**3.1 Albumin depletion in IVF cultivation medium**

The presence of albumin in cultivating medium is necessary for the normal development of embryos. However, its presence is a significant burden for proteomics analysis, and it must be removed prior to further analysis steps. The removal of albumin has been extensively discussed and described in a number of publications [1, 2, 7–9]. Different groups have used a number of methods such as immunodepleting chromatography, molecular weight cutoff filters, peptide libraries, size exclusion chromatography, etc. All these methods have some advantages but also show disadvantages. In case of depleting the albumin from IVF cultivating medium, the very low sample volume (max. of 40 μl) must be taken into consideration. Furthermore, depletion method must be performed fast and must be reproducible over a large number of samples, if intended to be used for fast analysis of clinical samples that shall help making the decision on which fertilized oocyte shall be transferred first and which ones shall be frozen for later procedures. The use of a novel immunoaffinity-based convective interaction media analytical columns (CIMac) for depletion of HSA (CIMac-HSA) was performed in this study, and it proved that it can be used for fast and reproducible albumin depletion from minute sample amounts. The column's architecture and the convective flow-through columns' channels enable a flow rate-independent binding capacity and excellent chromatographic resolution. These characteristics give CIMac-αHSA column some important analytical benefits like shorten time of analysis in comparison to common chromatographic depletion of albumin using silica-based columns, which

is an extraordinary important parameter for clinical use. The albumin content in different batches of cultivating medium differs strongly, and it also differs strongly between different suppliers. Therefore, the depletion method must be selected in a way that can be applied for a variety of samples. However, independently of the selected methods, the removal of albumin from the cultivating medium results with a number of identified proteins of which some can be of importance for embryo development and for later pregnancy development.

**Figure 1** shows two SDS-gel lanes for the separation of two media upon embryo transfer. Since albumin originates from different donors and is being mixed at the manufacturing site, it will certainly contain a number of other proteins that may interfere with proteins secreted from the developing embryo. Also, secreted proteins might bind on albumin and, therefore, be "invisible" for proteomics analysis.

In addition to that, the human proteome, as any proteome, is dynamic, and it is constantly changing through internal and external stimuli. Proteins being translated from RNA are directly responsible for cellular function, but different gene expression studies have, unfortunately, shown that a reliable prediction of proteins' function or abundance cannot be made. As for the human proteome, significant advances have been made for the analysis of the whole proteome and of the proteome of different disease states [3, 5, 10–20]. Bearing all of it in mind, the analysis of proteins obtained from the cultivating media is challenging, and the results are not always conclusive.

**Figure 2** shows the results obtained upon analyzing four different cultivating media used in routine IVF procedures and upon embryo transfer.

Dyrlund et al. [21] have performed the analysis of unconditioned commercial embryo culture media and have identified a number of proteins upon depletion and digestion of a total of 5 mL of media. The amount of 5 mL, however, will never be available if samples are retrieved during the actual IVF process. However, this analysis showed that this amount contains an astounding amount of 25 mg of albumin. Here, a total of 111 proteins with different concentrations in different batches of the media were identified in addition to albumin. The sample of unconditioned media also contained eight proteins previously suggested as possible markers of

### **Figure 1.**

*Lanes 2 and 4 show an example of the separation of proteins secreted in culturing media on silver-stained SDS gel. As expected, large spots for human serum albumin (HSA) are observed for both samples with significantly lower amounts of other proteins. Since the culturing media have been declared to contain only HSA, the proteins below are secreted from the fertilized oocyte (lane 2) and unfertilized oocyte (lane 4).*

**139**

protein, ubiquitin, etc.

*proteins according to Tarasova et al. [1].*

**Figure 2.**

*Proteomics as a Future Tool for Improving IVF Outcome DOI: http://dx.doi.org/10.5772/intechopen.89880*

embryonic viability, e.g., proteoglycan-4, serotransferrin, vitamin-D-binding

from the same batch in order to make valid comparison.

culture media for the offspring should also be evaluated.

Another study performed on IVF medium examined the batch-to-batch variations and showed that variation in both protein amounts and protein identifications can be expected [22]. When studying these media, it is important to pay attention to the fact that both media, the control and the media where embryos grow, must be

*Identified proteins and their relative quantities in albumin-free fractions after performing albumin depletion of culturing media samples on two different depletion columns. Proteins identified either in the CIMac-αHSA FT or the Seppro FT1 fractions are labeled with B and S, followed by Swiss-Prot protein ID. Median values of normalized spectral indexes (SI) and standard deviations are shown. Median SIHSA in non-depleted samples is marked with red dashes. More than two peptide-spectrum matches were assumed for each of the listed* 

Spent IVF medium is a valuable source for proteins that can be used as putative biomarkers in IVF. The major focus of researchers has been on secreted proteins, but the proteins already present in the medium can be equally valuable and must be considered in the pursuit for biomarkers of embryos' "quality." It is very possible that proteins already present in the medium may be necessary for embryo development, and the uptake or degradation of specific proteins might correlate to ascertain embryo development or lack thereof. Therefore, these media must be evaluated for their potential to differentiate embryos' success rates and track the proteins, which can be potential biomarkers. Additionally, the safety of these proteins in

### **Figure 2.**

*Innovations in Assisted Reproduction Technology*

proteomics analysis.

results are not always conclusive.

development and for later pregnancy development.

is an extraordinary important parameter for clinical use. The albumin content in different batches of cultivating medium differs strongly, and it also differs strongly between different suppliers. Therefore, the depletion method must be selected in a way that can be applied for a variety of samples. However, independently of the selected methods, the removal of albumin from the cultivating medium results with a number of identified proteins of which some can be of importance for embryo

**Figure 1** shows two SDS-gel lanes for the separation of two media upon embryo transfer. Since albumin originates from different donors and is being mixed at the manufacturing site, it will certainly contain a number of other proteins that may interfere with proteins secreted from the developing embryo. Also, secreted proteins might bind on albumin and, therefore, be "invisible" for

In addition to that, the human proteome, as any proteome, is dynamic, and it is constantly changing through internal and external stimuli. Proteins being translated from RNA are directly responsible for cellular function, but different gene expression studies have, unfortunately, shown that a reliable prediction of proteins' function or abundance cannot be made. As for the human proteome, significant advances have been made for the analysis of the whole proteome and of the proteome of different disease states [3, 5, 10–20]. Bearing all of it in mind, the analysis of proteins obtained from the cultivating media is challenging, and the

**Figure 2** shows the results obtained upon analyzing four different cultivating

Dyrlund et al. [21] have performed the analysis of unconditioned commercial embryo culture media and have identified a number of proteins upon depletion and digestion of a total of 5 mL of media. The amount of 5 mL, however, will never be available if samples are retrieved during the actual IVF process. However, this analysis showed that this amount contains an astounding amount of 25 mg of albumin. Here, a total of 111 proteins with different concentrations in different batches of the media were identified in addition to albumin. The sample of unconditioned media also contained eight proteins previously suggested as possible markers of

*Lanes 2 and 4 show an example of the separation of proteins secreted in culturing media on silver-stained SDS gel. As expected, large spots for human serum albumin (HSA) are observed for both samples with significantly lower amounts of other proteins. Since the culturing media have been declared to contain only HSA, the proteins below are secreted from the fertilized oocyte (lane 2) and unfertilized oocyte (lane 4).*

media used in routine IVF procedures and upon embryo transfer.

**138**

**Figure 1.**

*Identified proteins and their relative quantities in albumin-free fractions after performing albumin depletion of culturing media samples on two different depletion columns. Proteins identified either in the CIMac-αHSA FT or the Seppro FT1 fractions are labeled with B and S, followed by Swiss-Prot protein ID. Median values of normalized spectral indexes (SI) and standard deviations are shown. Median SIHSA in non-depleted samples is marked with red dashes. More than two peptide-spectrum matches were assumed for each of the listed proteins according to Tarasova et al. [1].*

embryonic viability, e.g., proteoglycan-4, serotransferrin, vitamin-D-binding protein, ubiquitin, etc.

Another study performed on IVF medium examined the batch-to-batch variations and showed that variation in both protein amounts and protein identifications can be expected [22]. When studying these media, it is important to pay attention to the fact that both media, the control and the media where embryos grow, must be from the same batch in order to make valid comparison.

Spent IVF medium is a valuable source for proteins that can be used as putative biomarkers in IVF. The major focus of researchers has been on secreted proteins, but the proteins already present in the medium can be equally valuable and must be considered in the pursuit for biomarkers of embryos' "quality." It is very possible that proteins already present in the medium may be necessary for embryo development, and the uptake or degradation of specific proteins might correlate to ascertain embryo development or lack thereof. Therefore, these media must be evaluated for their potential to differentiate embryos' success rates and track the proteins, which can be potential biomarkers. Additionally, the safety of these proteins in culture media for the offspring should also be evaluated.

### **3.2 Identification of proteins in IVF cultivation medium and their possible role for embryo development**

Proteins identified by Dyrlund et al. were also reported by Katze-Jaff upon analysis of the secretome of the individually cultured human embryos, and a hint was made that these proteins could be related to embryo's morphology and thus its viability [23–25]. They have reported that ubiquitin was upregulated in developing blastocytes as compared to degenerating blastocytes. However, there was no correlation between the ubiquitin upregulation and the observed pregnancy rates.

Cortezzi et al. [26] reported the identification of proteins in both positive and negative groups of embryos, i.e., embryos termed viable for transfer and does who were not selected. For positive-implantation group, protein, called Jumonji (JARID2), was reported. This protein composes a histone methyltransferase complex called polycomb repressive complex 2 (PRC2), which modifies chromatin methylation to silence many embryonic patterning genes, acting as a negative regulator of cell proliferation signaling. This results with a restricted gene expression to an appropriate cell population that is essential for development, differentiation, and maintenance of cell fates.

In the same study, the testis-specific gene 10 protein (TSGA10) was identified only in negative samples. TSGA10 is a perinuclear protein, and it was described to participate in actively dividing and fetal differentiating tissues in mice embryos.

Katz-Jaffe et al. reported the identification of heparin-binding epidermal growth factor EGF-like growth factor precursor (HB-EGF) [24]. This growth factor precursor belongs to the EGF family growth factors, and it is found in the membrane-anchored form (proHB-EGF) and the soluble form (sHB-EGF). The soluble form is produced from the proteolytic cleavage of proHB-EGF at the extracellular domain. Furthermore, soluble HB-EGF has been observed to be a growth stimulator, and it does significantly improve the blastocyst development and hatching when added to serum-free medium. However, other studies from an in vitro model system indicate that proHB-EGF might function in cell-to-cell signaling by a juxtacrine mechanism inhibiting growth activity [27]. Since this protein was identified in degenerating blastocytes, its upregulation might contribute to the lack of further development.

A cystatin-like precursor is needed for successful mammalian implantation for a controlled trophoblastic invasion of the maternal uterine epithelium. It must be available and functional [28] since this invasion involves the extracellular degradation of the uterine matrix by a variety of proteinases, and one of the crucial ones are cysteine proteinases. Cystatins are known to inhibit cysteine proteinases, and their upregulation will most probably contribute to failure of implantation of degenerating blastocysts. Therefore, it is not only of importance to identify these proteins, the possible biomarkers, but also to validate these findings in the human.

### **4. Conclusion**

Proteomics is a promising technology for the identification and validation of possible biomarkers for embryo selection in ART. As listed by Dyrlund et al., a growing list of secreted proteins has been identified that could further contribute to this field [21]. However, the challenge ahead of the research still includes the reliable and reproducible identification of a proteomics secretome signature. This signature shall be directly associated with embryo viability and the success of the procedure, i.e., successful pregnancy, and child's birth. This is a very challenging task not only due to the complexity, heterogeneity, and diversity of human embryos

**141**

**Author details**

Vienna, Austria

Goran Mitulović1,2\* and Tanja Panić-Janković1

provided the original work is properly cited.

1 Clinical Department of Laboratory Medicine, Medical University of Vienna,

© 2019 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/ by/3.0), which permits unrestricted use, distribution, and reproduction in any medium,

2 Proteomics Core Facility, Medical University of Vienna, Vienna, Austria

\*Address all correspondence to: goran.mitulovic@meduniwien.ac.at

*Proteomics as a Future Tool for Improving IVF Outcome DOI: http://dx.doi.org/10.5772/intechopen.89880*

proteins therein.

but also because of irreproducibility of used culturing media and contaminant

Another challenge for the clinical use of proteomics methods is the speed of the proteomics analysis. Sample preparation, measurement, and data analysis of a sample is currently not feasible within the time window needed for IVF. Current sample analysis methods require at least half a day for the fastest proteomics method available, which is too long. Nevertheless, proteomics methods can contribute to identification and validation of putative biomarkers, which once clinically confirmed, can be analyzed using other, faster, methods upon building corresponding antibodies.

*Proteomics as a Future Tool for Improving IVF Outcome DOI: http://dx.doi.org/10.5772/intechopen.89880*

*Innovations in Assisted Reproduction Technology*

**for embryo development**

and maintenance of cell fates.

development.

**4. Conclusion**

**3.2 Identification of proteins in IVF cultivation medium and their possible role** 

Proteins identified by Dyrlund et al. were also reported by Katze-Jaff upon analysis of the secretome of the individually cultured human embryos, and a hint was made that these proteins could be related to embryo's morphology and thus its viability [23–25]. They have reported that ubiquitin was upregulated in developing blastocytes as compared to degenerating blastocytes. However, there was no correlation between the ubiquitin upregulation and the observed pregnancy rates. Cortezzi et al. [26] reported the identification of proteins in both positive and negative groups of embryos, i.e., embryos termed viable for transfer and does who were not selected. For positive-implantation group, protein, called Jumonji (JARID2), was reported. This protein composes a histone methyltransferase complex called polycomb repressive complex 2 (PRC2), which modifies chromatin methylation to silence many embryonic patterning genes, acting as a negative regulator of cell proliferation signaling. This results with a restricted gene expression to an appropriate cell population that is essential for development, differentiation,

In the same study, the testis-specific gene 10 protein (TSGA10) was identified only in negative samples. TSGA10 is a perinuclear protein, and it was described to participate in actively dividing and fetal differentiating tissues in mice embryos. Katz-Jaffe et al. reported the identification of heparin-binding epidermal growth factor EGF-like growth factor precursor (HB-EGF) [24]. This growth factor precursor belongs to the EGF family growth factors, and it is found in the membrane-anchored form (proHB-EGF) and the soluble form (sHB-EGF). The soluble form is produced from the proteolytic cleavage of proHB-EGF at the extracellular domain. Furthermore, soluble HB-EGF has been observed to be a growth stimulator, and it does significantly improve the blastocyst development and hatching when added to serum-free medium. However, other studies from an in vitro model system indicate that proHB-EGF might function in cell-to-cell signaling by a juxtacrine mechanism inhibiting growth activity [27]. Since this protein was identified in degenerating blastocytes, its upregulation might contribute to the lack of further

A cystatin-like precursor is needed for successful mammalian implantation for a controlled trophoblastic invasion of the maternal uterine epithelium. It must be available and functional [28] since this invasion involves the extracellular degradation of the uterine matrix by a variety of proteinases, and one of the crucial ones are cysteine proteinases. Cystatins are known to inhibit cysteine proteinases, and their upregulation will most probably contribute to failure of implantation of degenerating blastocysts. Therefore, it is not only of importance to identify these proteins, the

Proteomics is a promising technology for the identification and validation of possible biomarkers for embryo selection in ART. As listed by Dyrlund et al., a growing list of secreted proteins has been identified that could further contribute to this field [21]. However, the challenge ahead of the research still includes the reliable and reproducible identification of a proteomics secretome signature. This signature shall be directly associated with embryo viability and the success of the procedure, i.e., successful pregnancy, and child's birth. This is a very challenging task not only due to the complexity, heterogeneity, and diversity of human embryos

possible biomarkers, but also to validate these findings in the human.

**140**

but also because of irreproducibility of used culturing media and contaminant proteins therein.

Another challenge for the clinical use of proteomics methods is the speed of the proteomics analysis. Sample preparation, measurement, and data analysis of a sample is currently not feasible within the time window needed for IVF. Current sample analysis methods require at least half a day for the fastest proteomics method available, which is too long. Nevertheless, proteomics methods can contribute to identification and validation of putative biomarkers, which once clinically confirmed, can be analyzed using other, faster, methods upon building corresponding antibodies.
