INTRODUCTION
Preterm prelabor rupture of membranes (PPROM) is a significant complication that profoundly impacts pregnancy. It is characterized by the untimely rupture of fetal membranes before the onset of labor and before reaching the 37th week of gestation [1, 2]. This clinical challenge contributes significantly to the prevalence of preterm births worldwide, posing substantial implications for neonatal morbidity and mortality [3, 4]. The etiology of PPROM is multifaceted, involving various risk factors such as infection, inflammation, mechanical stress, and hormonal imbalances. Among these, ascending bacterial infections, in particular, have been strongly associated with an increased risk of PPROM [5, 6]. Additionally, maternal and environmental factors, including smoking, maternal stress, and nutritional deficiencies, may also contribute to the occurrence of this condition [7]. Understanding these factors comprehensively is crucial to develop targeted interventions that can improve maternal and neonatal outcomes [5, 8]. Furthermore, the economic impact of PPROM-related preterm births, encompassing both immediate neonatal care and long-term medical support, calls for urgent attention from a public health perspective [8–12].
Galectins, a family of β-galactoside-binding lectins, play diverse roles in various physiological and pathological processes [13, 14]. During pregnancy, these multifunctional proteins are pivotal in regulating immune responses, cell adhesion, and tissue remodeling, significantly influencing key events such as implantation, placental development, and fetal growth [15, 16]. By interacting with glycoconjugates on the cell surface, galectins modulate various critical signaling pathways essential for successful pregnancies [17–19].
Among the galectins involved in pregnancy-related processes, galectin-1 and galectin-9 hold significant implications for pregnancy. Previous research has linked galectin-1 and galectin-9 to the establishment and maintenance of maternal-fetal immune tolerance, a fundamental component for successful pregnancies [20–23]. Additionally, both galectin-1 and galectin-9 have been implicated in promoting essential processes such as trophoblast invasion and angiogenesis in the placenta, thus exerting a notable influence on fetal growth and development [16, 24]. Investigating the expression and function of these galectins in the placentas of pregnancies affected by PPROM could provide novel perspectives on their potential contribution to the pathogenesis of this condition.
This study aims to elucidate the expression patterns of genes encodinggalectin-1 and galectin-9 in placental tissues of pregnancies affected by PPROM and explore their potential roles in the pathophysiology of this obstetric complication. Dół formularza
MATERIAL AND METHODS
This prospective, single-center study was conducted at the Gynecology and Obstetrics Clinical Hospital in Poznań, Poland, spanning from June 2021 to May 2023. A total of 25 women participated in the study, comprising 12 cases of preterm prelabor rupture of membranes occurring between 23 and 36 weeks of gestation, involving healthy women with a physiological course of pregnancy prior to PPROM. Additionally, 13 healthy mothers of full-term infants were included as the control group. Cases were recruited at the Delivery Ward following confirmation of PPROM and after a thorough assessment of the patients’ general and obstetrical history. The control group consisted of pregnant women admitted to the Delivery Ward during the same period, who had no obstetric complications or other serious medical conditions.
Exclusion criteria were strictly defined and encompassed maternal age under 18 years, history of drug abuse or cigarette smoking, and serious maternal diseases, such as hypertension, preeclampsia, diabetes, cholestasis of pregnancy, and unstable thyroid disease. Other factors leading to exclusion included intrauterine infection, multiple pregnancies, detected fetal or placental abnormalities, fetal growth restriction, and cervical insufficiency. All participants were required to provide written, informed consent before their inclusion in the study. Medical records of the participants were obtained to gather more comprehensive information about the patients.
Gestational age was determined either based on the last menstrual period or through fetal crown-rump length measurement during the first trimester. The diagnosis of PPROM was established through speculum examination, wherein the visualization of amniotic fluid in the vagina served as an indicator. Alternatively, in cases requiring further confirmation, a placental insulin growth factor binding protein-1 (IGFBP-1) test (Amnioquick, Biosynex Swiss SA) was employed.
Patients diagnosed with PPROM were promptly hospitalized and received comprehensive management according to a specified protocol. For those with gestational age below 34 weeks, corticosteroids were administered to promote lung maturation. Additionally, prophylactic antibiotics were administered, and daily cardiotocographic monitoring and fetal assessments were conducted. Throughout the hospitalization, close monitoring for any signs of intrauterine infection was maintained, involving regular complete blood count (CBC) and C-reactive protein (CRP) assessments every second day, or more frequently if necessary.
For analysis, each participant provided a one-time 1 cm3 sample of placenta, collected approximately 10 minutes after delivery. Two samples were acquired — one from the middle and one from the edge of the placenta, through its entire thickness. The samples were immediately stored in tubes containing RNAlater® (Sigma-Aldrich®, Merck Group, USA) and preserved at –20°C until analysis.
Total RNA was extracted from human placenta tissue samples using TRIzolTM Reagent (Thermo Fisher Scientific; Waltham; MA; USA) following the protocol. The quality and quantity of RNA samples were checked spectrophotometrically (DS-11; Wilmington, DE; USA), and each RNA sample was diluted to a final concentration of 100 ng/µl for cDNA synthesis using reverse PCR (High Capacity cDNA RT Kit; Thermo Fisher Scientific; Waltham; MA; USA). The expression of the LGALS1 (galectin 1) and LGALS9 (galectin 9) genes was evaluated by quantitative real-time PCR using the commercial TaqMan Gene Expression Assay (Hs00355202_m1 for LGALS1 and Hs00371321_m1 for LGALS9; Thermo Fisher Scientific; Waltham; MA; USA) and LightCycler 480 (Roche; Basel; Switzerland). The GAPDH (glyceraldehyde-3-phosphate dehydrogenase) and CYC1 (cytochrome c1) genes were selected as reference genes (Hs02758991_g1 for GAPDH and Hs00357717_m1 for CYC1; Thermo Fisher Scientific; Waltham; MA; USA). Relative quantification of the analyzed genes was performed based on the second derivative maximum method (Roche). All samples were analyzed in duplicate.
All analyses were conducted using PQStat Software 2022 (PQStat v.1.8.4, Poznan, Poland). To assess the distribution of all continuous variables, the Kolmogorov-Smirnov test was utilized. Variables with a normal distribution were estimated using means ± standard deviation (SD) and then compared using the independent samples Student’s t-test. On the other hand, non-normally distributed variables were analyzed using the Mann-Whitney U-test, and the results were expressed as median and interquartile range. For categorical variables, frequency counts and percentages were used. The correlations between variables were evaluated through Spearman’s correlation analysis. The one-way analysis of variance (ANOVA) was used to evaluate the differences between the groups. P value below 0.05 was considered statistically significant.
RESULTS
A prospective case-control study was undertaken, comprising 25 women, with 12 participants experiencing PPROM and 13 serving as controls. Age-matching was meticulously ensured between the cases and controls. Preterm prelabor rupture of membranes was specifically defined as the rupture of fetal membranes occurring between the 23rd and 36th week of gestation. The characteristics of the cases and controls are summarized in Table 1. Similar BMI at blood sampling, C-section rate, female neonate rate, and fetal outcomes at birth, as defined by the neonate’s pH, were observed between the two groups. However, statistically significant differences were found in terms of gestational age at delivery and birth weight. No statistical significance was found regarding the expression of both LGALS1 and LGALS9 in both groups (0.01099 vs 0.00913; p = 0.52, shown in Figure 1 and 0.00026 vs 0.00033; p = 0.4 shown in Figure 2, respectively).
|
Table 1. Demographic and clinical characteristics of the study population |
|||
|
PPROM (n = 12) |
Control (n = 13) |
p value |
|
|
Age [years] |
30.17 ± 1.38 |
30.46 ± 1.34 |
0.88 |
|
Gravida |
1 (1–3) |
3 (1–2.25) |
0.07 |
|
BMI at blood sampling [kg/m^2] |
28.95 ± 1.51 |
29.59 ± 1.52 |
0.38 |
|
GA at delivery |
34.5 ± 0.97 |
39.15 ± 0.39 |
0.00006 |
|
C-secection [%] |
33.33% |
23.07% |
0.57 |
|
Female neonate [%] |
16.67% |
30.77% |
0.41 |
|
Birth weight [g] |
2428 ± 208 |
3579 ± 132 |
0.00004 |
|
pH of the neonate at birth |
7.25 ± 0.02 |
7.24 ± 0.02 |
0.30 |
|
WBC count |
11.87 ± 0.72 |
10.79 ± 0.55 |
0.12 |
|
CRP |
4.4 ± 1.39 |
3.77 ± 1.11 |
0.39 |
|
LGALS1 |
0.01099 ± 0.00269 |
0.00913 ± 0.00111 |
0.52 |
|
LGALS9 |
0.00026 ± 0.00004 |
0.00033 ± 0.00007 |
0.40 |
|
BMI — body mass index; CRP — C-reactive protein; GA — gestational age; PPROM — preterm prelabor rupture of membranes; WBC — white blood cells |
|||
To further investigate the association between PPROM and LGALS1 and LGALS9 expression, we analyzed the correlations between galectins and other parameters (Tab. 2). However, no statistically significant correlations were found.
|
Table 2. Correlations between LGALS1 and LGALS9 transcript levels and other parameters |
||
|
LGALS1 |
LGALS9 |
|
|
Age |
R = –0.15 |
R = –0.25 |
|
p = 0.24 |
p–0.12 |
|
|
BMI |
R = 0.11 |
R = –0.16 |
|
p = 0.31 |
p = 0.23 |
|
|
GA at blood sampling |
R = 0.03 |
R = 0.17 |
|
p = 0.45 |
p = 0.21 |
|
|
Birth weight |
R = –0.07 |
R = 0.09 |
|
p = 0.37 |
p = 0.33 |
|
|
pH of the neonate at birth |
R = –0.15 |
R = –0.02 |
|
p = 0.24 |
p = 0.45 |
|
|
WBC |
R = –0.05 |
R = –0.30 |
|
p = 0.40 |
p = 0.07 |
|
|
CRP |
R = –0.16 |
R = 0.33 |
|
p = 0.27 |
p = 0.10 |
|
|
GA — gestational age; BMI — body mass index; WBC — white blood cells; CRP — C-reactive protein |
||
Discussion
The aim of this study was to investigate the placental expression of LGALS1 and LGALS9 in pregnancies complicated with PPROM. Our hypothesis was that the placental expression of LGALS1 and LGALS9 would decrease in pregnancies with PPROM since they both act as anti-inflammatory molecules, contributing to the maintenance of pregnancy [25, 26]. However, we were unable to confirm our hypothesis.
Galectin-1’s function has been extensively described in the literature due to its diverse impact on all stages of pregnancy. Abundant expression of LGALS-1 has been observed at the feto-maternal interface in humans, where it promotes maternal immune tolerance to the fetal semi-allograft. Galectin-1’s tolerance-promoting mechanisms have been well-established for adaptive immune cells, such as T cells and dendritic cells [25]. This remarkable process begins early on, as murine, bovine, and human models have shown widespread expression of LGALS1 in the endometrium during the implantation window. Galectin-1 plays a crucial role in decidualization and creates an immune-privileged environment at the maternal-fetal interface, contributing to the low cytotoxic activity of decidual natural killer (dNK) cells [20, 27–30]. Some authors suggest that low LGALS1 expression and concentration might be associated with unexplained infertility [31]. Notably, LGALS-1 is believed to be expressed not only in the decidua and placenta but also in human embryos on the trophectoderm and inner cell mass. Interestingly, circulating galectin-1 levels were significantly decreased in women who later experienced a miscarriage [32]. Additionally, low LGALS1 decidual expression is thought to play a role in initiating parturition, both term and preterm [33]. On the contrary, an increased LGALS1 mRNA expression in chorioamniotic membranes in PPROM was associated with chorioamnionitis, suggesting that gal-1 may be involved in regulating inflammatory responses to chorioamniotic infection [21].
Various pregnancy pathologies have been investigated concerning LGALS1 expression in placenta and serum concentration. For instance, LGALS1 exhibits low expression in the serum and placenta of pregnant women with fetal growth restriction (FGR) and is hypothesized to be involved in its pathogenesis [34]. Moreover, placental LGALS1 expression is higher in severe preeclampsia (PE) than in normal pregnancy, regardless of the presence of small for gestational age (SGA) fetuses. However, it is not altered in SGA without PE, which might be due to the fetal response to an exaggerated systemic maternal inflammation [35]. Another serious pregnancy complication, HELLP syndrome, was also associated with increased circulating levels of gal-1 [36].
In cases of PPROM, galectin-1 women’s serum concentration is believed to be decreased, similar to healthy individuals who deliver at term, thus facilitating the pro-inflammatory changes that lead to the onset of labor [33, 37]. However, the data are not consistent [38]. Considering the abovementioned reports and our own findings, in which we demonstrated that LGALS1 placental expression is not significantly different from healthy controls, we acknowledge that the studied group was relatively small to definitively confirm the hypothesis. Nonetheless, it does not discount the possibility, especially when considering all the previous data that supports it.
To date, galectin-9 has been less well-known, and the data regarding its exact function and characteristics are scarce. In addition to the endometrium, trophoblasts, and stromal cells of the decidua [29, 39], LGALS9 is also expressed by the placental endothelial cells and various types of immune cells [26, 40–42]. Several pregnancy pathologies have been linked to LGALS9 expression. Similar to galectin-1, galectin-9 concentrations were decreased in women with unexplained recurrent spontaneous abortion [26, 43, 44]. Moreover, the high expression of LGALS9 at uterodomes during the implantation window suggests that galectin9 can be considered as a marker of endometrial receptivity and may play an important role during the initial events of human embryo implantation [39]. It also immunomodulates the response in preeclampsia, with upregulation observed in decidual tissue and peripheral lymphocytes of preeclamptic pregnancies compared to normotensive pregnancies [45, 46]. During the course of pregnancy, the levels of galectin-9 in maternal blood rise in both concentration and expression, indicating its potential significance in maintaining the pregnancy [47]. Interestingly, some studies have indicated that galectin-9 serum levels are higher in women carrying a male fetus compared to a female fetus [48]. However, regarding PPROM, galectin-9 concentration did not show any significant differences in maternal serum [37]. In our study, we expected gal-9 to be downregulated similarly to gal-1, but we were unable to prove this.
This study is subject to several limitations, primarily due to the small number of participants and its single-center nature.
To conclude, there are no statistically significant differences in LGALS1 and LGALS9 expression in placentas with PPROM compared to healthy controls. However, further clinical studies on larger groups may be necessary to thoroughly investigate this topic.
Article information and declarations
Data availability statement
All data generated or analyzed during this study are included in this article. Further enquiries can be directed to the corresponding author.
Ethics statement
This study was approved by the Bioethics Committee of Poznan University of Medical Sciences, Poland, approval number 1158/19.
Author contributions
Conceptualization, Dorota Boron and Agnieszka Seremak-Mrozikiewicz; methodology, Dorota Boron; software, Joanna Mikolajczyk-Stecyna; validation, Joanna Mikolajczyk-Stecyna, Agata Chmurzynska and Grazyna Kurzawinska; formal analysis, Dorota Boron and Joanna Mikolajczyk-Stecyna; investigation, Dorota Boron; resources, Dorota Boron; data curation, Dorota Boron and Joanna Mikolajczyk-Stecyna; writing — original draft preparation, Dorota Boron; writing — review and editing, Agnieszka Seremak-Mrozikiewicz, Agata Chmurzynska; visualization, Dorota Boron; supervision, Agnieszka Seremak-Mrozikiewicz, Wieslaw Markwitz and Agata Chmurzynska; project administration, Dorota Boron; funding acquisition, Dorota Boron.
Funding
Research received no external funding.
Acknowledgments
None.
Conflict of interest
The authors have no conflicts of interest to declare.
Supplementary material
None.
