{"paper_id":"f2e17d51-931e-4d54-b156-1b0f5e11e4b9","body_text":"Infertility is a common condition which can effect \nmarital relationships, mental health and quality of life of \ncouples ( 1 - 3 ). Recent advances in assisted reproductive \ntechniques (ART), such as in vitro fertilization (IVF) and \nintra cytoplasmic sperm injection (ICSI), have resulted in \ndevelopment of effective methods for treating infertility. \nHowever, these methods are expensive and impose huge \ncosts on families, while a significant number of IVF/ICSI \nprocedures does not result in a live birth ( 2 ,  4 ).\nThe issue of endometrial preparation is largely overlooked\nsince the infertility clinics often focused on the\nprovision of appropriate quality embryos for transmission.\nThe existing relationship between maternal immune\nsystem and embryonic tissues at the time of implantation\nis considered quiet vital for a successful\nimplantation. This fact has been confirmed by one of the\nfirst letters of Betteridge ( 5 ). They discussed about the\nrole of endometrial receptivity in the embryo transfer\nprocess, while indicating that the existence of an accommodation\nbetween the embryo and endometrium is necessary\nfor pregnancy.\nSeveral studies have been conducted to determine the\neffective factors that seemed to be responsible for the\nsuccess of ARTs. One of these factors is the group of cytokines,\nproduced by fetus and uterine mucosa. They are\nresponsible for regulating the interaction between mother\nand fetus, ultimately causing the major influence on improvement\nof uterus receptivity ( 6 ).\nSome of the certain known cytokines and growth factors \nthat may contribute to increasing endometrial receptivity \ninclude interleukin-1 (IL-1), tumor necrosis factor-alpha\n(TNF-α), leukemia inhibitory factor (LIF), and transforming \ngrowth factor-beta (TGF-ß) ( 7 ). For the first time, in \n2003, a novel approach on the study of cytokines was presented \nby van der Gaast et al. ( 8 ), evaluating cytokines by \nanalyzing the endometrial secretions. In fact, the endometrial \ntissue itself is not ideal for assessing biomarkers, \ndue to the complexity of cell, its differentiation between \ndifferent individuals and even different stages of the cycle. \nFurthermore, the uterine fluid collection, by either \nlavage or aspiration, is less invasive than tissue biopsy \nand ultimately the observed changes in this fluid can be \nsign of a true microenvironment for implantation. These \ndiscoveries have been discussed in detail through a study \npublished by Salamonsen et al. ( 6 ).\nGiven the prevalence of infertility, high cost of fertility \ntreatments and crucial role of cytokines in the success \nof these methods, the importance of identifying these cytokines \nand determining the best time for performing the \nprocess of embryo transfer is quiet evident. Therefore, \nin this study, we have determined the levels of IL-1 and \nTNF-α in endometrial secretion and assessed their roles in \nthe success of embryo implantation.\n\nThis prospective study has been conducted in the Infertility \nCenter of Milad (Mashhad, Iran) from August \nto December 2017. Subsequent to performing sufficient \nexplanation and signing the informed consent, 79 \nwomen enrolled the experiment, with the mean age of \n32 years. They were candidates of obtaining their first \nIVF/ICSI due to tubal factor infertility caused by tubal \nobstruction (evidence of distal tubal obstruction in \nhysterosalpingography (HSG) was confirmed by laparoscopy).\nThe Research and Ethics Committee of Mashhad \nUniversity of Medical Sciences (Iran) (IR.MUMS.\nfm.REC.1395.329) approved the study protocol. To determine \nthe factors that are effective on the outcome of \nIVF, all subjects were provided with the following criteria \nfor enrollment in this study: normal menstrual cycles \nbetween 21 and 35 days, less than 40 years of age, BMI \nless than 30, TSH values less than 10, FSH values less \nthan 10 in the third day of the cycle and antral follicular \ncount (AFC) with at least 5-7 in each ovary on the third \nday of cycle using the vaginal sonography, normal sperm \nanalysis.\nThe exclusion criteria were also included: endocrine or \nmetabolic disorders, history of previous pelvic or gynecological \nsurgery (endometriosis, etc.). One or more than \none occurrence of previous IVF failure, cigarette smoking, \nrecurrent abortion, OHSS, inappropriate endometrium \nfor embryo transfer (echogenic/non triple line and less \nthan 7 mm), evidence of drosalpinx and uterine anomalies \nin HSG as well as transvaginal sonography (TVS) and \nother infertility causes.\nTo stimulate ovulation based on antagonist protocol, on \nthe 3 rd  day of menstrual cycle, we have subcutaneously \ninjected recombinant-follicle stimulating hormone (FSH) \n(Gonal-F) with a dosage of 150-225 IU/daily, depending \non the AFC of each person. During serial vaginal sonography \n(using PHILIPS, Affinifi 70W device, Netherlands), \nafter observing at least two follicles above 17 mm and follicular \ncohorts of 14-16 mm, 10000 U of urinary-human \nchorionic gonadotropin (hCG) were intramuscularly (IM) \ninjected to induce the final oocyte maturation. Thirty six \nhours after hCG injection, we have performed the oocyte \npick up process.\nThe luteal phase support was started from the day of \npick-up by injection of 50 mg progesterone daily/IM. On \nthe 4 th  day of progesterone, depending on the embryo grading, \nwe transferred one to three cleavage embryo for each \npatient through the employment of one infertility specialist, \nutilizing cook catheter under transabdominal guide. \nThe embryo grades were classified as follow: i. Embryo \nwith the stage-specific cell size, <10% fragmentation and \nno multi-nucleation, ii. Embryo with stage-specific cell \nsize for the majority of cells, 10-25% fragmentation and \nno evidence of multi-nucleation, and iii. Embryo with not \nstage- specific cell size, severe fragmentation (25%) and \nevidence of multi-nucleation ( 9 ) and the individuals conditions \n(such as the patients age).\nThe level of serum ß-hCG was checked 14 days after performance \nof embryo transfer. Upon observing a positive result \nand an appropriate increase in the titer within 48 hours \n(confirmation of successful implantation), the patients were \nsubjected to vaginal sonography between 4 and 5 weeks \nafter embryo transfer, to confirm the clinical pregnancy by \ndetecting the gestational sac and fetal heart rate.\nBefore beginning the pick-up and after washing the perineum \nand vagina with normal saline, we exposed the cervix \nthrough the utilization of a speculum. Subsequent to \nwashing the cervix with normal saline, a mannered catheter \nwas employed to administer 2-3 ml of normal saline \ninto the uterine cavity, using a 2-cc syringe. After 30 seconds \nof fluid infusion, the fluid was suctioned and transferred \ninto a microtubule. The specimen was inscribed \non the micro tube and stored in liquid nitrogen at a temperature \nof 80°C. This process was completely performed \non all of the 76 samples. After collecting the 76 samples \nwithin five months, standard kits (IBL, USA) were used \nto measure IL-1 and TNF-α biomarkers by ELISA method. \nIt should be noted that the safety of this method has \nbeen discussed and approved in previous studies.\nThe results of this study were collected in a coherent \nmanner. After completing the statistical data of the in\nvolved subjects, we performed the statistical analysis \nthrough application of SPSSII, version 23 and separately \nbased on chemical pregnancy (positive serum ß-hCG was \nchecked 14 days after embryo transfer and successful implantation \nwas confirmed by the appropriate increase) and \nclinical pregnancy (observing gestational sac and fetal \nheart rate (FHR) by TVS, 4-5 weeks after embryo transfer). \nIn this experiment, P<0.05 was considered statistically \nsignificant.\n\nThese 79 candidate women for their first IVF/ICSI \nwere enrolled based on the provided criteria. Three \ncases were excluded, one due to the occurrence of \nOHSS while the other two contained inappropriate \nendometrium, followed by freezing their embryos. \nAs the last step, the aspirated endometrial secretions \nof the enrolled candidates were evaluated for IL-1 \nand TNF-α mean levels by ELISA method. Findings \nshowed among the 76 patients, 33 of them carried positive \nß-hCG (chemical pregnancy) and 43 candidates \nhad negative ß-hCG. Based on positive FHR in TVS \n(clinical pregnancy) throughout the 76 patients, 23 of \nthem have shown FHR positive while 10 of the have \nresulted in FHR negative.\nThere was not any significant statistical difference between \nthese two groups in demographic characteristics \nincluding age, body mass index (BMI), duration of infertility, \nAFC and 3 rd  day FSH as well as number and grade \nof transferred embryos, which have been mentioned in \ndetails in Table 1.\nBaseline and clinical characterization of pregnant and non-pregnant groups\nData are presented as mean ± SD or n (%). a; Embryo with stage-specific cell size, <10% \nfragmentation and no multi-nucleation, b; Embryo with stage-specific cell size for the majority \nof cells, 10-25% fragmentation and no evidence of multi-nucleation, ß-hCG; Beta-\nhuman chorionic gonadotropin, BMI; Body mass index, FSH; Follicular stimulating hormone, \nand AFC; Antral follicular count.\nIn terms of IL-1, chemical pregnancy (positive ß-hCG) \ngroup significantly carrier higher level than that of the \nnegative ß-hCG group (P=0.000,  Table 2 ). Although \nthere were higher levels of IL-1 in FHR positive group \nin terms of clinical pregnancy (observing FHR in TVS), \nyet the difference has not been statistically notable \n(P=0.06,  Table 3 ).\nComparison of IL-1β and TNF-α levels in aspirated endometrial secretions in patients with positive and negative value of chemical pregnancy\nIL-1ß; Interleukin-1 beta, TNF-α; Tumor necrosis factor-alpha, and ß-hCG; Beta-human \nchorionic gonadotropin.\nComparison of IL-1 and TNF-α levels in aspirated endometrial secretions in patients with positive and negative value of clinical pregnancy\nIL-1ß; Interleukin-1 beta, TNF-α; Tumor necrosis factor-alpha, and FHR; Fetal heart rate.\n\nSeveral studies have confirmed the role of interaction \nbetween embryo-endometrium and cytokines in implantation. \nTo be stated as an example, in a study published \nby the Journal of Reproductive Biology in 2009, Haouzi \net al. ( 10 ) reported that successful implantation of fetus \nis highly dependent on the fetus quality and endometrial \nreception.\nNieuwenhuizen et al. ( 11 ) and Lieberman et al. ( 12 ) \nmentioned in their reviews that cytokines, which are produced \nby fetuses and mucous membranes, are responsible \nfor improving the endometrial receptivity. It has also been \nnoted in a study by Zhou et al. ( 13 ), that IL-1 stands as an \nimportant factor through interaction between embryo and \nendometrium, as a functional factor during implantation \nin both maternal and fetal sites. This particular review \nwas confirmed by other studies, such as Bourdiec and Akoum \n( 14 ), pointing out the effective role of IL-1 through \nthe success of embryo implantation process.\nIn accordance with the study done by Sequeira et al. \n( 15 ) the levels of IL-1 in maternal serum levels and median \nculture of developing embryos were significantly \nhigher in women with successful implantation. Moreover, \na study by Loetscher et al. ( 16 ) has stated that TNF-α was \nat a very high level in people with a history of recurrent \nabortion and infertility.\nAside from this fact, Reid et al. ( 17 ) has also discovered \nthat TNF-α is apparently associated with infertility \nand recurrent abortion, which can be considered as a confirmation \nof Clark’s study. However, in contrast to Clark \nand Reid studies, a review performed by Boomsma et al. \n( 18 ) from Netherlands in 2009, exhibited the existence of \na positive correlation between successful pregnancy and \nhigher levels of TNF-α and lower levels of IL-1 in endometrial \nsecretions.\nSimilar to the present work, Rahiminejad et al. ( 1 ), \nassessed the levels of IL-1 and TNF-α in the endometrial \nfluid along with their effects on implantation \nsuccess. They concluded that lower levels of TNF-α \nin endometrial secretions results in the improvement \nof endometrial reception. However, no significant difference \nbetween IL-1 of the two groups was observed \nin terms of increasing chance of performing successful \nimplantation.\nAt the end, next to the contradictions that are theoretically \nrelated to the relationship between the levels of \nthese cytokines and successful outcome of pregnancy, \nvarious limitations such as technical differences in the \naspiration-discharge procedure, aspiration scheduling, \nlow sample size, the leading cause of tubal factor and \nthe confounding effect of drugs could be the cause of \nthe existing differences between the obtained results of \ndifferent studies and the present investigation. In fact, \nevaluating the endometrial receptivity probably stands \nas one of the next steps that will be taken in infertility \nclinics. This particular test can provide clear information \nabout the inferiority of endometrial receptivity as a \nprimary cause of infertility and ultimately, contribute to \nthe probable prediction of embryo transfer outcome in \nIVF/ICSI cycles.\nThe present study has concluded that there is not any \nsignificant statistical relationship between higher levels of \nIL-1 in endometrial secretion and successful implantation \nin IVF/ICSI cycles. Furthermore, with regards to the case \nof TNF-α, we have not discovered any statistical significant \ndifference between the two groups with successful \nand unsuccessful implantation.\nWe believe that it would be quiet useful if researchers \ninvestigate some other categories, such as association \nbetween cytokine levels and ongoing pregnancy rate or \neven live birth rate, in addition to the relationship between \ncytokine levels and successful implantation with any specific \ninfertility etiology.\n\nThis study suggests that higher level of IL-1 in endometrial \nsecretions may associate with improved endometrial \nreceptivity and subsequently, this can be related to the \nimproved IVF/ICSI outcomes. In fact, this noninvasive \nmethod can enhance the understanding of immunological \nevents, which are involved in the implantation process of \nfetus.","source_license":"CC-BY-4.0","license_restricted":false}