The aim of the proteomic analysis of the oocyte environment is an in-depth comprehension of the molecular pathways behind oocyte maturation, development, and competence acquisition. The identification of protein pathways that participate in the communication between oocytes, cumulus cells, and the follicular fluid may be significant for the identification of key proteins which could serve as biomarkers indicating good oocyte quality. Most proteomic studies that have been conducted have focused on the analysis of the follicular fluid and oocyte surrounding granulosa cells (cumulus cells).

Proteomic analysis of follicular fluid

The follicular fluid (FF) is the natural environment in which the oocyte matures and becomes competent. Until now, only a small portion of the entire human FF proteome has been revealed. FF, due to its proximity and communication with the maturing oocyte, makes up a unique fluid for the study of the processes occurring during oocyte maturation [67]. Some studies were conducted to comprehend the molecular pathways that are involved in normal oocyte development through the proteomic analysis of the follicular fluid. In the study of Ambekar et al., the proteome of human FF was analyzed by SDS-PAGE, OFFGEL, and strong cation exchange (SCX)-based separation, followed by LC-MS/MS and 480 proteins were identified. These proteins of the FF belonged to functional categories such as growth factors, hormones, receptor signaling, enzyme catalysis, defense/immunity, and complement activity [68]. In the study of Twigt et al., after proteomic analysis of FF with SDS-PAGE, in tube gel digestion and prefractionation of proteolytic peptides, followed by LC-MS/MS, 246 proteins were identified, most of which are involved in coagulation and immune response pathways [69]. Moreover, a study done by Jarkovska et al., who performed two-dimensional gel electrophoresis, followed by MALDI mass spectrometry, showed that FF consists of proteins involved in the complement cascade, angiogenesis, and coagulation cascade [70]. Shen et al. performed reverse-phase high-performance liquid chromatography (RP-HPLC), followed by matrix-assisted laser desorption/ionization time of flight tandem mass spectrometry (LC-MALDI TOF/TOF MS), and found a total of 219 unique high confidence FF proteins through Swiss-Prot human database. The proteins he found were involved in complement, coagulation cascade, growth factor group, hormone group, immunity, and transportation [71]. Zakerkish et al. used mass spectrometry with the isobaric tags for relative and absolute quantification (iTRAQ) technology for isobaric tagging of peptides, which enables simultaneous identification and quantification of proteins, and analyzed the protein profiles of FF of the preovulatory and ovulatory phases and found 502 proteins, out of which 20 were overexpressed during ovulation. These proteins were inflammatory-related, coagulation factors, proteins in lipid metabolism, complement factors, and antioxidants. In addition, he found 5 proteins to be downregulated during ovulation, three of which were enzymes and two proteins of lipid metabolism and iron transport [72]. Poulsen et al. used liquid chromatography-mass spectrometry and found 400 proteins in FF, 40 of which showed significant change in their expression during ovulation. Among these were proteins involved in the immune and inflammatory system, secretion pathway, and proteins related to extracellular structure organization [73]. The involvement of the complement cascade in the folliculogenesis and oocyte maturation process was also shown by Jarkovska et al., who implemented 2DE, HPLC, and mass spectrometry in HFF of women undergoing IVF [74]. Angelucci et al. analyzed the follicular fluid and plasma from normo-ovulatory women undergoing assisted reproduction techniques, with 2DE and MALDI-TOF-MS, and found 183 HFF/plasma matched proteins and 27 unmatched. Many acute-phase proteins in high concentrations were identified, including transferrin, ceruloplasmin, afamin, hemopexin, haptoglobin, and plasma amyloid protein in the HFF, indicating that ovulation can be compared to an inflammatory event. Other proteins that were identified were some antioxidant enzymes such as catalase, superoxide dismutase, glutathione transferase, paraoxonase, heat shock protein 27, and protein disulfide isomerase. The above findings also indicate that during maturation the human follicle is protected against toxic injury due to oxidative stress [75]. Many studies have shown the importance of antioxidants in the normal oocyte growth. Nagy et al. showed that FF-HDL anti-oxidative function was related to a decrease in the odds of the oocyte undergoing normal fertilization [76]. Calonge et al. found that the activity of follicular fluid antioxidant enzymes was significantly lower in young women with reduced ovarian reserve compared with that in high responders and oocyte donors. Follicular fluid concentrations of oxidative stress marker malondialdehyde combined with 4-hydroxyalkenals and nitric oxide were higher in low responders than in high responders and oocyte donors [77]. In another study, Nishihara et al. showed that total GSH (glutathione) levels were lower in patients who had a low fertilization rate after ICSI, but it did not show a significant difference in pregnancy outcome. In addition, a total of 8-OHdG levels were higher in patients who had a low fertilization rate after ICSI and a low rate of good quality blastocysts. Total GSH and 8-OFdG in human FF may be potential markers for fertilization success in ART [78]. Lewandowska et al. used ultrafiltration to fractionate FF to high molecular weight (HMW) and low molecular weight (LMW) peptidome fractions. The HMW and LMW fractions were analyzed using LC-MS in sequential window acquisition of all theoretical (SWATH) data acquisition and processing methodology. A total of 158 proteins were identified out of which, 59 were never reported before as FF components. The concentrations of 11 proteins varied substantially among FF samples from single donors, and these proteins could be significant targets to identify biomarkers useful in oocyte quality assessment [79]. Bianchi et al. analysis of the follicular fluid suggests that effectors and inhibitors control and balance the induction and inhibition of inflammation, coagulation, and ECM degradation/remodeling. Such fine modulation of enzymatic activities plays an important role in follicle development and oocyte competence acquisition. Among the control proteins was alpha-1 antitrypsin, which is involved in 21 interconnections and may play a key role in balancing FF protease/anti-protease activity controlling ECM degradation, in inflammation, in wound response and coagulation cascade during follicle maturation, ovulation, and corpus luteum formation [80]. Klun et al. after implementing LC-MS/MS in oocytes found that tudor and KH domain-containing protein (TDRKH) is expressed in immature oocytes, while Wee2 (wee1-like protein kinase 2), PCNA (proliferating cell nuclear antigen), and DNMT1 (DNA (cytosine-5)-methyltransferase 1) were enriched in mature cells [81]. Bayasula et al. after implementing LC/MS/MS found that albumin and immunoglobulin families of proteins represent 80% of the total proteins. From the rest proteins that were identified, two were classified in the developmental process group, four in the signal transduction group, nine in the localization group, and 52 in the metabolic process group. Heparin sulfate proteoglycan percecan protein was upregulated in the group that resulted in the fertilized oocyte [82]. In the study of Zamah et al., 742 follicular fluid proteins were identified after implementation of high and low pH HPLC peptide separation, followed by mass spectrometry. Among them, 413 were not previously reported. The proteins belonged to insulin growth factor and insulin growth factor binding protein families, growth factor and related proteins, receptor signaling, defense/immunity, antiapoptotic proteins, matrix metalloproteinase related proteins, and complement activity. Moreover, after quantitative analysis, 17 follicular proteins were found at significantly altered levels between pre-hCG and post-hCG samples. These proteins belong to functional processes such as protease inhibition, inflammation, and cell adhesion [83].

Potential biomarkers of female infertilityPotential biomarkers in follicular fluid

Hashmitabar et al. compared human follicular fluid from younger and older women with normal FSH levels with 2DE and MALDI-TOF-TOF mass spectrometry and identified twenty-three proteins differentially expressed. Five were downregulated in the older group which were serotransferrin, hemopexin precursor, complement C3, C4, and kininogen and are proteins involved in complement cascade pathway, immunity response, iron transport, and angiogenesis [84]. Estes et al. compared the proteome profile of follicular fluid in women under 32 years old between samples that did not lead to pregnancy and others that resulted in a live birth. After LC-MS/MS was performed, 11 potential protein candidates that were haptoglobin alpha, predominantly fetal expressed T1 domain, mitochondrial integrity genome (ATPase), apolipoprotein H (beta-2 glycoprotein I), dihydrolipoyl dehydrogenase, lysozyme C, fibrinogen alpha-chain, and immunoglobulin heavy chain V-III (region BRO) were increased in the live birth group, whereas antithrombin, vitamin D-binding protein, and complement 3 were decreased [85]. Kushnir et al., after depletion of abundant proteins from HFF samples and analysis with nano LC-QTOF, identified a total of 75 proteins, out of which 4 (paraoxonase 1, PRP6 pre-mRNA processing factor 6 homolog, complement component 6, and rho guanine nucleotide exchange factor 37) were present only in the IVF cycles that resulted in delivery, 7 proteins (complement component 2, hypothetical protein LOC64762, complement component c8 alpha-chain, apolipoprotein B, carboxypeptidase N, similar to Lg gamma-1 chain C region, alpha 2 globin) were present only in cycles that resulted in miscarriage, and finally, 2 proteins (growth inhibition and differentiation-related protein 88 and PHD finger protein 16) were identified only in IVF cycles that did not achieve pregnancy. In general, the proteins that were identified in the FF belong to the acute response signaling, coagulation system, neutroprotective role of THOP1, FXR/RXR activation, role of tissue factor, and growth hormone pathways. Proteins associated with biosynthesis were more abundant in the FF samples of oocytes that resulted in pregnancy, as well as 7 that were associated with steroidogenesis [86]. Severino et al. found 89 proteins, 30 of which were differentially expressed in hFF with successful compared to unsuccessful IVF outcomes. In particular, 2 were found to be downregulated. These were actin cytoplasmic 1 (ACTB), a structural constituent of the cytoskeleton, and tubulin polyglytamylase (TTLL7), involved in cell differentiation, while 28 were upregulated in hFFs with positive IVF outcomes [87]. Chen et al., after performing LC-MS/MS, identified 7 peptides as potential biomarkers for positive IVF outcomes. These were derived from insulin-like growth factor binding protein-5 (IGFBP5), alpha 2-antiplasmin (A2AP), complement component 3 (C3), inter-alpha-trypsin inhibitor heavy chain H1 (ITIH1), serum albumin (ALBU), protein diaphanous homolog 1, and plastin-3, which belong to different functional groups like growth factors, negative regulation of plasminogen activation, complement cascade, and stabilization of cumulus mass [88].

Biomarkers in granulosa/cumulus cells

Other studies have analyzed the granulosa/cumulus cells environment and have searched for potential biomarkers related to female infertility. Braga et al. compared protein expression of human cumulus cells of embryos that reached and did not reach the blastocyst stage. They found 87 different proteins in samples from the blastocyst and non-blastocyst groups, of which 30 were exclusively expressed in the blastocyst group and 19 in the non-blastocyst group. The proteins were binding proteins, enzymes, as well as structural proteins, transport proteins, contraction, and DNA repair proteins. Among the 72 proteins that were detected in the pregnancy group, 19 were exclusively expressed in the positive and 16 were exclusively expressed in the negative-pregnancy group [89]. Luddi et al., after implementing western blotting and immunofluorescence, showed the significant role of metalloproteinase, especially MMP2 (abbreviation) and MMP9, in fertilization. They found that MMP9 is expressed only in granulose cells, whereas MMP2 is more expressed in cumulus and granulose cells in cases of reduced ovarian response and decreased fertilization rate [90].

The above studies show that several molecular pathways in normal oocyte function are impaired in IVF cycles that did not have a positive outcome. Among the pathways are the inflammation pathway, complement and coagulation cascade, cell differentiation, and cytoskeleton organization (Table 2). Identifying biomarkers that indicate female infertility could help in the early prevention of an unsuccessful result or even in the monitoring of each cycle by a prospective therapeutic invasive approach, with the aim to achieve the desired outcome.

Table 2 Potential biomarkers of female infertilityPotential biomarkers for embryo quality

Until now, only a few studies have been conducted which search for biomarkers that indicate good embryo quality, either in an invasive manner where blastocoel fluid (blastocele fluid) is analyzed or in a non-invasive way (embryo secretome) through analysis of the embryo’s culture media.

Starting with the invasive studies, Katz-Jaffe et al., after lysis of blastocyst-stage embryos, studied the protein profile of the embryo using anion exchange chromatography, followed by SELDI-TOF-MS. Six proteins were found to be upregulated in arrested embryos compared to non-arrested ones. Candidate IDs for these proteins were heparin-binding EGF-like growth factor precursor (HB-EGF), cystatin-9-like precursor, CART/NADH-ubiquinone oxireductase, beta-catenin-interacting protein1, cytochrome c oxidase subunit, caspase-1 precursor, and inhibitor of growth protein 1 ING1-like tumor suppressor protein, X-linked. Many of these proteins play a role in implantation [91]. Poli et al. used an invasive shotgun proteomic analysis of blastocoel fluid to compare normal and aneuploid embryos and found two proteins to be differentially expressed among the two groups. These were GAPDH (glyceraldehyde 3-phosphate dehydrogenase) that was underexpressed in euploid and H2A (histone H2A) that was overexpressed in aneuploid embryos [92].

Moving on to non-invasive studies, Dyrlund et al. searched for biomarkers which are detectable in good quality embryo’s secretome. He used eight different commercial culture media and performed in-solution sample digestion with trypsin and LC-MS/MS after albumin depletion. A total of 110 proteins other than HSA (human serum albumin) were identified. Among them, eight have previously been suggested as biomarkers for embryonic viability. These are afamin, apolipoprotein A-I, epidermal growth factor receptor, haptoglobin, haptoglobin-related protein, peroxiredoxin-I, serotransferrin, and serum albumin. Based on biological processes, these proteins were grouped into three major groups: inflammatory response, innate immune response, and response to peptide hormone stimulus [93]. Kaihola et al. analyzed the proteome profile of embryo culture media using multiplex proximity extension assay (PEAs). He found that day-2 cultured embryos resulting in pregnancy after IVF treatment secreted significantly lower levels of caspase-3 in correlation with those that did not result in pregnancy. However, no differences were found in HRG (histidine-rich glycoprotein) levels between the two groups, although these were higher in culture media of embryos that reached the morula stage faster. The same analysis was also carried out for blastocysts, but no significant differences were found. The above results would make sense as caspase-3 is a protein involved in apoptosis (programmed cell death through DNA fragmentation). HRG inhibits the apoptotic effect of caspase-3 via interactions with thrombospondins. Normally, thrombospondins bind to CD36 and thereby initiate a cascade of events, ending in the activation of caspases to initiate apoptosis [94]. The lower levels of caspase-3 in high- versus low-quality blastocysts were also shown in the study of Lindgren et al. performing a multiplex proximity assay in human day-2 cryopreserved embryos. The study also found that embryos developing into blastocysts had higher levels of extracellular matrix metalloproteinase inducer protein (EMMPRIN) secreted in their culture media. Finally, the levels of VEGF-A, IL-6, and EMMPRIN were higher in embryos which reached the morula stage in a shorter time [95]. In another study by Katz-Jaffe et al., protein expression of human embryos in culture media was again analyzed using anion exchange chromatography, followed by SELDI-TOF-MS. Ubiquitin was identified as a potential biomarker for embryo developmental potential [96]. Butler et al. performed ELISA and MALDI TOF-MS in embryos culture media and found that hCG, hCGh, and hCGΒ could be potential biomarkers of embryo viability and of their implantation potential [97]. Montsko et al. used LC coupled MS in culture media of in vitro fertilized embryos to correlate the alpha-1 chain of human haptoglobin (HPT) concentration and morphological score. He found that HPT concentration could predict the outcome of the embryo transfer [98]. In the study of Montsko et al., haptoglobin alpha-1 fragment was also found to be a potential biomarker for viable embryos. A significant correlation was also found among the presence of the peptide in the culture media and their achievement of pregnancy [99]. Mains et al. used embryo culture media to search for potential biomarkers showing good quality in embryos. He applied two-dimensional gel electrophoresis and mass spectrometry and found that apolipoprotein A1 is increased in culture media of blastocysts with higher morphologic grade [100]. McReynolds implemented LC-MS/MS to compare euploid and aneuploid blastocysts secretome and found nine potential biomarkers for aneuploidy, with the most significant being lipocalin-1 [101]. In the study by Domiquez et al., culture media from blastocysts that were implanted and culture media from blastocysts that were not implanted were analyzed using protein array technology, and it was found that CXCL13 (BLC) and granulocyte-macrophage colony-stimulating factor (GM-CSF) was significantly decreased in the implanted blastocyst media compared to the non-implanted. The above proteins are involved in functions like a response to wounding, response to external stimulus/response to stress, response to pathogen or other organisms, inflammatory response, cell communication, immune response, and chemotaxis. Moreover, the soluble TNF receptor 1 and IL-10 were significantly increased, while MSP-A, also called hepatocyte growth factor-like (HGFL), SCF (stem cell factor), CXCL13 (C-X-C motif chemokine), TRAILR3 (tumor necrosis factor receptor superfamily member 10C), and MIP1b (macrophage inflammatory protein beta) was significantly decreased in culture media containing blastocysts (both implanted and non-implanted) in comparison with the control ones [102]. Cortezzi et al. implemented the nano-UPLC with nano-electrospray ionization (nano-ESI) in culture media samples from embryos that achieved and those that did not achieve pregnancy and found 18 proteins in the group that achieved implantation. Among them, protein Jumonji (JARID2), which composes a histone methyltransferase complex called polycomb chromatin methylation that silences many embryonic patterning genes, that normally serve as negative regulators of cell proliferation and may also be related to cell differentiation. Eleven more proteins were identified in the negative implantation group, with TSGA10 (testis-specific protein 10) being the most abundant protein. TSGA10 is a perinuclear protein which has structural activity and is detected in actively dividing and fetal differentiating tissues during developmental of mouse embryos [103]. Brison et al. tried to correlate the presence of specific amino acids with the potential of successful pregnancy after performing liquid chromatography. Three amino acids were identified, ASn, Gly and Leu, and were found to be significantly related to the achievement of pregnancy [104].

Pathways of the immune system, energy metabolism, apoptosis, structural pathways, and also implantation process play an important role in the regulation of embryo development. The list of the potential biomarkers of good embryo quality is presented below (Table 3). Proteomics could help in the single embryo transfer (sET) approach, as it could contribute to the selection of the best embryos, in combination with the morphological approach as well as the implementation of PGD.

Table 3 Proteomic biomarkers of competent embryosEndometrial proteomics

The identification of a receptive endometrium could contribute to the prevention of implantation failure and pregnancy loss and could therefore lead to an increase in ART success rates. There is a need for a practical, non-invasive test to predict the receptivity of the endometrium for embryo implantation. The crosstalk between the embryo and the endometrium is time- and location-sensitive, occurring during a short time span, usually between days 16 and 22 of a 28-day normal menstrual cycle, known as the “window of implantation” 5–10 days after the luteinizing hormone (LH) surge [105]. Currently, there are no markers of endometrial receptivity [106]. The proteomics approach to endometrial receptivity can either be studied by analyzing endometrial tissue or uterine fluid.

Endometrial tissue

Studies that utilized endometrial tissue to compare receptive and non-receptive endometrium are presented below. Hood et al. performed a proteomic analysis of the endometrial tissue using LC-MS/MS, followed by immunochemistry, in order to compare the proliferative and secretory endometrium. A total of 318 proteins were found to be differentially expressed between the two phases in the epithelial cells and 19 in the stroma compartment. Proteins identified from glandular epithelial cells included progesterone receptor B, expressed in the proliferative phase, and glycodelin A (PAEP), expressed in the secretory phase. In addition, CPM, paladin (PALLD), minichromosome maintenance complex component 6 (MCM6), ENPP3, periplakin (PPL), homogentisate 1,2-dioxygenase (HGD), and polymeric immunoglobulin receptor (PIGR) were also significantly differentially abundant in the glandular epithelium of the proliferative as compared to the secretory phase. The above proteins indicate an upregulation of cellular growth and proliferation molecular pathways in the proliferative compared to the secretory endometrium [107]. DeSouza et al. used isotope-coded affinity tags, three stages of chromatographic separation, and online tandem mass spectrometry (MS/MS) to also analyze the proteomic profile differences between the secretory and proliferate endometrial tissue. He found five proteins to be consistently differentially expressed between the two phases, with glutamate NMDA receptor subunit zeta 1 precursor and FRAT1 being the most frequent in the secretory endometrium. The first protein is known to be involved with synaptic plasticity in neurons. In a recent paper, it is suggested that it may also play a role in glutamate-mediated toxicity to mitochondria, leading to apoptosis. FRATI is known to inhibit c-Jun activity, thereby inhibiting subsequent apoptosis [108]. Differences in mid-secretory and proliferative phases of the endometrial tissue were also found in the study of Parmar et al., who used two-dimensional protein maps, followed by MALDI-TOF-TOF to compare MSE (mid-secretory endometrium) with PROE (proliferative phase endometrial tissues) as well as with MSU (mid-secretory phase uterine fluids) and found Calreticulin precursor, fibrinogen, adenylate kinase isoenzyme 5 (KAD5), and transferrin to be upregulated in the proliferative phase endometrium. The above proteins participate in cellular activities such as calcium-binding, blood clotting, energy metabolism, and blood plasma protein, respectively, while annexin V (ANXA5), peroxidoxin 6 (PRDX6), a1-antitrypsin (AAT), and creatine kinase were upregulated in the mid-secretory phase, whose functions include apoptosis, antioxidant, protease inhibitor, and energy metabolism [109]. Another study by Chen et al. that aimed to identify proteins that were differentially expressed in the human endometrium tissue between proliferative and secretory phase with 2D differential in-gel electrophoresis (DIGE) showed enhanced expression of proteins in the secretory endometrium. The differentially expressed isoforms of the same proteins were identified by MALDI-TOF/TOF MS. These isoforms belonged to 4 proteins, three of which were increased in the mid-secretory phase. These were annexin A4 (ANXA4), keratin 8 (KRT8), and heat shock protein beta 1 (HSPB1), while albumin (ALBU) was decreased. Their functions are differentiation, apoptosis and inhibition of proliferation (ANXA4), cellular assembly and organization (KRT8), and heat stress (HSPB1) [110]. Annexin A2 (ANXA2) and stathmin I (STMNI) were found to be differentially expressed in non-receptive (day 2) versus receptive (day 7) endometrium in the study of Dominguez et al. after implementation of 2DE and MALDI-MS on endometrial tissue. Both proteins play a role in cytoskeletal development. Stathmin I is also involved in the intracellular signaling cascade [111]. Garrido-Gomez et al., after performing DIGE and MALDI mass spectrometry in receptive and non-receptive endometrial biopsies, found 24 differentially expressed proteins. He applied in silico analysis and identified the pathways that were most different between the two groups. These were the carbohydrate biosynthetic pathway and the rearrangement of the cytoskeleton pathway. After immunochemistry was performed, it was found that PGRMCI (progesterone receptor membrane component I) and ANXA6 (Annexin A6) play an important role in endometrial receptivity [112]. Another study by Berkova et al., who performed HPLC and immunoblotting, found that the concentration of haptoglobin was significantly higher in deciduas graviditatis in comparison with non-pregnant endometrial tissue and higher in the stroma in contrast to the epithelium of the proliferative endometrium. In the secretory phase, it was found to be in moderate concentrations in the stroma in contrast with the epithelium. Haptoglobin in the uterus may bind with hemoglobin but could also be involved in the multi-factorial mechanism protecting the fetus from a maternal allograft-like immune response [113].

Endometrial fluid

More non-invasive studies analyzing the proteome of the uterine fluid have been conducted. Casado-Vela et al. set three different proteomic approaches (in-solution tryptic digestion and SDS-PAGE, followed by HPLC-MS/MS and 2D-PAGE, followed by MALDI-TOF/TOF) in order to analyze the proteomic profile of endometrial fluid and found in total 803 proteins, including albumin, IgGs, transferrin, fibrinogen, antitrypsin, complement C3, haptoglobin, apolipoprotein, ceruloplasmin, and complement factor B [114]. Matorras et al., in another study, analyzed endometrial fluid right before embryo transfer after 2DE MS/MS and found 23 proteins differentially expressed between successful embryo implantation cycles and failed cycles. Most of these proteins were downregulated in the group that achieved implantation, and these were heat shock cognate (HSP cognate), heat shock (HSP), plastin-2, protein disulfide-isomerase A3 (PDIA3), arginase-1, F-actin-capping protein subunit alpha-1 (CAPZA-1), putative beta actin-like protein 3 (ACTBL3), actin, cytoplasmic1, proteasome subunit beta type 4 (PSMB4), protein deglycase DJ-1, (Parkinson disease protein7), superoxide dismutase[Mn], mitochondrial (SOD2), cell division control protein 42 homolog (CDC42), cofilin-1, stathmin, myeloid-derived growth factor (MYDGF), tubulin-specific chaperone A (TBCA), glyceraldehydes-3-phosphate dehydrogenase (GAP-DH), F-actin capping protein subunit beta (CAPZB), and annexin A2 (ANXA2). The above proteins are involved in cell growth, signal transduction, metabolism, cell communication, blood coagulation, barbed-end actin filament capping, and regulation of nucleobase, nucleoside, nucleotide, and nucleic acid metabolism. Four proteins were upregulated, which were catalase, serum albumin (ALBU), serotransferrin, and Lg kappa chain V. These proteins are involved in immune response and transport. The last three should be considered to have a blood origin or nonspecific source [115]. Hannan et al. analyzed the endometrial secretions and compared receptive and non-receptive states in fertile and infertile women after 2D-DiGE and found 7 spots significantly decreased in the MP (mid proliferative) phase compared to the MS (mid-secretory) of the endometrium and 18 spots between fertile and infertile women. Moreover, after immunostaining of the endometrial tissue, antithrombin III was found to be localized in glandular and luminal epithelium with higher levels in MP in contrast to the MS phase. The higher concentrations of antithrombin iii were also found in the infertile endometrium in comparison to the fertile one. Another protein that was found to be upregulated in the MS phase and in moderate levels in the MP phase was a2 macroglobulin; however, there was no difference in the intensity of A2macroglobulin immunostaining between fertile and infertile women. ANT3 has anti-inflammatory properties, while A2M deactivates of matrix metalloproteinases (MMPs), limiting trophoblast invasion [106]. Kasvandik et al. analyzed the proteome of early (ESE) and mid-secretory endometrium (MSE) of fertile and infertile women after performing LC/MS/MS and found 367 proteins that undergo significant proteomic changes while transitioning from the early to mid-secretory endometrial phase. Twenty-one proteins were found to display similar levels between control ESE and RIF (repeated implantation failure) MSE, indicating the displacement of WOI. Four proteins had similar levels to control ESE than to control MSE, potentially indicating a pre-receptive EM in the RIF cohort [116]. Scotchie et al. analyzed the luteal endometrial secretome (lh+4, lh+9) by performing 2DE and MS/MS and found 82 proteins to be differentially expressed. Increased expression in the secretory phase showed proteins involved in host defense and molecule transport. Among them were haptoglobin precursor, anti-TNFα antibody, apolipoprotein A1 fragment, transferrin precursor, and vitamin D binding protein variant, while proteins with decreased expression had many functions, like apoptosis regulation, cell proliferation, and host defense. Among them were heat shock protein b1, clusterin precursor, cofilin1, haptoglobin precursor, and others [117]. Al-Rumaih et al. attempted to identify uterine markers in early and late proliferative phases of the menstrual cycle with respect to estrogen levels using 2D-PAGE protein maps and found that nineteen spots were differentially expressed in the two groups; five of them were identified and were also increased in the high E2 group. These were serotransferrin (STF), hemopexin precursor, fibrinogen β chain, a-1 antichymotrypsin (AACT), and complement component C4. The first two are involved in the transport of iron. Hemopexin is also involved in acute-phase reactions. Fibrinogen and AACT are involved in blood coagulation, complement activation, programmed cell death, and development [118]. Fitzgerald et al. analyzed with LC-MS/MS endometrial fluid of the proliferative phase from fertile and infertile women and found four proteins to be downregulated in the infertile women. These were extracellular matrix protein 1 (ECM1), transforming growth factor–beta-induced protein ig-h3 (TGFB1), secreted frizzled-related protein 4 (SFRP4), and CD44 antigen. Their role is negative regulation of signaling, cell communication, and tissue development. Upregulated were found to be two proteins, protein-glutamine gamma-glutamyltransferase 2 (TGM2) and Lg gamma-4 chain C region (IGHG4). In addition, seven proteins were unique in the fertile group. These were filamin A (FLNA), pregnancy zone protein (PZP), oviduct specific glycoprotein (OVGP1), endoplasmin (HSP90B1), annexin A6 (ANXA6), 40s ribosomal protein S3 (RPS3), and septin-2 (SEPT2). These proteins take part in cilium assembly, actin filament organization, and macromolecule catabolic process. Finally, six proteins were found to be unique in the infertile group; these were neuroblast differentiation-associated protein (AHNAK), isoaspartyl peptidase/L-asparaginase (ASRGL1), UMP-CMP kinase (CMPK1), phosphoglucomutase-1 (PGM1), cornulin (CRNN), and suprabasin (SBSN). These proteins are involved in carbohydrate metabolism and other cellular processes [119]. Bhutada et al. used endometrial fluid and tissue of the early secretory or secretory phase. After iTRAQ analysis and immunoblotting, he identified HMGBI (amphoterin), a DNA binding non-histone protein, in pre-receptive and receptive endometrium. The protein was downregulated in the receptive phase compared to the pre-receptive phase of the endometrium [120]. Gillot et al., after analysis of endometrial flushing of the proliferative phase endometrium from thirty-one women undergoing assisted reproduction, using 2DPAGE, found a statistical difference in the expression of two leucine-rich alpha2-glycoprotein (LRG) isoforms, which were higher in women who got pregnant. These isoforms may be involved in the infiltration of decidua by uterine natural killer cells, which actually differentiate into granular forms during early pregnancy [