personnel. Personal identifiers will not be used in any dissemination materials.

The findings of this study will be disseminated through peer-reviewed journals, national and international conferences, and academic forums. Additionally, summary results may be shared with participants and stakeholders upon study completion. The final dataset will be archived according to institutional policies and applicable regulations.

Any substantial protocol amendments will be submitted for additional ethics review and communicated to all investigators and relevant parties.

Consent for publication

Not applicable.

Competing interests

The authors declare no competing interests.