{"paper_id":"5b8fc541-965d-4fb7-9c3b-e37e5bcf650f","body_text":"Assisted reproductive technologies (ART) are not only\nused to overcome fertility issues in infertile cases but\nare also key tools in preventing genetic abnormalities\nin fertile couples. However, the efficacy of ARTs is\nstill suboptimal and the low rates of transferred embryo\nimplantation in ART cycles remains the main challenge\nfor achieving successful pregnancies. This limitation\npartly results from inadequate knowledge about the\ncellular and molecular basis of germ cells, embryos, and\nendometrium physiology ( 1 ). Despite recent advances in\nembryo development, selection, and transfer techniques,\nimplantation failure occurring in approximately 75% of\ncases is a major limiting factor for pregnancy following\n in vitro  fertilization (IVF) attempts ( 2 ). The receptive\nendometrium is one of the most important factors for\nthe outcome of pregnancies following ART cycles\nand optimizing endometrial receptivity is imperative\nto improving the success rate of ART. During the\nimplantation window, a unique timeframe in which\nimplantation is possible, the endometrium plays a crucial\nrole in successful implantation ( 3 ).\nEndometrial receptivity and subsequent embryo\nimplantation can only happen after a complex series\nof histological, cellular and molecular changes in the\nendometrium. It has been shown that successful embryo\nimplantation depends on an ideal endometrium-embryo\ncross-talk through the known crucial growth factors and\ncytokines which are secreted from endometrial cells ( 4 ).\nPrevious studies have indicated that differential\nexpression of a variety of genes including those involved\nin immune response, the complement cascade pathway,\ncell adhesion and exosome biogenesis may influence endometrial receptivity ( 5 ). Hence, in the past decade,\nmany global transcriptomic studies have been designed\nto find potential biomarkers or molecular signatures\nfor a receptive endometrium. There are, however,\nmany disagreeing reports due to differences in the\nanalysis of gene expression and sample selection ( 6 ,  7 ).\nMoreover, different studies have suggested that the use\nof supplements such as vitamins, hormones and minerals\nmay improve endometrial receptivity and increase the\nchance of implantation in ART procedures ( 8 ,  9 ) .\nDue to the complex nature of endometrial receptivity,\nit seems the use of a cocktail of supplements can be\nmore effective than individual supplements. Seminal\nplasma (SP) and Follicular fluid (FF) can be considered\nas cocktails of various natural biocompounds. SP is\na reach medium comprised of different biologically\nactive factors including cytokines, chemokines,\nprostaglandins, growth factors, angiogenic factors,\nvitamins, zinc, etc ( 10 ). According to recent proteomic\nstudies, high concentrations of these cytokines and\nprostaglandins in the SP of fertile men and aberrant\nconcentrations of these components in the SP of\ninfertile men demonstrate that SP constituents may\nplay a key role in human reproduction. Furthermore,\nmost of these important compounds which have been\nidentified in the female reproductive tract (FRT)\nsuggest their involvement in the regulation of FRT\nfunctions and successful reproduction ( 11 ). From\nthe mechanistic point of view, different studies in\nmice, pigs and humans have shown that SP may\nplay a critical role in endometrial receptivity and the\nimprovement of implantation chances ( 12 ,  13 ). In\naddition to SP, FF which provides critical factors for\noocyte development, contains important cytokines,\nhormones and growth factors that may mediate\nparacrine/autocrine interactions during the process of\nimplantation ( 14 ).\nDuring ART cycles such as IVF or ICSI, unlike\nwhat happens naturally in the body of mammals, the\nblastocyst is transferred without any SP and the FF is\nalso discarded during ovum pick-up. As these fluids\ncontain a vast range of natural elements including\ngrowth factors that possibly improve receptivity of the\nendometrium, we hypothesize that using the blastocyst\nalone might cause limited implantation rates after ART\ncycles. We evaluated this hypothesis through the study\nof the  in vitro  influence of SP and FF on the expression\nlevels of the genes  HOXA10, HOXA11, LIF, ITGB3, \nand  ITGAV , whose role in the successful implantation\nof blastocysts have been established, in endometrial\nstromal cells.\n\nIn this experimental study, twelve fresh endometrial\ntissue samples were collected from childbearing-age\nwomen (ages 23-35) undergoing hysterectomy for\nbenign conditions. Six of the participants were receiving\nhysterectomies for fibroids, three for adenomyosis, one\nfor uterine prolapse and one for heavy menstrual bleeding.\nAll of them were in the secretory phase of their menstrual\ncycle. Malignancy, drug and hormone therapy, and\npregnancy were the exclusion criteria for the participants\nin our study. Written informed consent was obtained from\nall participants and the study was approved by the Ethics\nCommittee of Tehran University of Medical Sciences (IR.\nTUMS.MEDICINE.REC.1396.4258). Immediately after\nsampling, the endometrium specimens were placed in\nHank’s Balanced Salt Solution (HBSS, Sigma-Aldrich,\nUSA) with 1% penicillin/streptomycin (Pen/Strep, Gibco,\nUSA) and transported to the laboratory within two hours.\nTissue samples were then transferred to sterile 10 cm petri\ndishes and rinsed with phosphate buffered saline (PBS,\nMerck, Germany). After washing, the tissue samples were\ntransferred to another petri dish containing pre-warmed\nHBSS and were cut into small pieces with a sterile scalpel.\nThe dissected specimens were transferred into sterile 15\nml tubes containing HBSS and 3 mg/ml collagenase type\nI (Sigma-Aldrich, USA) for enzymatic digestion. After\n60 minutes of incubation at 37˚C, the solutions were resuspended in Dulbecco’s Modified Eagle Medium and\nHam’s F-12 (DMEM/F12, Gibco, USA) supplemented\nwith 10% fetal bovine serum (FBS, Gibco, USA) for\nenzyme neutralization. Following complete tissue\ndigestion, cell suspensions were filtered through 70\nmicron cell strainers to remove the undigested fragments\nfrom the suspension. Afterwards, the cell suspensions\nwere filtered through a 40 micron cell strainer to isolate\nthe endometrial stromal cells from endometrial epithelial\ncells.\nFor further purification and removal of red blood cells\n(RBCs), fresh DMEM/F12 culture medium supplemented\nwith 10% FBS was added to the collected cells and\ncentrifuged at 1500 rpm for 10 minutes. After removing\nthe supernatant, the cell pellet was resuspended and\nadded to Ficoll-Paque media solution in 15 ml tubes, then\ncentrifuged for 20 minutes at 1200 rpm. Afterward, the\nupper layer containing stromal cells was transferred to\na new tube and washed twice with PBS ( 15 ) including\nosteocytes and adipocytes. Here, the potency of EnSC\nin neural differentiation has been investigated. Flow\ncytometric analysis showed that they were positive for\nCD90, CD105, OCT4, CD44 and negative for CD31,\nCD34, CD133. The characterized cells were induced into\nneural differentiation by bFGF (basic fibroblast growth\nfactor\nThe stromal cells were transferred to T 25  culture flasks\ncontaining DMEM/F12, 10% FBS and 1% Pen/Strep and\nwere incubated at 37˚C and 5% CO 2 . After 24 hours of\nincubation, the culture medium and nonadherent cells\nwere discarded and the attached cells were washed twice\nwith PBS, then fresh culture medium was added and\nincubated at 37˚C and 5% CO 2 . The medium was changed every 3 days until passage 3. Cells of passage 3 were used\nfor the experiments\nFibrin gel was used to provide a three-dimensional\nmatrix for culturing endometrial stromal cells. The 3D\ncultures of for MTT (3-( 4 , 5 -Dimethylthiazol-2-yl)-\n2,5-diphenyltetrazolium bromide) assays were carried\nout in 96-well culture plates while other cultures took\nplace in 24-well plates for optimized treatment. Fibrin\ngel, were produced by dissolving 3 mg of fibrinogen\n(Sigma, USA) in 1 ml of M199 medium (Sigma,\nUSA). Stromal cells (2×10 5  cells/ml) were added to the\nprepared fibrinogen solution and carefully mixed with\n2 μl of a thrombin solution (120 U/ml in 1 M sodium\nbuffer, Sigma, USA), 1.5 μl of CaCl 2  (1%). Then\n100μl of cell containing fibrinogen medium was added\nto each well of a 96-well cell culture plate. The plate\nwas incubated at 37˚C for 1-2 hours to form a threedimensional structure. Following fibrin gel formation,\n0.1 ml of DMEM/F12 medium supplemented with\n10% FBS was added to each well and the plate was\nreturned to the 37˚C incubator. The culture medium\nwas refreshed every 3 days following a previously\npublished protocol ( 16 ).\nSemen samples were collected from 15 fertile\ndonors, aged 27-41 years old (mean age of 34), with\nnormal spermogram from the Reproductive Health\nCenter of Tabriz Alzahra Hospital. The samples were\ncentrifuged at 3000 rpm for 20 minutes. Supernatants\nwere collected and centrifuged again at 10000 rpm\nfor 15 minutes in order to remove. The supernatants\nwere pooled, filtered through 0.22 μm filters for the\nprevention of microbial contamination and stored at\n-20˚C until used.\nFF was obtained by puncturing ovarian follicles\nfrom 15 ovum donors, aged 23-32 (mean age of 27), at\nReproductive Health Center of Tabriz Alzahra Hospital.\nMacroscopically clear FF samples were centrifuged at\n6000 rpm for 20 minutes to remove cellular components.\nThe supernatants were pooled, filtered through 0.22 μm\nfilters and stored at -20˚C until used.\nThe MTT assay was used to determine the nontoxic doses and effects of semen plasma and FF on\nviability of the fibrin gel-encapsulated endometrial\nstromal cells. The MTT assay is a reliable colorimetric\nreaction, which is widely used to measure cell viability\nand cytotoxicity. The principle of this assay is the\nreduction of MTT dye to formazan crystals by the\nmitochondrial dehydrogenases of viable cells. There\nis a linear correlation between the amount of formazan\nand cell viability. So, determination of formazan\nquantity can be used as an estimate of the population\nof living cells.\nA total of 10 4  endometrial stromal cells isolated\nfrom three different samples were encapsulated in\nfibrin gel (3 mg/ml) in separate wells of 96-well plate.\nDifferent concentrations of FF and semen plasma (1%,\n5%, 10%, 20%, 50% and 100%) were added to each\nwell as treatment groups. These concentrations were\nachieved through dilution with DMEM/F12 culture\nmedium containing 2% FBS. The control group was\nendometrial stromal cells encapsulated in fibrin gel\nwithout any treatments.\nAfter treatment with SP for 3, 6, 24, 48 and 72 hours\nor FF for 3, 6, 24, 48 72, and 96 hours, the culture\nmedia were removed and 100 µl of MTT solution (0.5\nmg/ml in PBS, Sigma, USA) was added to each well\nand incubated at 37˚C for 4 hours. Afterward, 100 µl\nof dimethyl sulfoxide (DMSO, Sigma Aldrich, USA)\nwas added to each well and incubated for 15 minutes\nin a dark room at room temperature to dissolve the\nformazan crystals. Finally, absorbance was measured\nat 570 nm using a spectrophotometric plate reader Asys\nExpert 96 (Biochrom, UK) according to established\nguidelines. These experiments were repeated five\ntimes. Cell survival was calculated as the percentage\nof test absorbance compared to the control absorbance.\nTotal RNA was extracted from cultured endometrial\nstromal cells using TriPure Isolation Reagent (Roche,\nSwitzerland) according to the manufacturer’s instructions.\nConcentration and purity of extracted RNA samples were\nchecked with a NanoDrop 2000C spectrometer (Thermo\nScientific, USA) and RNA integrity was evaluated by\nrunning 1 µl of total RNA samples through 1% agarose\ngel. A total 1 μg of extracted RNA was retrotranscribed to\ncomplementary DNA (cDNA) with random hexamer and\noligo dT primers, using a cDNA synthesis kit (Takara,\nJapan). The final cDNA product was used as the template\nfor qRT-PCR.\nQuantitative real-time PCR was done in duplicates on a\nlight cycler 96 real-time PCR system (Roche, Switzerland).\nThe qRT-PCR reactions were run with the following\nsettings: 95˚C for 10 minutes, followed by 45 cycles of\n95˚C for 10 seconds and 60˚C for the 30 seconds. At\nthe end of each qRT-PCR run, the melting curve program\nwas run to make sure the PCR product’s specificity. Each\nqRT-PCR reaction contained 10 µl RealQ Plus Master Mix\nGreen (Ampliqon- Denmark), 1 µl (100 ng/µl) of cDNA,\n1µl mixed forward and reverse primers and 8 µl ddH 2 O.\nExpression levels of the target genes were evaluated by\nnormalizing to the expression of the  GAPDH  gene as the\nreference gene. The relative expression levels of target\ntranscripts were calculated through the 2 −ΔΔCt  method.\nPrimer sequences and characteristics are summarized in\nTable 1.\nPrimer sequences used for quantitative real-time polymerase chain reaction\nThe obtained data were analyzed statistically using\nthe SPSS 20.0 software package (SPSS, Chicago, IL,\nUSA). Paired t test was applied to assess statistical\nsignificance differences as appropriate. For all\nstatistical analyses, a P<0.05 was considered to be\nstatistically significant.\n\nBefore analyzing of the effects of semen plasma and\nFF on the expression levels of receptivity genes in\nendometrial stromal cells, we determined the optimum\nFF and SP concentrations and treatment time that had\nno dose- or time-dependent cytotoxic effects on the\n3D- cultured endometrial stromal cells obtained from 3\ndifferent women.\nAs shown in Figures  1  and  2 , respectively, we found that a\n72-hour incubation with 20% FF and a 48-hour incubation\nwith 10% SP resulted in the highest proliferation rate and\nhad no cytotoxic effects on the cultured cells. Therefore,\nthese non-toxic conditions were chosen for subsequent\nqRT-PCR analyses.\nTo evaluate the capacity of SP and FF, to modulate\nexpression of endometrial receptivity genes, cultured\nendometrial stromal cells were exposed to either vehicle\ncontrol medium alone, 10% SP for 48 hours or 20% FF\nfor 72 hours. mRNA expression levels of  LIF, ITGB3,\nITGAV, HOXA11,  and  HOXA10  were analyzed with qRTPCR.\nThe exposure of endometrial stromal cells to FF\nresulted in elevated expression of  HOXA10  (fold\nchange=2.6, P=0.02),  HOXA11  (fold change=3.3,\nP=0.002),  LIF  (fold change=4.6, P=0.0003), ITGB3\n(fold change=3.5, P=0.012) and  ITGAV  (fold\nchange=2.8, P=0.001) compared to vehicle control\nmedium alone ( Fig.3 ).\nIn addition, we found that in SP treated endometrial\nstromal cells, only the mRNA levels of the  LIF  gene was\nincreased (fold change=2.5, P=0.008) compared with\nvehicle control medium while the expression of  ITGB3,\nITGAV, HOXA11,  and  HOXA10  did not change ( Fig.4 ).\nAnalysis of the dose- and time-dependent effect of follicular fluid (FF) on the viability of endometrial stromal cells using the MTT assay. The\nendometrial cells were exposed to different concentrations of FF (0, 1, 5, 10, 20, 50 and 100%) for 3, 6, 24, 48, 72 and 96 hours. The numbers on the bars\nshow mean absorbance at 570 nm.\nAnalysis of the dose- and time-dependent effect of follicular fluid (FF) on the viability of endometrial stromal cells using the MTT assay. The\nendometrial cells were exposed to different concentrations of FF (0, 1, 5, 10, 20, 50 and 100%) for 3, 6, 24, 48, 72 and 96 hours. The numbers on the bars\nshow mean absorbance at 570 nm.\nThe relative mRNA expression levels of endometrial receptivity genes following follicular fluid (FF) treatment. The line in each box indicates the mean\nexpression level and bars represent confidence interval (CI) 95%. Gene expression levels are indicated in Log scales. *; P≤0.05, **; P≤0.01, and ***; P≤0.001.\nThe relative mRNA expression levels of endometrial receptivity genes following follicular fluid (FF) treatment. The line in each box indicates the mean\nexpression level and bars represent confidence interval (CI) 95%. Gene expression levels are indicated in Log scales. *; P≤0.05, **; P≤0.01, and ***; P≤0.001.\n\nDuring the past two decades, ART has become a key\nmedical procedure to help infertile women achieve\npregnancy. However, embryo implantation failure in ART\ncycles remains the main obstacle for achieving a successful\npregnancy. The implantation of the blastocyst is a complex\nprocess involving reciprocal communication between the\nembryo and the uterus, which is mainly dependent on the\nfunction and receptivity of the endometrium. It is thought\nthat suboptimal endometrial receptivity is the reason for\ntwo-thirds of implantation failures. Thus, different clinical\nstrategies have been employed to improve implantation\nrates following ART cycles ( 17 ).\nMany different histological, biochemical, and\nmolecular genetics studies have been conducted with\nthe aim of defining endometrial receptivity markers.\nIdentification of these markers can be very useful in using\nthem in diagnostic setting and improving ART methods.\nOf these markers, molecular markers are of a great\nimportance due to their high sensitivity and specificity\n( 7 ). That is why many transcriptomic and proteomic\ninvestigations have been performed in the past decade to\nfind reliable molecular markers that reflect the level of\nthe endometrium’s receptivity ( 18 ). Discovery of these\nmarkers and the key molecular pathways involved in the\nimplantation process can facilitate improvement of the\nendometrium receptivity methods ( 19 ). However, due to\nconsiderable differences among the results of these studies,\nthere is no consensus on the genes that can be used as\nbiomarkers in clinical diagnostic tests for determining of\nthe level of endometrial receptivity ( 7 ). Nevertheless, the\ncentral roles of some gene families and signaling pathways\nthat are involved in implantation have been established\nin endometrial cells. These gene families are mainly\ngrowth factors, cytokines, chemokines and cell adhesion\nmolecules. The fact that a variety of molecular mechanisms\nsuch as disrupted growth factor, cytokine, and hormone\nsignaling are thought to be involved in the suppression of\nendometrial receptivity biomolecules that lead to a reduced\nimplantation rate further confirms this ( 20 ).\nIt therefore seems that one main approach for\nenhancing endometrial receptivity can be exposure of the\nendometrium to a biological cocktail containing various\nbiologically active factors. It is believed that human semen\nplasma and FF naturally contain the critical signaling\ncomponents that have major functions in the process of\nimplantation. Furthermore, recent studies have indicated\nthat SP and FF which can enter the uterine cavity during\nsexual intercourse ovulation respectively, may affect and\nregulate endometrial signaling mechanisms to induce\nimplantation and increase reproductive success ( 21 ).\nSeveral studies have assessed the  in vitro  and  in vivo \neffects of SP and FF on the functions and gene expression\nof the endometrium. Chen et al. ( 22 ) examined the  in vitro \neffects of SP on the transcriptome of human endometrial\ncells. Their results indicated that SP exposure leads to upregulation\nof genes involved in proliferation, viability,\nand migration in the endometrial stromal cells. Gutsche et\nal. ( 23 ) indicated that SP has an  in vitro  stimulatory effect\non the expression levels of pro-inflammatory cytokines\nincluding IL-1b, IL-6 and, LIF in human endometrial\ncells. In agreement with the previous studies, a recent\nstudy by Rodriguez-Caro et al. ( 24 ) has proved that  in\nvitro  interaction between SP extracellular vesicles and\nendometrial stromal cells may improve endometrial\nreceptivity by inducing prolactin secretion, which is a key\nhormone in implantation, and enhancing decidualization.\nA recent systematic review and meta-analysis conducted\nby Saccone et al. ( 11 ) indicated a higher clinical pregnancy\nrate after intra-vaginal/cervical injection of SP at the time\nof oocyte pickup suggesting that SP plays an important\nrole on endometrial function and the maternal immune\nsystem, and thereby supports implantation. A prospective\nrandomized study by Hashish et al. ( 25 ) showed that\nflushing of the endometrial cavity with FF in patients\nundergoing intracytoplasmic sperm injection (ICSI) did\nnot significantly improve implantation rates. However, FF\nis rich in growth factors and its potential for increasing the\nrate of implantation and subsequent successful pregnancy\nshould be the focus of further research.\nTo address these challenges, in the current study,\nwe determined the  in vitro  influence of FF and SP on\nthe expression levels of main endometrial receptivity\ngenes with established roles in the implantation process\nincluding  HOXA10, HOXA11, ITGAV, ITGB3  and  LIF , in\nendometrial stromal cells\nWe have applied a fibrin three-dimensional cell culture\nsystem for endometrial stromal cells culture to provide\ninformation that is more physiologically relevant to\ncells  in vivo . This polymer has recently become widely\nused in tissue engineering. Fibrin gel is naturally formed\nin the body, through the natural process of fibrinogen\nmixing with thrombin ( 26 ).\nWe first ensured that the response to FF or SP was\nnot due to their cytotoxic effects by performing precise\ncytotoxicity assays. We found that a 48 hours incubation\nwith 10% SP and a 72 hours incubation with 20% FF\nshowed they had no dose- or time-dependent cytotoxic\neffects on the endometrial stromal cells and interestingly,\nthe maximum viability for cells was seen in these\nconditions.\nExposure of endometrial stromal cells to FF resulted in\nelevated expression of all of the analyzed genes including\nan increase of 2.6 fold in  HOXA10 , 3.3 fold in  HOXA11 ,\n4.6 fold in  LIF , 3.5 fold in  ITGB3 \nand 2.8 fold in  ITGAV \nexpression levels. In addition, we found that SP-treated\nendometrial cells only showed an increase of 2.5 fold in\nthe mRNA levels of the  LIF  gene compared to untreated\ncontrols.\nIn this study, we selected genes whose essential\nrole in fetus implantation has been proven by different\nstudies, and are involved in the key signaling pathways.\n HOXA10  and  HOXA11  are two important members of\nthe homeobox gene family, and encode very important\ntranscription factors. It has been proven that expression\nof  HOXA10 , a member of a family of homeobox\ngenes, is critical for FRT development and endometrial\nreceptivity. Moreover, up-regulation of  HOXA10  at the\ntime of implantation improves endometrial receptivity\nby regulating downstream genes such as ITGB3 ( 27 ). A\nsupporting study, Wang et al. ( 28 ) reported that 5-Aza-\n20-deoxycytidine (AZA) might improve endometrial\nreceptivity through the induction of  HOXA10  expression.\nLike  HOXA10, HOXA11  expression plays an\nimportant role in implantation and the down regulation\nof this gene leads to female infertility. Therefore, upregulation of\nboth  HOXA10  and  HOXA11  in the receptive\nendometrium indicate that these genes play important\nroles in decidualization. Women with abnormal expression\nof  HOXA10  and  HOXA11  genes show lower rates of\nimplantation indicating that these genes are important\nfor blastocyst implantation because they regulate the\nexpression of molecular and cellular markers needed for\nembryo implantation ( 29 ).\nITGAV  and  ITGB3  encode essential cell adhesion\nmolecules.  ITGAV , a member of the integrin gene\nfamily that encodes integrin αv, heterodimerizes with\nthe integrin β3 chain that leads to improvement of\nangiogenesis and embryo attachment. Accordingly,\nderegulation of  ITGAV  gene has been reported in a variety\nof reproductive disorders suggesting its essential role in\nthe human reproduction processes. It is reported that\nthe expression of αvβ3 integrin, a cell surface adhesion\nmolecule, was elevated during implantation in humans\nand its endometrial expression was reduced in infertile\nwomen suggesting that it is important for the process of\nimplantation ( 30 ).\nThe last gene,  LIF , encodes a multi-functional cytokine\nthat plays a key role in the implantation process.  LIF ,\nbelongs to the IL-6 family and plays a clear role in\nthe process of implantation by regulating a variety of\nbiological processes during blastocyst implantation\n( 31 ). The absence of  Lif  in mice reduces blastocyst\nimplantation. A recent study by Shokrzadeh et al. ( 32 )\nindicated that administration of calcitonin during the\nimplantation window improves endometrial receptivity\nin mice by up-regulating  LIF  and Le‐7a miRNAs and\ndown-regulating that of Muc‐1. Increased expression\nof endometrial  LIF  in the secretory phase is essential\nfor implantation in humans, because multiple critical\nevents during implantation are regulated by  LIF  such as\npromoting the endometrial receptive state, endometrialembryo interaction, decidualization of stromal cells, and\ndevelopment of the blastocyst. In addition, leukemia\ninhibitory factor-mediated adhesion molecules require\nintegrin αvβ3 and αvβ5 for the adhesion of trophoblast cells\nto endometrial cells. Gremlich et al. ( 33 ) showed in their\nstudy that IVF patients with lower plasma concentrations\nof  LIF  had an increased risk of implantation failure.\nMost previous studies have only investigated the\nbiochemical compounds of FF and its relation with\nthe quality of the ovum. In contrast, the present study\ninvestigated the effect of FF on endometrial cells and\nit was observed that FF induces the expression of the\ngenes that are involved in Embryo implantation. FF is\nrich in steroidal hormones ( 34 ), especially estrogen and\nprogesterone, and previous studies have shown that the\nexpression level of  HOXA10  and  HOXA11  in endometrial\ncells are upregulated by these hormones ( 35 ). It seems\nthat these steroidal hormones have induced the expression\nof the  HOX  genes that, in turn, have functioned as\ntranscription factors leading to the upregulation of  LIF \nin the endometrial cells. Many related studies have\nsuggested  LIF  as a positive regulator of integrins αv\nand β3. Therefore,  LIF  is a key regulator of the fetus\nimplantation, as it has in turn induced the expression of\nthe adhesion molecules  ITGAV  and  ITGB3  ( 36 ).\nUnlike the study of Hashish et al. ( 25 ) that used FF\nfrom a single mature oocyte, we used pooled FF obtained\nfrom 15 mature follicles to treat the endometrial stromal\ncells. Our results showed a higher expression level of\nendometrial receptivity genes suggesting that FF can be\nan inexpensive, more readily available source of cytokine\nand growth factors and may be applicable for improving\nimplantation rates in ART cycles.\nIn case of SP, unlike with FF, we only observed\na significant up-regulation in the level of  LIF  gene.\nConsidering the fact that the cytokine LIF has multiple\nroles in the implantation process and functions as a\nmediator in some other pathways ( 37 ), it seems that\nit may be necessary to study other  LIF -related genes.\nMany studies have indicated the positive effect of SP\nin the implantation process ( 23 ,  38 - 40 ) . Therefore, it is\nnecessary to perform systematic studies to investigate the\ngenes that are affected by SP to decipher the underlying\nmolecular mechanisms.\n\nTaken together, our results provide evidence that SP\nand FF may contribute to the regulation of endometrial\nfunction by upregulating endometrial receptivity genes\nin human endometrial cells. Accordingly, the application\nof SP and FF can be considered a potential natural\nsupplement to IVF. More research is required to evaluate\nthe clinical importance of SP and FF in the rate of embryo\nimplantation such as intravaginal or intracervical uses of\nSP and FF in IVF therapies.","source_license":"CC-BY-4.0","license_restricted":false}