- Email: [email protected]
Embryology in the era of proteomics
Embryology in the era of proteomics Mandy G. Katz-Jaffe, Ph.D.,a,b and Susanna McReynolds, Ph.D.a a
National Foundation for Fertility Research, and b Colorado Center for Reproductive Medicine, Lone Tree, Colorado
Proteomic technologies have begun providing evidence that viable embryos possess unique protein profiles. Some of these potential protein biomarkers have been identified as extracellular and could be used in the development of a noninvasive quantitative method for embryo assessment. The field of assisted reproductive technologies would benefit from defining the human embryonic proteome and secretome, thereby expanding our current knowlUse your smartphone edge of embryonic cellular processes. (Fertil SterilÒ 2013;99:1073–7. Ó2013 by American Soto scan this QR code ciety for Reproductive Medicine.) and connect to the Key Words: Omics, embryo, ART, proteomics, secretome Discuss: You can discuss this article with its authors and with other ASRM members at http:// fertstertforum.com/katzjaffek-embryology-proteomics/
T
he ability to select the most competent embryo(s) for transfer is a crucial component in the success of assisted reproductive technologies (ART). Detailed morphologic assessment is routinely used to select the embryos with the highest implantation potential (1, 2). Although this method is relatively successful and has led to improvements in pregnancy rates, it has its limitations, with >70% of IVF embryos failing to implant. The field of human ART would therefore benefit from more quantitative methods of embryo viability determination to further improve pregnancy rates and allow for routine single embryo transfer (3). Recent advances in omics technologies (genomics, transcriptomics, proteomics, and metabolomics), including improvements in platform sensitivity, have allowed for the investigation of new molecular methods for embryos selection. Assessment of the embryonic proteome has been of particular interest and provides a snapshot of the cellular function and physiology of an embryo. In addition, analysis of the embryonic secretome (proteins that are produced
and secreted by the developing embryo) could provide a noninvasive approach of embryo assessment (4). In this review we discuss the role of proteomics in defining the human embryonic secretome and its potential applicability and importance to the IVF field. Defining and characterizing the embryonic secretome will also expand our knowledge of early embryogenesis and advance our understanding of the embryo's role during implantation.
PROTEOMICS AND THE EMBRYO The human proteome consists of more than 1 million proteins and still counting. The processes involved in protein translation, posttranslational modification and interactions, as well as protein activity, are constantly changing, influenced by both internal and external stimuli. The proteome is responsible for cellular function and represents all the proteins translated from a cell's specific gene expression products. Studies investigating transcription do not always predict the proteome owing to targeted proteolysis and mechanisms
Received August 30, 2012; revised December 19, 2012; accepted December 20, 2012; published online January 30, 2013. M.G.K.-J. has nothing to disclose. S.M. has nothing to disclose. Reprint requests: Mandy G. Katz-Jaffe, Ph.D., National Foundation for Fertility Research, 10290 RidgeGate Circle, Lone Tree, Colorado 80124 (E-mail: [email protected]). Fertility and Sterility® Vol. 99, No. 4, March 15, 2013 0015-0282/$36.00 Copyright ©2013 American Society for Reproductive Medicine, Published by Elsevier Inc. http://dx.doi.org/10.1016/j.fertnstert.2012.12.038 VOL. 99 NO. 4 / MARCH 15, 2013
discussion forum for this article now.*
* Download a free QR code scanner by searching for “QR scanner” in your smartphone’s app store or app marketplace.
that degrade messenger RNA (mRNA) transcripts before translation. Consequently, to fully comprehend biological processes and understand cellular function, an investigation of the proteins themselves is vital. Despite recent advances in proteomic technologies, the knowledge of the human embryonic proteome remains very limited. The combined effects of limited template, low protein concentration, deficient platform sensitivity, and limited protein database information continue to be the main hurdles. The secretome, defined as those proteins produced by cells and secreted at any given time, is of particular interest to researchers trying to identify proteins involved in specific disease states (5, 6). In ART, the secretome includes those proteins that are produced by embryos and secreted into the surrounding micro drop of culture medium. Proteomic assessment of these secreted embryonic proteins could lead to a noninvasive method of embryo selection and improved ART outcomes (7). To date, this has proven to be a challenging task but holds promise with recent developments in proteomic technology.
SECRETOME AND SINGLE PROTEIN ANALYSIS In initial investigations of the human embryonic secretome, targeted 1073
THE EMBRYO analysis of individual proteins or molecules was mainly performed. One of the first molecules to be identified was the soluble factor, 1-o-alkyl-2-acetyl-sn-glycero3-phosphocholine (PAF) (8). PAF acts as a survival factor in an autocrine fashion and is produced and secreted by mammalian embryos during preimplantation development. The release of PAF also influences a range of maternal physiology alterations, including maternal immune function and platelet activation (8). Leptin, a 16-kDa small pleotrophic peptide, was discovered in blastocyst conditioned media while studying the interaction between the embryo and endometrial epithelial cells (9). During the window of implantation, leptin is thought to initiate and establish a molecular dialogue with leptin receptors on the maternal side (10). Competent human blastocysts were shown to secrete higher leptin concentrations than arrested embryos during in vitro culture. HOXA10 is another protein that is involved in the reciprocal embryo–endometrial interaction that could impact both embryo development and the transformation of the uterine environment. It has been shown that epithelial endometrial cells express HOXA10, which in turn is regulated by an unknown soluble molecule secreted by human blastocysts (11). Human oocytes and embryos have been shown to produce human leukocyte antigen G (HLA-G) at both the mRNA and protein levels (12–14). Several publications have reported an association of the presence of soluble HLA-G (sHLA-G) in embryo spent culture media and successful pregnancy outcome. It has been suggested that sHLA-G in conjunction with current morphological embryo assessment represents a noninvasive marker for prediction of embryo quality and implantation success (15). However, these results have not been absolute, with conflicting studies revealing undetectable levels of sHLA-G in embryo spent culture media and clinical pregnancies established from sHLA-G–negative embryos (15–17). In a recent multicenter study, a wide range of sHLA-G concentrations was detected, and a significant association between successful implantation and sHLA-G–positive embryo spent culture media was only established at one of the three participating clinics (18). Another study was unable to find any association between sHLA-G expression and implantation rates but did observe that miscarriage rates were significantly lower when embryos were selected according to a graduated embryo morphology score and sHLA-G levels vs. the morphology score alone (19). There are several factors that could influence the presence of sHLA-G in embryo spent culture media, including the culture system itself, the extent of cumulus removal, single vs. group embryo culture, media composition, microdrop volume, and the day of media collection (15, 16). The lack of sensitivity of the current ELISA used for most sHLA-G analysis could be another explanation for the lack of reproducibility and association observed to date. Perhaps a more sensitive (picogram level) and reproducible quantitative method for analysis is required to determine the significance of sHLA-G in relation to embryo development and implantation outcome (16). 1074
PROTEIN PROFILING OF THE EMBRYONIC SECRETOME Considering the multifactorial nature of human embryos it would be reasonable to assume that more than one molecule would be required to predict developmental competence and/ or implantation potential. With increased sensitivity and recent advancements in proteomic technologies, it is now possible to investigate deeper into the human embryonic secretome. Mass spectrometry (MS) has rapidly become an important technology in the field of proteomics. The use of MS to search for reliable and reproducible changes in protein expression between groups of samples has revealed important molecular mechanisms associated with both normal physiologic processes and disease states (20). Mass spectrometry includes an ion source for production of a charged species in the gas phase and an analyzer that can separate the ions by their mass-to-charge ratio. Several ionization methods exist, including electrospray ionization and matrix-attenuated laser desorption/ionization, which are then coupled to analyzers such as time-of-flight, quadrupoles, and ion trap analyzers. The ions that are properly aligned by the mass analyzer are then detected and amplified. The resulting data can then be processed through databases to predict the identity of the molecules according to the mass-to-charge ratio (Fig. 1). Tandem mass spectrometry (MS/MS) offers further information about specific ions and is commonly used to sequence proteins (Fig. 1). Biological samples are often complex and exhibit large dynamic concentration ranges between the analytes/proteins. Methods of separation, such as gas or liquid chromatography, are therefore commonly used to separate the molecules/proteins before MS analysis. To obtain reliable and reproducible proteomic data strict quality control is a crucial requirement, including running samples in replicates, routinely performing internal and external calibrations, and including suitable control samples with every run (21). Additionally, because of the dynamic and sensitive nature of the human proteome, tight protocols need to be followed for sample collection, storage, and handling. Using a surface enhanced laser desorption/ionization platform coupled to a time of flight analyzer, Katz-Jaffe et al. (22) were the first to successfully analyze the protein secretome profiles of individual human embryos. Distinct profiles were observed every 24 hours during preimplantation development, from the time of fertilization to the blastocyst stage. During the first 24 hours of embryonic development unique maternal proteins were observed. On day 3 of development with the activation of the embryonic genome, unique embryonic proteins were detected in the human embryonic secretome. A clear association between protein expression profiles, stages of development, and embryo morphology was observed (22). The protein secretome profiles of degenerating embryos exhibited significant up-regulation of several biomarkers that were predicted by molecular weight and database searches to be associated with apoptotic and growth-inhibiting pathways (22). In addition, an 8.5-kDa protein was observed with increased expression in the protein secretome of only VOL. 99 NO. 4 / MARCH 15, 2013
Fertility and Sterility®
FIGURE 1 Mass Spectrometer (MS) ESI MALDI Ion Source
TOF Ion Trap Quadrupoles Mass Analyzer
Ion Detector
Computer
Tandem Mass Spectrometer (MS/MS)
Ion Source
MS1
Fragmentation
MS2
Ion Detector
Computer
Description of the basic components of a mass spectrometer and a tandem mass spectrometer. ESI ¼ electrospray ionization; MALDI ¼ matrixattenuated laser desorption/ionization; TOF ¼ time of flight. Katz-Jaffe. Embryology in the era of proteomics. Fertil Steril 2013.
developing blastocysts, potentially indicating an association between this protein and developmental competence. Protein identification using MS/MS with peptide sequencing indicated that the best candidate for this 8.5-kDa protein was ubiquitin. Ubiquitin is involved in a number of physiologic processes, including proliferation, apoptosis, and implantation (23). Secreted ubiquitin has also recently been shown to be up-regulated in body fluids in certain disease states, providing evidence for an association with an increase in protein turnover (24, 25). In addition to MS technology, there are other proteomic platforms that show promise in the characterization of the embryonic protein secretome. Protein microarrays offer complementary information to transcriptome studies and eliminate the need for follow-up protein identification. In 2008 Dominguez et al. (26) used protein microarrays containing 120 antibody targets to analyze the protein secretome of the human blastocyst (26). The proteins CXCL13 (BCL, B lymphocyte chemoattractant), stem cell factor, TRAILR3, MIP-1b, and MSP-a were significantly decreased in the blastocyst culture media when compared with control media. The authors hypothesized that the decrease in expression of the proteins observed in conditioned media of human blastocysts indicates consumption of these proteins by the blastocyst. In contrast, soluble tumor necrosis factor receptor 1 and interleukin (IL)-10 significantly increased in media when a blastocyst was present. Further, when comparing pooled conditioned media from implanted vs. nonimplanted blastocysts after single embryo transfer, two proteins, CXCL13 and granulocytemacrophage colony-stimulating factor, were found to have significantly decreased expression in conditioned media of implanted blastocysts. Indeed, when granulocytemacrophage colony-stimulating factor is added to either human or murine blastocyst culture media, this protein has been shown to promote mammalian embryonic development and VOL. 99 NO. 4 / MARCH 15, 2013
implantation potential (27). Interestingly, no proteins were found to be significantly increased in expression in conditioned media of implanted blastocysts (26). Follow-up research by Dominguez et al. (28) compared protein secretome profiles from an endometrial epithelial cell (EEC) coculture system with profiles from a sequential microdrop culture media system (28). Several proteins showed altered expression in the EEC system, including IL-6, PLGF, and BCL (CXCL13), which were increased, and FGF-4, IL-12p40, VEGF, and uPAR, which were decreased. Interleukin-6 was the most abundant protein in the EEC coculture system secreted by the endometrial cells themselves. Using an IL-6 ELISA assay on individual microdrops of spent culture media, viable blastocysts displayed an increased uptake of IL-6 compared with blastocysts that failed to result in a pregnancy, suggesting a potential role for IL-6 in blastocyst development and implantation (28). Two-dimensional gel electrophoresis in combination with MS/MS has also been used to identify proteins in spent culture media from human preimplantation embryos. Apolipoprotein A1 (ApoA1) was observed in the embryonic protein secretome using this platform and validated by ELISA analysis. Increased levels of ApoA1 were associated with blastocysts of higher morphologic grade (29). However, no association with IVF outcome was observed with ApoA1 levels in the embryonic secretome. MicroRNAs (miRNAs) are short single-stranded noncoding RNA molecules (20–24 nucleotides) that regulate gene and protein expression typically by either mRNA degradation and/or translational repression, respectively. MicroRNAs have been associated with numerous biological processes, including development (30), cell growth (31), differentiation (32), and infertility (33). During mouse embryogenesis miRNAs are maternally inherited, with the loss of approximately 60% between the one- and two-cell stages at the maternal 1075
THE EMBRYO zygotic transition (30). A dynamic degradation and synthesis of miRNAs coexists during the development of the preimplantation mouse embryo, with an overall increase in miRNAs toward the blastocyst stage (34). Our group further investigated the possibility of miRNA secretion to assist in protein regulation during the embryo–maternal dialogue. MicroRNA profiling of euploid blastocyst spent culture media revealed an absence of expression of 377 miRNAs suggesting that these miRNAs are not secreted during blastocyst development. Chromosome aneuploidies, defined as the gain or loss of an entire chromosome, contribute to the vast majority of pregnancy losses in both natural and ART conceptions. Currently, selection of euploid embryos (correct number of chromosomes) for transfer involves testing of embryonic cells after an invasive embryo biopsy, which could compromise subsequent development. The potential to develop a noninvasive assay to include both viability and aneuploidy screening would be an important addition to embryo selection methods. Initial protein secretome studies were promising, with a clear difference between individual euploid and anueploid blatocysts (4). Using a liquid chromatography–MS/MS platform, deeper investigation of the blastocyst protein secretome in relation to chromosome aneuploidy was performed. From a cohort of transferable-quality blastocysts, nine novel candidate biomarkers in the protein secretome characteristically classified chromosome aneuploidy. One of these potential biomarkers, lipocalin-1, was identified as the first potential biomarker for noninvasive aneuploidy screening (35). Lipocalin-1 has a large variety of ligands and is overproduced under conditions of stress, inflammation, and infection. Examining both pooled and individual embryo spent culture media using an ELISA, the increased expression of lipocalin-1 was confirmed (35). The increased secretion of lipocalin-1 from an aneuploid blastocyst could represent an overall compromised state of the embryo itself, reflecting the aneuploid chromosome complement. Ongoing research by this group is focused on continuing validation of differential biomarkers to reliably discriminate between euploid viable, euploid nonviable, and aneuploid blastocysts (Fig. 2). Once clinically confirmed, proteomic analysis using immunodetection techniques can be highly sensitive, high-throughput, and costeffective for routine testing in a clinical IVF laboratory. Despite these promising results and advances in proteomic technologies, the knowledge of the protein secretome of preimplantation embryos remains limited. As outlined above, the combined effects of limited template, low protein expression, and lack of sensitivity of current proteomics platforms remain main hurdles. Other challenges in the search of noninvasive viability biomarkers include the overwhelming presence of albumin, immunoglobins, and other serum proteins in the culture media, making it difficult to identify the low expressed secreted embryonic proteins. Chromatographic approaches to remove such abundant proteins do exist and in combination with multidimensional fractionation allow for the detection of proteins secreted by the embryo (35, 36). In general, other difficulties in biomarker discovery include the sources of variability, such as the experimental design and data interpretation, differences in methodology, lack 1076
FIGURE 2 Blastocyst
Individual Blastocyst Microdrop Culture
Analysis of Spent Media
Euploid Implanted Blastocyst Secretome
Euploid nonimplanted Blastocyst Secretome
Aneuploid Blastocyst Secretome
Ongoing research is focused on validation of identified biomarkers to reliably discriminate between euploid implanted, euploid nonimplanted, and aneuploid blastocyst secretome. Katz-Jaffe. Embryology in the era of proteomics. Fertil Steril 2013.
of standardized sample collection, and storage (37). Deficiencies in follow-up studies for biomarker validation remain another limited factor, primarily owing to technological limitations and sample collection (38). Further, proteomic analysis of the spent culture media requires embryos to be individually cultured. In the past, debate has existed regarding the impact on developmental competence with single embryo culture. However, recent studies have shown that high implantation rates and live birth rates are correlated with individual embryo culture for comprehensive chromosome screening (39, 40). Even with these limitations, proteomic analysis of embryonic development is still considered a promising platform aimed to improve ART success. To date there is no noninvasive platform, including noninvasive proteomics, that has proven to be of true clinical predictive value or been examined in prospective randomized control trials to be better than current morphology-based selections methods. Perhaps it is not the viability biomarkers in the culture media alone but the combination of a quantitative noninvasive assay examining the embryonic secretome alongside morphology assessment that would represent the greatest improvement in embryo selection techniques VOL. 99 NO. 4 / MARCH 15, 2013
Fertility and Sterility® and possibly lead to routine healthy singleton transfers and deliveries.
20. 21.
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National Foundation for Fertility Research, and b Colorado Center for Reproductive Medicine, Lone Tree, Colorado
Proteomic technologies have begun providing evidence that viable embryos possess unique protein profiles. Some of these potential protein biomarkers have been identified as extracellular and could be used in the development of a noninvasive quantitative method for embryo assessment. The field of assisted reproductive technologies would benefit from defining the human embryonic proteome and secretome, thereby expanding our current knowlUse your smartphone edge of embryonic cellular processes. (Fertil SterilÒ 2013;99:1073–7. Ó2013 by American Soto scan this QR code ciety for Reproductive Medicine.) and connect to the Key Words: Omics, embryo, ART, proteomics, secretome Discuss: You can discuss this article with its authors and with other ASRM members at http:// fertstertforum.com/katzjaffek-embryology-proteomics/
T
he ability to select the most competent embryo(s) for transfer is a crucial component in the success of assisted reproductive technologies (ART). Detailed morphologic assessment is routinely used to select the embryos with the highest implantation potential (1, 2). Although this method is relatively successful and has led to improvements in pregnancy rates, it has its limitations, with >70% of IVF embryos failing to implant. The field of human ART would therefore benefit from more quantitative methods of embryo viability determination to further improve pregnancy rates and allow for routine single embryo transfer (3). Recent advances in omics technologies (genomics, transcriptomics, proteomics, and metabolomics), including improvements in platform sensitivity, have allowed for the investigation of new molecular methods for embryos selection. Assessment of the embryonic proteome has been of particular interest and provides a snapshot of the cellular function and physiology of an embryo. In addition, analysis of the embryonic secretome (proteins that are produced
and secreted by the developing embryo) could provide a noninvasive approach of embryo assessment (4). In this review we discuss the role of proteomics in defining the human embryonic secretome and its potential applicability and importance to the IVF field. Defining and characterizing the embryonic secretome will also expand our knowledge of early embryogenesis and advance our understanding of the embryo's role during implantation.
PROTEOMICS AND THE EMBRYO The human proteome consists of more than 1 million proteins and still counting. The processes involved in protein translation, posttranslational modification and interactions, as well as protein activity, are constantly changing, influenced by both internal and external stimuli. The proteome is responsible for cellular function and represents all the proteins translated from a cell's specific gene expression products. Studies investigating transcription do not always predict the proteome owing to targeted proteolysis and mechanisms
Received August 30, 2012; revised December 19, 2012; accepted December 20, 2012; published online January 30, 2013. M.G.K.-J. has nothing to disclose. S.M. has nothing to disclose. Reprint requests: Mandy G. Katz-Jaffe, Ph.D., National Foundation for Fertility Research, 10290 RidgeGate Circle, Lone Tree, Colorado 80124 (E-mail: [email protected]). Fertility and Sterility® Vol. 99, No. 4, March 15, 2013 0015-0282/$36.00 Copyright ©2013 American Society for Reproductive Medicine, Published by Elsevier Inc. http://dx.doi.org/10.1016/j.fertnstert.2012.12.038 VOL. 99 NO. 4 / MARCH 15, 2013
discussion forum for this article now.*
* Download a free QR code scanner by searching for “QR scanner” in your smartphone’s app store or app marketplace.
that degrade messenger RNA (mRNA) transcripts before translation. Consequently, to fully comprehend biological processes and understand cellular function, an investigation of the proteins themselves is vital. Despite recent advances in proteomic technologies, the knowledge of the human embryonic proteome remains very limited. The combined effects of limited template, low protein concentration, deficient platform sensitivity, and limited protein database information continue to be the main hurdles. The secretome, defined as those proteins produced by cells and secreted at any given time, is of particular interest to researchers trying to identify proteins involved in specific disease states (5, 6). In ART, the secretome includes those proteins that are produced by embryos and secreted into the surrounding micro drop of culture medium. Proteomic assessment of these secreted embryonic proteins could lead to a noninvasive method of embryo selection and improved ART outcomes (7). To date, this has proven to be a challenging task but holds promise with recent developments in proteomic technology.
SECRETOME AND SINGLE PROTEIN ANALYSIS In initial investigations of the human embryonic secretome, targeted 1073
THE EMBRYO analysis of individual proteins or molecules was mainly performed. One of the first molecules to be identified was the soluble factor, 1-o-alkyl-2-acetyl-sn-glycero3-phosphocholine (PAF) (8). PAF acts as a survival factor in an autocrine fashion and is produced and secreted by mammalian embryos during preimplantation development. The release of PAF also influences a range of maternal physiology alterations, including maternal immune function and platelet activation (8). Leptin, a 16-kDa small pleotrophic peptide, was discovered in blastocyst conditioned media while studying the interaction between the embryo and endometrial epithelial cells (9). During the window of implantation, leptin is thought to initiate and establish a molecular dialogue with leptin receptors on the maternal side (10). Competent human blastocysts were shown to secrete higher leptin concentrations than arrested embryos during in vitro culture. HOXA10 is another protein that is involved in the reciprocal embryo–endometrial interaction that could impact both embryo development and the transformation of the uterine environment. It has been shown that epithelial endometrial cells express HOXA10, which in turn is regulated by an unknown soluble molecule secreted by human blastocysts (11). Human oocytes and embryos have been shown to produce human leukocyte antigen G (HLA-G) at both the mRNA and protein levels (12–14). Several publications have reported an association of the presence of soluble HLA-G (sHLA-G) in embryo spent culture media and successful pregnancy outcome. It has been suggested that sHLA-G in conjunction with current morphological embryo assessment represents a noninvasive marker for prediction of embryo quality and implantation success (15). However, these results have not been absolute, with conflicting studies revealing undetectable levels of sHLA-G in embryo spent culture media and clinical pregnancies established from sHLA-G–negative embryos (15–17). In a recent multicenter study, a wide range of sHLA-G concentrations was detected, and a significant association between successful implantation and sHLA-G–positive embryo spent culture media was only established at one of the three participating clinics (18). Another study was unable to find any association between sHLA-G expression and implantation rates but did observe that miscarriage rates were significantly lower when embryos were selected according to a graduated embryo morphology score and sHLA-G levels vs. the morphology score alone (19). There are several factors that could influence the presence of sHLA-G in embryo spent culture media, including the culture system itself, the extent of cumulus removal, single vs. group embryo culture, media composition, microdrop volume, and the day of media collection (15, 16). The lack of sensitivity of the current ELISA used for most sHLA-G analysis could be another explanation for the lack of reproducibility and association observed to date. Perhaps a more sensitive (picogram level) and reproducible quantitative method for analysis is required to determine the significance of sHLA-G in relation to embryo development and implantation outcome (16). 1074
PROTEIN PROFILING OF THE EMBRYONIC SECRETOME Considering the multifactorial nature of human embryos it would be reasonable to assume that more than one molecule would be required to predict developmental competence and/ or implantation potential. With increased sensitivity and recent advancements in proteomic technologies, it is now possible to investigate deeper into the human embryonic secretome. Mass spectrometry (MS) has rapidly become an important technology in the field of proteomics. The use of MS to search for reliable and reproducible changes in protein expression between groups of samples has revealed important molecular mechanisms associated with both normal physiologic processes and disease states (20). Mass spectrometry includes an ion source for production of a charged species in the gas phase and an analyzer that can separate the ions by their mass-to-charge ratio. Several ionization methods exist, including electrospray ionization and matrix-attenuated laser desorption/ionization, which are then coupled to analyzers such as time-of-flight, quadrupoles, and ion trap analyzers. The ions that are properly aligned by the mass analyzer are then detected and amplified. The resulting data can then be processed through databases to predict the identity of the molecules according to the mass-to-charge ratio (Fig. 1). Tandem mass spectrometry (MS/MS) offers further information about specific ions and is commonly used to sequence proteins (Fig. 1). Biological samples are often complex and exhibit large dynamic concentration ranges between the analytes/proteins. Methods of separation, such as gas or liquid chromatography, are therefore commonly used to separate the molecules/proteins before MS analysis. To obtain reliable and reproducible proteomic data strict quality control is a crucial requirement, including running samples in replicates, routinely performing internal and external calibrations, and including suitable control samples with every run (21). Additionally, because of the dynamic and sensitive nature of the human proteome, tight protocols need to be followed for sample collection, storage, and handling. Using a surface enhanced laser desorption/ionization platform coupled to a time of flight analyzer, Katz-Jaffe et al. (22) were the first to successfully analyze the protein secretome profiles of individual human embryos. Distinct profiles were observed every 24 hours during preimplantation development, from the time of fertilization to the blastocyst stage. During the first 24 hours of embryonic development unique maternal proteins were observed. On day 3 of development with the activation of the embryonic genome, unique embryonic proteins were detected in the human embryonic secretome. A clear association between protein expression profiles, stages of development, and embryo morphology was observed (22). The protein secretome profiles of degenerating embryos exhibited significant up-regulation of several biomarkers that were predicted by molecular weight and database searches to be associated with apoptotic and growth-inhibiting pathways (22). In addition, an 8.5-kDa protein was observed with increased expression in the protein secretome of only VOL. 99 NO. 4 / MARCH 15, 2013
Fertility and Sterility®
FIGURE 1 Mass Spectrometer (MS) ESI MALDI Ion Source
TOF Ion Trap Quadrupoles Mass Analyzer
Ion Detector
Computer
Tandem Mass Spectrometer (MS/MS)
Ion Source
MS1
Fragmentation
MS2
Ion Detector
Computer
Description of the basic components of a mass spectrometer and a tandem mass spectrometer. ESI ¼ electrospray ionization; MALDI ¼ matrixattenuated laser desorption/ionization; TOF ¼ time of flight. Katz-Jaffe. Embryology in the era of proteomics. Fertil Steril 2013.
developing blastocysts, potentially indicating an association between this protein and developmental competence. Protein identification using MS/MS with peptide sequencing indicated that the best candidate for this 8.5-kDa protein was ubiquitin. Ubiquitin is involved in a number of physiologic processes, including proliferation, apoptosis, and implantation (23). Secreted ubiquitin has also recently been shown to be up-regulated in body fluids in certain disease states, providing evidence for an association with an increase in protein turnover (24, 25). In addition to MS technology, there are other proteomic platforms that show promise in the characterization of the embryonic protein secretome. Protein microarrays offer complementary information to transcriptome studies and eliminate the need for follow-up protein identification. In 2008 Dominguez et al. (26) used protein microarrays containing 120 antibody targets to analyze the protein secretome of the human blastocyst (26). The proteins CXCL13 (BCL, B lymphocyte chemoattractant), stem cell factor, TRAILR3, MIP-1b, and MSP-a were significantly decreased in the blastocyst culture media when compared with control media. The authors hypothesized that the decrease in expression of the proteins observed in conditioned media of human blastocysts indicates consumption of these proteins by the blastocyst. In contrast, soluble tumor necrosis factor receptor 1 and interleukin (IL)-10 significantly increased in media when a blastocyst was present. Further, when comparing pooled conditioned media from implanted vs. nonimplanted blastocysts after single embryo transfer, two proteins, CXCL13 and granulocytemacrophage colony-stimulating factor, were found to have significantly decreased expression in conditioned media of implanted blastocysts. Indeed, when granulocytemacrophage colony-stimulating factor is added to either human or murine blastocyst culture media, this protein has been shown to promote mammalian embryonic development and VOL. 99 NO. 4 / MARCH 15, 2013
implantation potential (27). Interestingly, no proteins were found to be significantly increased in expression in conditioned media of implanted blastocysts (26). Follow-up research by Dominguez et al. (28) compared protein secretome profiles from an endometrial epithelial cell (EEC) coculture system with profiles from a sequential microdrop culture media system (28). Several proteins showed altered expression in the EEC system, including IL-6, PLGF, and BCL (CXCL13), which were increased, and FGF-4, IL-12p40, VEGF, and uPAR, which were decreased. Interleukin-6 was the most abundant protein in the EEC coculture system secreted by the endometrial cells themselves. Using an IL-6 ELISA assay on individual microdrops of spent culture media, viable blastocysts displayed an increased uptake of IL-6 compared with blastocysts that failed to result in a pregnancy, suggesting a potential role for IL-6 in blastocyst development and implantation (28). Two-dimensional gel electrophoresis in combination with MS/MS has also been used to identify proteins in spent culture media from human preimplantation embryos. Apolipoprotein A1 (ApoA1) was observed in the embryonic protein secretome using this platform and validated by ELISA analysis. Increased levels of ApoA1 were associated with blastocysts of higher morphologic grade (29). However, no association with IVF outcome was observed with ApoA1 levels in the embryonic secretome. MicroRNAs (miRNAs) are short single-stranded noncoding RNA molecules (20–24 nucleotides) that regulate gene and protein expression typically by either mRNA degradation and/or translational repression, respectively. MicroRNAs have been associated with numerous biological processes, including development (30), cell growth (31), differentiation (32), and infertility (33). During mouse embryogenesis miRNAs are maternally inherited, with the loss of approximately 60% between the one- and two-cell stages at the maternal 1075
THE EMBRYO zygotic transition (30). A dynamic degradation and synthesis of miRNAs coexists during the development of the preimplantation mouse embryo, with an overall increase in miRNAs toward the blastocyst stage (34). Our group further investigated the possibility of miRNA secretion to assist in protein regulation during the embryo–maternal dialogue. MicroRNA profiling of euploid blastocyst spent culture media revealed an absence of expression of 377 miRNAs suggesting that these miRNAs are not secreted during blastocyst development. Chromosome aneuploidies, defined as the gain or loss of an entire chromosome, contribute to the vast majority of pregnancy losses in both natural and ART conceptions. Currently, selection of euploid embryos (correct number of chromosomes) for transfer involves testing of embryonic cells after an invasive embryo biopsy, which could compromise subsequent development. The potential to develop a noninvasive assay to include both viability and aneuploidy screening would be an important addition to embryo selection methods. Initial protein secretome studies were promising, with a clear difference between individual euploid and anueploid blatocysts (4). Using a liquid chromatography–MS/MS platform, deeper investigation of the blastocyst protein secretome in relation to chromosome aneuploidy was performed. From a cohort of transferable-quality blastocysts, nine novel candidate biomarkers in the protein secretome characteristically classified chromosome aneuploidy. One of these potential biomarkers, lipocalin-1, was identified as the first potential biomarker for noninvasive aneuploidy screening (35). Lipocalin-1 has a large variety of ligands and is overproduced under conditions of stress, inflammation, and infection. Examining both pooled and individual embryo spent culture media using an ELISA, the increased expression of lipocalin-1 was confirmed (35). The increased secretion of lipocalin-1 from an aneuploid blastocyst could represent an overall compromised state of the embryo itself, reflecting the aneuploid chromosome complement. Ongoing research by this group is focused on continuing validation of differential biomarkers to reliably discriminate between euploid viable, euploid nonviable, and aneuploid blastocysts (Fig. 2). Once clinically confirmed, proteomic analysis using immunodetection techniques can be highly sensitive, high-throughput, and costeffective for routine testing in a clinical IVF laboratory. Despite these promising results and advances in proteomic technologies, the knowledge of the protein secretome of preimplantation embryos remains limited. As outlined above, the combined effects of limited template, low protein expression, and lack of sensitivity of current proteomics platforms remain main hurdles. Other challenges in the search of noninvasive viability biomarkers include the overwhelming presence of albumin, immunoglobins, and other serum proteins in the culture media, making it difficult to identify the low expressed secreted embryonic proteins. Chromatographic approaches to remove such abundant proteins do exist and in combination with multidimensional fractionation allow for the detection of proteins secreted by the embryo (35, 36). In general, other difficulties in biomarker discovery include the sources of variability, such as the experimental design and data interpretation, differences in methodology, lack 1076
FIGURE 2 Blastocyst
Individual Blastocyst Microdrop Culture
Analysis of Spent Media
Euploid Implanted Blastocyst Secretome
Euploid nonimplanted Blastocyst Secretome
Aneuploid Blastocyst Secretome
Ongoing research is focused on validation of identified biomarkers to reliably discriminate between euploid implanted, euploid nonimplanted, and aneuploid blastocyst secretome. Katz-Jaffe. Embryology in the era of proteomics. Fertil Steril 2013.
of standardized sample collection, and storage (37). Deficiencies in follow-up studies for biomarker validation remain another limited factor, primarily owing to technological limitations and sample collection (38). Further, proteomic analysis of the spent culture media requires embryos to be individually cultured. In the past, debate has existed regarding the impact on developmental competence with single embryo culture. However, recent studies have shown that high implantation rates and live birth rates are correlated with individual embryo culture for comprehensive chromosome screening (39, 40). Even with these limitations, proteomic analysis of embryonic development is still considered a promising platform aimed to improve ART success. To date there is no noninvasive platform, including noninvasive proteomics, that has proven to be of true clinical predictive value or been examined in prospective randomized control trials to be better than current morphology-based selections methods. Perhaps it is not the viability biomarkers in the culture media alone but the combination of a quantitative noninvasive assay examining the embryonic secretome alongside morphology assessment that would represent the greatest improvement in embryo selection techniques VOL. 99 NO. 4 / MARCH 15, 2013
Fertility and Sterility® and possibly lead to routine healthy singleton transfers and deliveries.
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