PTX3/NF-κB/TLR4 Pathway Evaluation in the Follicular Fluid to Successfully Predict Blastocyst Implantation: A Pilot Study

Abstract

Background: The implantation process is complex and involves numerous factors that can affect its success. In artificial reproductive treatments (ARTs), chronic inflammation seems to be associated with implantation failure, largely contributing to reproductive dysfunction. Pentraxin 3 (PTX3) is overexpressed in several pathological conditions by exerting a pivotal role both as a regulator and indicator of inflammatory response. Some literature data have shown that PTX3 could have an impact on follicle growth and development, influencing women's fertility. This study aimed to detect PTX3 in follicular fluids collected during an ART protocol in relation to implantation outcomes. Methods: The PTX3/NF-kB/TLR4 pathway and other cytokines were assessed in the follicular fluid of 169 subjects, under the age of 40 years, undergoing IVF cycles, including females without achieved implantation (n = 98) and those with implantation (n = 71). Furthermore, subgroup analyses were performed to evaluate PTX3 values according to age difference. Results: From our data, PTX3 emerged as a strong predictor, more than TNFα and IL-1β, of implantation failure and related inflammatory follicular state. Overall, the results point to PTX3 as a potential biomarker for ART success, and their testing may be helpful in women whose successful implantation remains unexplained. Conclusions: Therefore, PTX3 could constitute a reliable biomarker and a valuable target to improve ART outcomes.

Keywords: Pentraxin 3 (PTX3); artificial reproductive treatments (ARTs); blastocyst implantation; follicular fluid; inflammation; pregnancy outcome.

Figure 1

Follicular fluid PTX3 levels among no implantation women (n = 98) and women with successful implantation (n = 71). ELISA kits in follicular fluid to evaluate PTX3 (A), TLR4 (B), IkBα (C), and NF-κB levels (D). All data in the figure are shown as median with Interquartile Range (IQR). Data were not distributed normally. Statistical analysis was performed by using Mann–Whitney test. *** p < 0.001 vs. “No implantation”.

Figure 2

Follicular fluid levels of TNFα, IL-1β, and IL-6 among no implantation women (n = 98) and women with successful implantation (n = 71). ELISA assays executed for TNFα (A), IL-1β (B), and IL-6 (C). All data in the figure are shown as median with IQR. Data were not distributed normally. Statistical analysis was performed by using Mann–Whitney test. * p < 0.05 vs. “No implantation”; ** p < 0.01 vs. “No implantation”.

Figure 3

Follicular fluid PTX3, TLR4, IkBa, and NF-kB levels among under-30 no implantation women (n = 6) and women with successful implantation (n = 12). ELISA kits in follicular fluid to evaluate PTX3 (A), TLR4 (B), IkBα (C), and NF-κB levels (D). All data in the figure are shown as median with IQR. Due to the small sample size, statistical analysis was performed using Mann–Whitney test. *** p < 0.001 vs. “No implantation”.

Figure 4

Cytokine levels of TNFα, IL-1β, and IL-6 in follicular fluid from younger women who did not achieve implantation (n = 6) and those with successful implantation (n = 12). ELISA assays were conducted to measure TNFα (A), IL-1β (B), and IL-6 (C). Results are expressed as median with IQR. Due to the small sample size, statistical comparisons were carried out using the Mann–Whitney test. ns: not significant vs. “No implantation”.

Figure 5

Follicular fluid levels of PTX3, TLR4, IKBα, and NF-κB in women over 30 with and without successful implantation. ELISA assays were conducted to measure PTX3 (A), TLR4 (B), IKBα (C), and NF-κB (D) concentrations in follicular fluid from women over 30 who experienced failed implantation (n = 92) and those with successful implantation (n = 59). Results are expressed as median with IQR. Non-normal distribution of data was detected. Statistical comparisons were performed using the Mann–Whitney test. *** p < 0.001 vs. “No implantation”.

Figure 6

Cytokine levels of TNFα, IL-1β, and IL-6 were analyzed in follicular fluid from women over 30 years old who did not achieve implantation (n = 92) and those with successful implantation (n = 59). ELISA assays were used to measure TNFα (A), IL-1β (B), and IL-6 (C). Results are expressed as median with IQR. Non-normal distribution of data was found. Statistical analysis was performed using the Mann–Whitney test. * p < 0.05 vs. “No implantation”.

Figure 7

PTX3 and cytokines correlation with implantation rate. Only a p-value of less than 0.05 was considered significant.