{"paper_id":"5cf990b5-dcac-44c8-93d5-e31ff994db16","body_text":"Alzaidi et al. Egypt J Med Hum Genet           (2021) 22:85  \nhttps://doi.org/10.1186/s43042-021-00201-9\nRESEARCH\nThe effect of cytokine leukemia-inhibitory \nfactor (LIF) and interleukin-11 (IL-11) gene \nexpression on the primary infertility related \nto polycystic ovary syndrome, Tubal factor, \nand Unexplained infertility in Turkish women\nZahraa Alzaidi1* , Şule Menziletoğlu Yildiz2, Çetin Saatçi3, Hilal Ünlü Akalin4, Iptisam Ipek Muderris5, \nBuşra Aynekin3, Izem Olcay Şahin3 and Munis Dündar3 \nAbstract \nBackground: Successful implantation of blastocysts is indeed an important path in mammalian reproduction that is \ngoverned by a complicated web of cytokines interactions. Leukemia inhibitory factor (LIF) and interleukin-11 (IL-11) \npart of the interleukin (IL)-6 groups are cytokines that are needed for effective implantation and prevent infertility \nsymptoms. This study aimed to determine the expression level (LIF, IL-11) genes in patients with primary infertility \nrelated to polycystic ovary syndrome (PCOS), tubal factor infertility (TFI), and unexplained infertility (UI).\nResults: In this study, 75 infertility women and 40 controls were involved. The expressions of LIF and IL-11 genes \nwere evaluated by quantitative real-time polymerase chain reaction qRT–PCR Light Cycler in patients and healthy \ncontrols. PCOS, TFI, and UI groups showed promising results regarding LIF gene, which appeared at very small levels \ncompared to the control (p < 0.0001). Regarding IL-11, the two groups TFI and UI were significantly linked to the lower \nlevel of gene expression, while the PCOS group has no significant difference when it is compared to the control group \n(p < 0.0001, < 0.05, 0.19), respectively.\nConclusion: The current findings show that low levels of LIF and IL-11 gene expression are linked to various primary \ninfertility conditions, including PCOS, tubal factor, and unexplained infertility since they play a fundamental role in \nembryo implantation.\nKeywords: Leukemia inhibitory factor (LIF), PCOS, Interleukin-11 (IL-11), Primary infertility, Tubal factor infertility (TFI), \nUnexplained infertility (UI)\n© The Author(s) 2021. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which \npermits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the \noriginal author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or \nother third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line \nto the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory \nregulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this \nlicence, visit http:// creat iveco mmons. org/ licen ses/ by/4. 0/.\nBackground\nReproduction is a prerequisite for maintaining the \nexistence of any species. In humans, fertility refers to a \nwoman’s capacity to become pregnant [1]. Infertility is \na prevalent human condition, affects millions of repro -\nductive-age individuals worldwide, and it impacts their \nfamilies and communities [2]. According to the Turkey \nDemographic and Health Survey, the prevalence of infer -\ntility in women in Turkey is significantly between 10 and  \n15% [3]. One of the main causes of hormone dysregula -\ntion is polycystic ovary syndrome (PCOS), a cluster of \nclinical symptoms found within a phenotypically hetero -\ngeneous group of women linked to ovarian dysfunction \nOpen Access\nEgyptian Journal of Medical\nHuman Genetics\n*Correspondence:  biotech.zahraa@gmail.com\n1 Department of Biotechnology, Faculty of Applied Science, Cukurova \nUniversity, Adana, Turkey\nFull list of author information is available at the end of the article\n\nPage 2 of 5Alzaidi et al. Egypt J Med Hum Genet           (2021) 22:85 \n[4]. Another main frequent reason for primary fertility \nproblems is tubal factor infertility (TFI), related to block -\nages, destruction, tissue damage, congenital abnormali -\nties, as well as other triggers that hinder a fertilized or \nunfertilized ova from descending into the uterus via the \nfallopian tubes and preventing a successful pregnancy \n[5]. Correspondingly, fertility aberrations are likely to \nappear in unexplained infertility (UI) but are undetect -\nable with the existing technologies. Aberrations in endo -\ncrinological equilibrium and the immune system as well \nas the defect of the genetic and reproductive physiology \nhave all been suggested as possible explanations of unex -\nplained infertility [6]. However, defects in endometrial \nreceptivity may be one cause of fertility issues in these \nwomen, in which embryos cannot implant in the uterus if \nthe endometrium is damaged [7]. The implant process or \nimplantation is the action through which the blastocyst \nconnects to the underlying endometrial layer and eventu-\nally penetrates, which is a dynamic and flexible process. \nIt is essential to create an efficient ‘interference’ between \nmaternal and embryonic tissues that entails many endo -\ncrines, paracrine, and autocrine influences [8]. A com -\nprehensive framework of molecules is activated at the \nmaternal–fetal interacting together under the control of \novarian hormones, which play a critical role in facilitat -\ning the events [9]. LIF (leukemia inhibitory factor) is one \nof the interleukin-6 (IL-6) families of cytokines; IL-6 and \nIL-11 are also part of the group. LIF is related to the sub -\nsequent activities throughout implantation: the receptive \ncondition of the endometrium, the connection between \nthe embryo and the endometrium, stromal decidualiza -\ntion, trophoblast invasion, blastocyst development and \ngrowth, and uterine leukocyte infiltration, according to \nevidence [10]. Interleukin-11 (IL-11), the second mem -\nber of interleukin-6 (IL-6), is thought to be essential for \ndecidualization in the endometrium, which promotes \nblastocyst adhesion and implantation by acting primarily \non uterine luminal epithelium and blastocyst [11]. Given \nthis, this work aimed to study the predictable pathophysi-\nology of the cytokine, leukemia inhibitory factor (LIF), \nand interleukin-11 (IL-11), in women suffering from \nprimary infertility related to polycystic ovary syndrome \n(PCOS), tubal factor infertility (TFI), and unexplained \ninfertility (UI).\nMethods\nStudy design\nThis prospective case study was conducted in ERCİYES \nUniversity hospitals, Department of Medical Genetics, \nKayseri, Turkey, between June 2019 and June 2021. A \ntotal of 75 patient women between 21 and 45 years of age \nhad been examined by a gynecologist, and they were suf -\nfering from 2 to 7 years of primary infertility divided into \nthree categories depending on the related case polycys -\ntic ovary syndrome (PCOS), tubal factor infertility (TFI), \nand unexplained infertility (UI). 25 women free from \nsigns and symptoms of PCOS, tubal factor dependent on \nthe medical criteria, clinically healthy, had a regular men-\nstrual cycle, exhibited normal ovulation and without any \ninfertility signs have involved as a control group. To com-\nplete the study, all patients and control groups were not \ntaking any medication or fertility drugs known to affect \novulation for at least 3 months before the study, and they \nare having irregular periods, no periods, or abnormal \nbleeding.\nBody mass index and waist circumference\nBody mass index (BMI) testing is recommended by the \nWorld Health Organization (WHO) to determine over -\nweight and obesity. Furthermore, BMI determines wom -\nen’s fertility in a significant direction, reduced female \nreproduction is linked to higher and lower levels of BMI \n[12]. BMI value, unhealthily thin weight (< 18), regular \nweight (18–24.9), overweight (25–29.9), obesity (30–\n39.9), and severe obesity (> 40) were classified by utiliz -\ning equation BMI = weight (kg)/height2 (m) [13]. When \nthe subject was standing up, the waist circumference was \ncalculated at the closest part of the torso width-wise, in \ngeneral directly just over the abdominal button, and the \nstandard value 88 cm in females [13].\nSamples of blood\nPeripheral blood collections (10 mL) were taken from the \npatient volunteer who signed a written informed consent \nform in ERCİYES University hospitals, Kayseri, Turkey. \nEach blood sample was saved in EDTA tubes and stored \nat the refrigerator temperature between 2 and 8  °C for \nmolecular studies.\nRNA isolation and PCR amplification\nTotal RNA was extracted using TRIZOL reagent (Thermo \nFisher Scientific, USA). Trizol ensures RNA integrity \nwhile lysing cells and dissolving cell components during \nhomogenization or lysis. Total RNA (1  µg) was reverse \ntranscribed using the EvoScript Universal cDNA Master \nStrand Kit according to the manufacturer’s instructions. \nThe mRNA expression level of LIF and IL-11 genes was \nexamined by quantitative real-time qRT–PCR Light -\nCycler 480 kit from Roche. The temperature and times \nprograms for expression in LightCycler 480 II software \nare shown in Additional file  1: Table 1. A pair of specific \nprimers was provided by Light Cycler 480 Probes Master \n(Roche) for each marker (LIF, IL-11) shown in Additional \nfile 2: Table 2. Gene expression levels were normalized to \nbeta-actin (ACTB).\n\nPage 3 of 5\nAlzaidi et al. Egypt J Med Hum Genet           (2021) 22:85 \n \nStatistical analysis\ncDNA synthesis and qPCR results were independently \nreplicated twice. Statistical significance levels of mRNA \nexpressions were analyzed using the GraphPad Prism \ntest. ANOVA repeated measures were used to com -\npare the mean of physiological parameters between \nthe patients’ groups. Statistical significance was deter -\nmined by p  ≤ 0.05 values.\nResults\nThe findings of this current study indicated a considera -\nble relationship (p  < 0.05) in the physical and physiolog -\nical measurements between infertile and control groups \nshown in Table  1. Age and BMI were significantly \nhigher (p  < 0.05) in the infertile patients compared with \nthe control group. There was no meaningful association \nbetween the remaining diagnostic indicators regarding \nwaist/hip ratio (p  0.25) and infertility duration (p  0.29). \nThe age limit of the patient groups was 32–45 years; the \nmean value was 32.87 ± 7.41, 33.98 ± 8.11, 32.80 ± 7.10 \nfor POCS, tubal factor, unexplained infertility groups \nsequentially. The age limit of the control group was \n23–43 years, and the average age was 28.45 ± 6.14 with \np value < 0.05 as in Table 1 .\nIn terms of genetic analysis, the research data revealed \nthat LIF gene expression level in the infertile patient \ngroups was considerably lower than that in the con -\ntrol group 0.230 ± 0.029, 0.190 ± 0.022, 0.138 ± 0.021 \nfor POCS, tubal factor, unexplained infertility groups \nsequentially and mean average (0.230 ± 0.043) for control \nhealthy group, demonstrating a highly significant correla-\ntion with p value < 0.0001 and < 0.001 (Table 2, Fig. 1).\nConsecutively the expression gene level of IL-11 \nwas remarkably higher in the healthy control group \n0.223 ± 0.04 than in the unexplained infertility group \n0.190 ± 0.09 with a significant result (p  value < 0.05), \nthe second group (tubal factor) also showed a highly \nnotable correlation (p  value < 0.0001) compared to the \ncontrol group (0.167 ± 0.026, 0.223 ± 0.04) sequentially. \nThere was no significant correlation concerning PCOS \ngroup 0.239 ± 0.08 (p value 0.19) (Table 3 , Fig. 1).\nTable 1 Physical parameters of normal controls and infertility subjects (women)\n*p < 0.05 = Significant\nParameters PCOS (n = 25) Tubal factor (n = 25) Unexplained infertility \n(n = 25)\nControl (n = 25) p value\nAge (years) 32.87 ± 7.41 33.98 ± 8.11 32.80 ± 7.10 28.45 ± 6.14 *< 0.05\nBMI (kg/m2) 32.73 ± 2.88 31.73 ± 2.36 31.88 ± 2.99 20.73 ± 2.36 *< 0.05\nWaist/hip ratio (WHR) 0.82 ± 0.06 0.80 ± 0.04 0.81 ± 0.06 0.80 ± 0.07 0.25\nInfertility duration (years) 3.78 ± 1.22 2.80 ± 1.01 4.09 ± 2.11 – –\nTable 2 Mean, standard deviation, and p values of study groups \nfor LIF gene\n*p < 0.05 = Significant\nStudy group (n = 75) Average and \nstandard \ndeviation\nControl p value\nPCOS 0.230 ± 0.029 0.230 ± 0.043 *< 0.0001\nTubal factor 0.190 ± 0.022 *< 0.001\nUnexplained (no reason) 0.138 ± 0.021 *< 0.001\nFig. 1 Mean, standard deviation, and p values of study groups for LIF \nand IL-11 gene\nTable 3 Mean, standard deviation, and p values of study groups \nfor IL-11 gene\n*p < 0.05 = Significant\nStudy group (n = 75) Average and \nstandard \ndeviation\nControl (n = 25) p value\nPCOS 0.239 ± 0.08 0.223 ± 0.04 0.19\nTubal factor 0.167 ± 0.026 *< 0.0001\nUnexplained (no \nreason)\n0.190 ± 0.09 *< 0.05\n\nPage 4 of 5Alzaidi et al. Egypt J Med Hum Genet           (2021) 22:85 \nDiscussion\nThe findings of this research showed that increased BMI \nhad a discernible effect with infertility patient groups \n(PCOS, tubal factor, and unexplained infertility), indicat -\ning that a high level of BMI affects the reproductive func-\ntions through dysregulation of several pathways, which \nincludes androgen receptors, leptin, or even pro-inflam -\nmatory cytokines like interleukins (IL-1), tumor necrosis \nfactor (TNF), cytokine leukemia-inhibitory factor (LIF), \ninterleukin-11 (IL-11), insulin-like growth factor (IGF)-I \nand II, as well as transforming growth factor (TGF)-I and \nII [14].\nIn this clinical study, the relationship of infertility with \ncytokines gene was examined by assessing the mRNA \nexpressions of leukemia-inhibitory factor (LIF) and inter-\nleukin-11 (IL-11), an important factor in the embryo \nimplantation process in women who had primary infer -\ntility related to PCOS, tubal factor and unexplained \ninfertility.\nThe results revealed that LIF gene expression level in \nPCOS patients was considerably lower than that in the \ncontrol group (p < 0.0001); these outcomes were equiva -\nlent to a study by Hussein et al. [15] which revealed that \nany increase or reduction in LIF gene expression levels \nis significantly useful in predicting reproductive out -\ncomes in infertile women with PCOS than for non-PCOS \nfemales.\nThis gene is supposed to be an endometrial receptivity \nindicator, and its mutational expressions may aid in the \nidentification of females who have experienced implanta-\ntion failure [16].\nAdditionally, in comparison with the control fertile \nhealthy group, LIF expression level was significantly \nlower in the second group who are suffering from infer -\ntility related to tubal factor (p < 0.001); this result shows \nthat the occurrence of the blockage in the fallopian tubes \nmay lead to a decrease in the LIF gene expression in the \nluminal epithelium of the fallopian tube, the central place \nfor the process of the pre-implantation embryo [17]. \nAccording to Li et  al. [18] research, the intensity of LIF \ngene expression in the embryonic culture medium could \nbe used as a non-invasive supplementary biomarker for \nclinical pregnancy prediction in infertile females diag -\nnosed with tubal problems, which are undergoing a sin -\ngle blastocyst transfer process.\nThe LIF gene expression also has been detected in a \nmanner of significantly low level in women with unex -\nplained infertility. Otherwise, the fertile control group \nshowed higher levels of LIF expression (p < 0.001). This \nresult suggests that infertile women’s LIF gene expres -\nsion may be dysregulated in both the proliferative and \nsecretory phases, leading to a defect in the endometrial \nLIF activity which can be the main cause of unexplained \ninfertility and recurrent implantation failures. The find -\nings were identical to those of the research of Steck et al. \n[19].\nFurthermore, along with interleukin-11 (IL-11) gene \nexpression, there was no statistical difference between \ninfertility PCOS females and the control fertile group, \nwhile this correlation was discovered and it is linked to \nadipocyte proliferation by Zhuang et al. [20] study. This \ndiscrepancy may be related to different inclusion criteria \nand the impossibility to rule out all factors that influence \nIL-11 gene expression levels.\nHowever, the highly significant different results in \nthe levels of IL-11 gene expression between the control \ngroup and the second patient group (infertility females \nwith tubal factor) reinforce the findings of Cakmak et al. \n[21] in their research, which reported that IL-11 secre -\ntion is destructed by tubal epithelial cells in response to \nChlamydia trachomatis infection, the main cause of tubal \nfactor infertility, which causes extensive destruction of \nthe ciliated cells. It confirms the direct role of IL-11 in \nthe pathogenicity of tubal factor infertility women.\nPatients with the unexplained infertility group also \nrecorded a significant correlation concerning the level \nof the IL-11 gene (p < 0.05). This result hypothesized that \nIL-11 may be dysregulated in the glandular epithelium, \nwhich leads to preventing the facilitation of its secretory \nand prevents the attachment or adhesion of the blasto -\ncyst on the endometrial uterine epithelium, leading to \nthe formation main cause for unexplained infertility [22].\nConclusions\nOur findings show that low levels of LIF and IL-11 gene \nexpression are linked to a variety of primary infertil -\nity conditions, including PCOS, tubal factor, and unex -\nplained infertility, since they play a fundamental role in \nembryo implantation. We note that this was merely a \npreliminary study so that more LIF and IL-11 gene low \nexpression infertile women are required for some further \nresearch. Investigation of the probability of mutations \nwithin IL-11 and LIF genes and their relation to infertility \nare recommended for more confirming results.\nAbbreviations\nACTB: Beta-actin; BMI: Body mass index; EDTA: Ethylenediamine tetraacetic \nacid; IGF-I: Insulin-like growth factor; IL-6, 11: Interleukin-6, 11; LIF: Leukemia \ninhibitory factor; PCOS: Polycystic ovary syndrome; qRT–PCR: Quantitative real-\ntime polymerase chine reaction; TFI: Tubal factor infertility; TGF-I: Transforming \ngrowth factor; TNF: Tumor necrosis factor; UI: Unexplained infertility; WHO: \nWorld Health Organization.\n\nPage 5 of 5\nAlzaidi et al. Egypt J Med Hum Genet           (2021) 22:85 \n \nSupplementary Information\nThe online version contains supplementary material available at https:// doi. \norg/ 10. 1186/ s43042- 021- 00201-9.\nAdditional file 1: Supplementary Table 1. Temperature and times \nprograms for expression in LightCycler 480 II software.\nAdditional file 2: Supplementary Table 2. Primer Sequences.\nAcknowledgements\nThe authors extend their gratitude to all of the contributors.\nAuthors’ contributions\nZA, ŞY, ÇS, HA, IM, and MD contributed to infertility diagnosis, genetic analysis \nsection and interpreting the patient data, regarding hormonal and blood test \nas well as the major contribution in writing the manuscript by ZA, BA, and IŞ. \nAll authors read and approved the final manuscript.\nFunding\nThe authors have not declared a specific grant for this research from any fund-\ning agency in the public, commercial, or not-for-profit sectors.\nAvailability of data and materials\nData are available upon request.\nDeclarations\nEthics approval and consent to participate\nThe study was approved by Erciyes University Hospitals, Medical Genetics \nDepartment, Kayseri, Turkey. Reference No. (ERC-305), all the patients in the \nstudy had been signed for a written consent before the procedure.\nConsent for publication\nNot Applicable.\nCompeting interests\nThe authors did not report any conflict of interest.\nAuthor details\n1 Department of Biotechnology, Faculty of Applied Science, Cukurova Univer-\nsity, Adana, Turkey. 2 The Blood Center of Balcali Hospital, Faculty of Medicine, \nCukurova University, Adana, Turkey. 3 Department of Medical Genetics, Faculty \nof Medicine, Erciyes University, Kayseri, Turkey. 4 Department of Medical \nGenetics, Faculty of Medicine, Kırşehir Ahievran University, Kırşehir, Turkey. \n5 Department of Obstetrics and Gynecology, Erciyes University Medicine \nFaculty, Kayseri, Turkey. \nReceived: 9 July 2021   Accepted: 3 October 2021\nReferences\n 1. Chua SJ, Danhof NA, Mochtar MH et al (2020) Age-related natural fertility \noutcomes in women over 35 years: a systematic review and individual \nparticipant data meta-analysis. 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