Sina Baharaghdam
1,2 
, Özkan Yildirim
3, Mohammadbagher Pirouzpanah
4, Danial Abbas kabiri
3, Shima Karimi
1,2, Mohammad Esfini Farahani
5, Amirhossein Aghapour
1,2, Ali Hassanzadeh
6, Amin Kamrani
1,2, Reza Mousavi Ardehaie
4, Amir Mehdizadeh
7, Parvin Hakimi
8, Javad Ahmadian Heris
9, Leili Aghebati-Maleki
1,2, Mehdi Yousefi
4,2 1 Immunology Research Center, Tabriz University of Medical Sciences, Tabriz, Iran
2 Department of Immunology, Faculty of Medicine, Tabriz University of Medical Sciences, Tabriz, Iran
3 Department of Histology and Embryology, Institute of Health Sciences, Atatürk University, Erzurum, Türkiye
4 Molecular Medicine Research Center, Tabriz University of Medical Sciences, Tabriz, Iran
5 Department of Toxicology and Cancer Biology, University of Kentucky, Lexington, USA
6 Department of Applied Cell Sciences, School of Advanced Technologies in Medicine, Tehran University of Medical Sciences, Tehran, Iran
7 Hematology and Oncology Research Center, Tabriz University of Medical Sciences, Tabriz, Iran
8 Women’s Reproductive Health Research Center, Tabriz University of Medical Sciences, Tabriz, Iran
9 Department of Allergy and Clinical Immunology, Pediatric Hospital, Tabriz University of Medical Sciences, Tabriz, Iran
Abstract
Introduction: Recurrent implantation failure (RIF) is a complex condition that makes it one of the most challenging cases in the field of infertility. The diagnosis of this condition with the immunological etiology may be aided by determining the endometrial immune profile for the classification of these patients. Current diagnostic approaches are based on invasive endometrial biopsies to classify patients into balanced, low, or over-immune activation profiles, which have limitations for routine use and serial monitoring. This study aimed to develop and validate a minimally invasive peripheral blood-based classification system using immunological and metabolic markers to mirror endometrial immune profiles in RIF patients.
Methods: Endometrial tissue and peripheral blood samples were collected during the mid-luteal phase from 163 RIF patients and 28 fertile controls. Endometrial immune profiles were determined via RT-qPCR for IL-18, IL-15, TWEAK, Fn-14, and CD56, classifying RIF into balanced, low, and over-immune activation subgroups, with sample sizes of 32, 47, and 84 women, respectively. Peripheral blood was analyzed by flow cytometry to determine the Th1/Th2 ratio and NK cell percentage; by ELISA to measure nuclear antibodies (ANA, anti-dsDNA), phospholipid-related antibodies (anticardiolipin, anti-β2-glycoprotein I, antiphospholipid antibodies), thyroid-related antibodies (anti-TPO, anti-TG), anti-tissue transglutaminase (anti-TTG), and metabolites (S1P, adiponectin, leptin, PGE2, phosphatidylserine, IGF-1); and by spectrophotometry to quantify total phospholipids.
Results: The over-immune activation group showed significantly elevated Th1/Th2 ratios, NK-cell percentages, and autoantibodies (ANA, anti-phospholipid, anti-β2-glycoprotein I, anti-TG, anti-TPO) compared to balanced and low-immune activation groups. Metabolic profiles revealed higher leptin, and total phospholipids but lower adiponectin, S1P, and PGE2 in over-immune activation group. The low-activation group exhibited lower Th1/Th2 ratios, reduced leptin, but elevated adiponectin, S1P, and PGE2 versus balanced and over-immune activation groups. No significant differences were found in phosphatidylserine or IGF-1 across groups.
Conclusion: Our results demonstrate that peripheral blood immunological and metabolic markers can effectively distinguish RIF immune endotypes, offering a non-invasive alternative to endometrial biopsy for personalized assisted reproductive technology (ART) management and potentially improving implantation success through targeted therapies.