**Abstract**

New technical and methodical and more efficient approaches beyond preimplantation genetic screening (PGS) are needed to elevate success rates in in vitro fertilization (IVF). One new approach could be the characterization of the embryos' proteome during the IVF process. This means that specific proteins secreted by the embryo in the surrounding cultivating medium can be analyzed and compared between embryos in order to identify potential markers for a successful embryo transfer and resulting pregnancy. Furthermore, this procedure could result with understanding the processes during the whole time of incubation, from the moment of oocyte fertilization until embryo transfer and subsequently implantation by analyzing the culturing medium used in multiple culture medium exchange during the cultivation period. This procedure of embryo transfer to a new culture medium is essential for the embryo's development and is performed daily or at least when the embryos reached the stage of embryoblast at day 4. The remaining medium after embryo removal is routinely discarded. However, this medium still can be useful for a detailed analysis of proteins and lipids that were secreted by the embryo during the previous incubation process and could help gaining information on the embryos' current developmental status.

**Keywords:** in vitro fertilization, proteomics, pregnancy, oocyte, pregnancy outcome

### **1. Introduction**

The European Society of Human Reproduction and Embryology (ESHRE) report from February 2018 shows that Europe continues to lead the world with approximately 50% of all reported in vitro fertilization (IVF) cycles. The latest figures are available for 2014, and these show that 39 European countries reported almost 800.000 cycles, which compare to 150.000 cycles in the USA or 65.000 cycles in Australia and New Zealand (https://www.eshre.eu/~/media/ sitecore-files/Guidelines/ART-fact-sheet\_vFebr18\_VG.pdf?la=en). Although there are no official reports from China, it is estimated that this country performs more than 800.000 IVF cycles with a tendency to grow. In summary, the ESHRE estimates that around 2.5 million IVF cycles is performed every year and that about 500.000 babies are born following the IVF.

The pregnancy rate depends on the embryo quality, and in Europe in 2014, the mean pregnancy rate per embryo transfer was 35% after IVF, 33% after ICSI, 30% after frozen embryo transfer, and 59% after egg donation. It is observed that pregnancy rates are higher in younger patients (<35 years).

There are large differences between countries concerning the number of embryos transferred and the resulting multiple births that can occur following the multiple embryo transfer. Multiple pregnancies are classified as high-risk pregnancies and pose a significant health risk to both the mother and baby. However, regardless of that, the practice of multiple embryo transfer is still present, and it is being widely used in order to increase the chances of pregnancy. Currently, efforts are made to reduce the number of multiple embryo transfer, and a mean of 1.81 embryos per transfer is being reported. In Europe, according to the report of the European Society of Human Reproduction and Embryology, the number of multiple delivery rates for embryo transfers dropped from 26.9% in year 2000 to 17.5% in 2014 (latest data available), and it is expected to decline further. Some countries, e.g., Sweden, has reached a very low multiple delivery rate, and the single embryo transfer was performed in more than two-thirds of all transfers.

The importance of IVF in society is also highlighted by the 2010 Nobel Prize which has been awarded to Robert Edwards who is, together with Robert Steptoe, the "father" of the fist "IVF-Baby."

The IVF process is an utmost stressful, both physically and psychologically, for both parents but primarily for the mother-in-being. A number of physical examinations and medical procedures are being performed in order to ensure that the process will lead to a successful pregnancy.

During the IVF process, especially when decisions are made which embryo shall be transferred in order to ensure the greatest chance for success, the best possible decision, based on the fast and accurate information on the status of the embryo, is needed. Due to the increasing age of mothers-to-be, during the past years, a steady increase in pregnancies following artificial reproductive techniques (ART), namely, IVF and intra-cytoplasmatic sperm injection (ICSI), was observed. The reasons for this development must be seen in economic, educational, and social factors, which lead to steadily rising rates of elderly patients. With increasing age, fecundability and fertility decrease. However, the advances made in reproductive medicine itself might be one of the reasons, why an increasing number of women delay childbearing, hoping for late babies and consciously making decisions to use the help of reproductive medicine.

The elevation of pregnancy rates for IVF procedures is certainly needed in order to reduce the burden on patients and lower costs affiliated with the procedures, which very often must be repeated until a pregnancy is reached and the embryo develops to successful birth. New technologies and methods more efficient than the current approach using PGS are needed. One new approach could be the analysis of the embryos' proteome during the IVF process. This means to analyze the specific proteins that are secreted by the embryo in the IVF surrounding of the culture medium. Routinely, the medium left after embryo transfer is being discarded. The multiple embryo transfer occurring during the IVF procedure is necessary after the fertilization step, and the spent media are a rich source of biological material that can be used for diagnostic purpose. This procedure of embryo transfer to a new culture medium is essential for the embryo's development and occurs daily or at least when the embryos have achieved the stage of embryoblast at day 4.

The spent medium can be useful for a detailed examination of proteins and lipids that were secreted by the embryo during each stage of development and can be used for the estimation of the "embryo quality" and selection of corresponding embryos for primary transfer.

An overview of technologies and approaches being used and a simplified experimental approach are presented in this chapter. The method used in our laboratory is used just as an example of the technologies used, and a number of different approaches will be discussed.

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and optimized.

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

**2.1 Analyzed embryo cultivation medium: source of the material**

Embryo cultivation medium is usually being discarded during the IVF procedure although it can be used for multiple diagnostic and prognostic procedures. Certainly, ethical and moral guidelines and procedures must be observed, and the ethical board or other corresponding bodies must approve of the use of this material. The following experiments have been performed by using cultivation media from IVF media upon approval from the ethical boards of the Medical University in Vienna and the University of Linz in Austria. Samples were collected during different stages of embryo development and analyzed using methods established for

Trypsin for protein digestion was purchased from Promega Inc. (Vienna, Austria). Solvents for HPLC—methanol (MeOH), acetonitrile (AcN), 2,2,2-trifluoroethanol (TFE), formic acid (FA), heptafluorobutyric acid (HFBA), iodoacetamide (IAA), triethylammonium bicarbonate (TEAB), and dithiothreitol—were

One of the most important steps when analyzing embryo cultivation media is the depletion of serum albumin present in these samples. Tarasova et al. [1] described the innovative method of using immobilized antihuman albumin antibodies for depletion of small sample volumes. Briefly, for depletion of culture media samples from selected embryos, the sample was diluted using the phosphate buffered saline buffer (pH 7.4) consisting of 0.01 M phosphate buffer, 0.0027 M KCl, and 0.14 M NaCl. This buffer was further also used as a washing buffer A upon the sample loading. In order to ensure full sample recovery from the depletion column, a ready-to-use elution buffer from Agilent (pH 2.25) (Agilent Technologies, CA, USA) was used as buffer B. We have developed a new column for the depletion of human albumin by immobilizing the antihuman albumin antibodies to the monolithic support disk, the CIMac-HSA column, especially for analysis of small sample amounts, which also occur in IVF samples. This column was used in an ICS-5000 inert HPLC system (Dionex-Thermo Scientific, Germering, Germany) for albumin depletion with a column flow rate of 0.3 mL/min. Upon sample injection, the loading and washing buffer A was pumped through the column for 5 min, and the flow-through fraction was collected (V = 350 μL). This fractions' volume corresponds to the full absorbance peak and contains all proteins that were not trapped on the column. The albumin was trapped by the interaction with the antibodies on the column's surface, and it was eluted by increasing the amount of the eluting mobile phase from 5 to 10 min. The column was, finally, flushed with the loading buffer A for additional 4 min, and this step was followed by an additional washing step with buffer B and, finally, equilibrating step using, again, the loading buffer A for 13 min. The total time for completing this depletion protocol is 30 min when applying the column flow rate of 0.3 mL/min. During this time, the very important column wash step and the complete re-equilibration of the column preceding the next depletion run is being perfromed. The flow rate used was selected for maximizing the protein's interaction time and was a compromise between the speed and efficacy of operation. If desired, higher column flow rates can be used without losing much of the column's performance [2], but this shall be carefully examined

**2. Materials and methods**

analysis of low samples amounts.

**2.2 Proteomics sample preparation**

purchased from Sigma-Aldrich (Vienna, Austria).
