Premature delivery, as a common obstetric occurrence, refers to childbirth occurring at 28–36 weeks⁺⁶ of gestation, which may lead to neonatal organ dysfunction and may seriously affect the pregnancy outcome. Cervical insufficiency (CIC), as a major risk factor for premature delivery, is defined as abnormal cervical structure and impaired cervical function caused by congenital developmental factors or acquired injury, whose clinical findings include cervical canal shortening and cervical dilation in the second and third trimesters of pregnancy [1]. Cervical cerclage is the only confirmed effective operation for treating CIC, with surgical closure of the cervical canal being performed to prevent further dilation. Thereby mitigating the fetal pressure on the internal cervical orifice and effectively prolonging the time to delivery of women with CIC [2]. However, recent research has shown that some pregnant patients still experience adverse outcomes such as premature delivery after cervical cerclage [3]. Actively searching for the risk factors of premature delivery following cervical cerclage in CIC pregnant patients is of great significance in order to lower the incidence of adverse pregnancy outcomes.

Depending on the presence or absence of infective factors, premature delivery occurring in cervical cerclage patients can be divided into infectious and non-infectious types. Early diagnosis of premature delivery secondary to infection can provide a reference value in guiding early anti-infective therapy for pregnancy prolongation [4, 5]. Research of serological cytokines can offer reference for the clinical diagnosis of infectious diseases. High mobility group box 1 (HMGB1) is a chemotaxis related factor, whose infiltrating effect on uterine smooth muscle cells can induce the contraction of the cells, increasing the risk for premature delivery [6, 7, 8, 9]. Soluble tumor necrosis factor receptor 1 (sTNFR-1) can induce the activation of downstream tumor necrosis factor alpha (TNF-$\alpha$), strengthen the recruitment of downstream inflammatory factors, and promote the damage of fetal membrane and placental tissue [10, 11]. Further research has demonstrated that placental inflammation is closely associated with the pregnancy conditions such as premature delivery, and that the blood neutrophil-to-lymphocyte ratio (NLR) can serve as a hematological parameter reflecting the inflammation, whose correlation with placental inflammation has received growing attention in recent clinical studies [12]. The predictive value of serum indicators for the risk of premature delivery after cervical cerclage in CIC pregnant patients remains unknown.

To this end, this study demonstrated the correlations of the serum HMGB1, sTNFR-1 and peripheral blood NLR expressions with premature delivery secondary to infection in cervical cerclage patients, which provides a reference for the early diagnosis and timely intervention of premature delivery secondary to infection among pregnant patients undergoing cervical cerclage, with a view to providing a certain level of protection for maternal and fetal health for pregnant patients with CIC.

Premature birth is one of the primary causes of perinatal mortality. While cervical cerclage can potentially prevent premature birth due to cervical laxity, a certain number of patients still experience premature birth following cervical cerclage. Infection is a significant risk factor for premature birth, particularly amniotic cavity infection triggered by pathogenic microorganisms in amniotic fluid [14]. This type of infection is an asymptomatic intrauterine infection that can only be detected during pathological examination. Therefore, actively seeking biological indicators to evaluate the occurrence of chorioamnionitis is of great significance for the diagnosis and treatment of potential infection and the prevention of premature birth caused by infection after cervical cerclage.

As a member of the chemokine family, HMGB1 induces the softening and relaxation of the internal cervical orifice by recruiting the downstream complement or chemokine C3a; low HMGB1 level can recruit the immune cells and promote the proliferation and differentiation of endometrial stromal cells required for normal pregnancy, while high HMGB1 level is associated with pregnancy failure [15, 16]. Endotoxins generated from the metabolism of pathogenic microorganisms after infection, along with various inflammatory factors released during the inflammatory response, can trigger the release of HMGB1, which mediates the inflammatory response. Conversely, HMGB1 can also stimulate monocytes to secrete pro-inflammatory factors, such as sTNFR-1 and interleukin 1 (IL-1), thereby exacerbating the inflammatory condition [17]. sTNFR-1 is a member of the TNFR family, whose regulatory effects on different TNF-$\alpha$ receptors can enhance the pathological role of inflammatory factors, intensifying their damage to amniotic sac wall cells to increase the risk of premature delivery [18, 19]. NLR is a sensitive inflammatory index discovered in current research, which not only indicates the role of neutrophils in infection, but also correlates with the lymphocyte count in the body. Neutrophils primarily exert their role in non-specific inflammatory responses, while lymphocytes play a role in immune system regulatory pathways. Upon the bodily infection by bacteria, the lymphocytes in the body diminish, accompanied by a quantitative increase in neutrophils. The latter can secrete a variety of inflammatory mediators, including chemokines and cytokines, to stimulate the chemotaxis, as well as the production and release of substantial neutrophils in the bone marrow, resulting in gradual local and systemic increases in the number of neutrophils. Thus, NLR is the ratio of two different but complementary immune pathways, which is considered a crucial indicator reflecting the inflammatory and immune states of the body [20] . Researchers have found in their studies on the early diagnosis of bacterial infectious diseases and blood infections [20, 21, 22] that NLR is closely associated with the early diagnosis and severity of bacterial infectious diseases. When the bodily infection by bacteria is at an early stage, NLR is the most rapid, simple and inexpensive early indicator available, which can more accurately reflect the infection status than conventional inflammatory indicators (e.g., leukocyte count, central granulocyte ratio). However, the correlation of NLR with premature delivery has rarely been reported, and studies on its association with premature delivery secondary to infection caused by cervical cerclage are even rarer.

In this study, based on the changes in the serum levels of HMGB1, sTNFR-1 and NLR detected among pregnant patients with premature delivery following cervical cerclage, those positive for pathogenic microorganisms in cervical secretion culture were assigned to the infectious group, while those negative to pathogenic microorganisms were assigned to the non-infectious group. It was found that the levels of HMGB1, sTNFR-1 and NLR in the infectious group were all significantly higher than those in the non-infectious group, suggesting that the high expression of serum HMGB1, sTNFR-1 and NLR could all affect the occurrence of premature delivery secondary to infection in cervical cerclage patients. Findings of relevant cause analysis were as follows: (1) increased expression of HMGB1 could increase the degree of oxytocin receptor upregulation on the uterine smooth muscle cell membrane by inducing the activation of downstream nuclear factor $\kappa$B (NF-$\kappa$B) signaling pathway, thereby triggering contraction of uterine smooth muscle cells to promote the occurrence of premature delivery. According to a study by Wallenstein et al. [23], the serum concentration of HMGB1 in preterm or infected pregnant patients could increase by approximately 40% on average, and such increase was more significant among those with younger gestational age or complicated by adverse neonatal clinical outcomes. (2) Increased expression of sTNFR-1 could aggravate the damage of fetal membrane and the occurrence of preterm membrane rupture by increasing the spread of inflammatory cascade response, thereby heightening the risk of premature delivery [24]. (3) The reason for the increase of NLR level is considered to be the intrauterine infection. The stimulation of chorionic and decidual inflammatory regions provoke the release of cytokines, which leads to changes in the number of leukocyte subsets in the body to promote the increase of neutrophil count and the decrease of lymphocyte count, thereby inducing premature delivery.

Through the multivariate logistic regression analysis of serological indicators in pregnant patients having premature delivery after cervical cerclage, this study found that the serum HMGB1 level and NLR were independent risk factors for premature delivery, which had certain value in the independent diagnosis of premature delivery secondary to infection among pregnant patients undergoing cervical cerclage. ROC analysis revealed that the peripheral blood NLR had high sensitivity and specificity for predicting premature delivery secondary to infection in cervical cerclage patients, with an AUC of 0.825. Consistent with previous findings, close monitoring of NLR levels facilitated early prediction of premature delivery [25]. Additionally, joint prediction by serum HMGB1, sTNFR-1 and NLR exhibited the maximum AUC, which was nearly 0.9. In clinical practice, microbes are usually cultured to determine whether infection is present. However, given the long cycle of microbial culture and the fast progression of premature delivery, the optimal intervention time may be missed. Serological indicators are characterized by fast detection. Through the detection of serum HMGB1, sTNFR-1 and NLR levels in pregnant patients with signs of premature delivery, early screening of and anti-infective therapy for premature delivery secondary to infection are possible, which can control the patient’s condition as soon as possible, prolong the gestational age, and improve the maternal and fetal pregnancy outcomes. Due to the small specimen size of this study and the failure to compare preoperative serological indicators, there are still limitations in our conclusions. In future research, more clinical data should be included to reduce experimental errors, factors associated with premature delivery secondary to infection in pregnant patients undergoing cervical cerclage should be further analyzed, relevant measures should be formulated, and the pregnancy outcome of cervical cerclage patients after receiving active intervention should be included to further corroborate these findings. Although explicit external infections have been excluded, some individual patients may have latent infections outside the reproductive tract or amniotic cavity. These infections could potentially introduce bias into the research results, particularly in studies with small sample sizes.

When the pregnant patients undergoing cervical cerclage have premature delivery secondary to infection, their serum levels of HMGB1, sTNFR-1 and NLR are significantly upregulated. Joint prediction by serum HMGB1, sTNFR-1 and NLR has a crucial referential value in diagnosing premature delivery secondary to infection among the cervical cerclage patients. Early diagnosis of the risk for premature delivery secondary to infection and initiation of early anti-infective therapy are necessary, in order to avoid premature delivery in cervical cerclage patients, gain time for early treatment, control the disease as early as possible, prolong the gestational age, and improve both the maternal and fetal pregnancy outcomes.

All data generated or analysed during this study are included in this published article.

SC: experimental design, case collection and article writing. YW: case collection and data analysis. YD: cervical cerclage operator. LZ: experimental design, review of experimental process. All authors contributed to editorial changes in the manuscript. All authors read and approved the final manuscript. All authors have participated sufficiently in the work and agreed to be accountable for all aspects of the work.

All subjects gave their informed consent for inclusion before they participated in the study. The study was conducted in accordance with the Declaration of Helsinki, and the protocol was approved by the Ethics Committee of Affiliated Matern & Child Care Hospital of Nantong University (approval number: Y2017031).

We would like to express our gratitude to all those who helped us during the writing of this manuscript. Thanks to all the peer reviewers for their opinions and suggestions.

This study was funded by Nantong Science and Technology Bureau Social Livelihood Science and Technology Plan Project (Grant No: MSZ2022035) and Nantong Youth Talent Project (Grant No: YQY202310) and Nantong Innovation Team Project (Grant No: NTXK202302).

The authors declare no conflict of interest.