**4.1 Contaminant proteins in urinary-derived samples**

As for the contaminants in urinary-derived products, uromodulin, which is also the major urinary protein, was a major hit following the major component. However, other proteins, such as alpha-1-microglobulin or apolipoprotein D, and prostaglandin were identified with high confidence. The contaminants in urinaryderived hCG formulations resemble urinary proteins identified in urine-only samples, which were described in a recently published study on stress-induced urinary incontinence [23].

Keratin was one of the major alien contaminants, and its presence suggests a contamination during the sample production and the origin might be the insufficient air-conditioning or the contamination of the packaging units, which might be traced back to the use of latex-made gloves, dust containing skin particles, etc. We can exclude the contamination in our lab since all sample preparation steps have been performed using the laminar flow and using nitrile gloves. The blank samples show no identifications of keratin or of other contaminations.

Additional distinction between the urinary and the recombinant products can be seen when looking at the GO analysis of the biological processes where these proteins are involved, as shown in **Figure 4**.

### **4.2 Contaminant proteins in recombinant samples**

Products originating from the production process employing recombinant method have significantly less contaminant proteins of human origin. To our best knowledge, until now, there are no reports describing contaminations for recombinant hCG. Upon analysis, the hCG was the major component identified with contaminants such as keratin and human serum albumin (HSA). These contaminants are easy to explain, we have given an explanation for keratin in previous section, and HSA is most probably a contaminant from the culturing medium of CHO that was not completely removed upon hCG extraction.

#### **Figure 4.**

*GO term analysis of proteins identified in both urinary and recombinant formulations showing differences in their abundancies in biological processes.*

*Embryology - Theory and Practice*

success rate.

health agencies and qualified bodies and from the European Medicines Agency. Companies producing medicines and medical devices must follow strict and detailed guidelines and secure that all operations are performed under governing GMP and GLP rules. These precautions shall secure the quality of the product and the safe use for the patients. Thennati et al. has described the method for the quality control of the recombinant product is ensured through analytical steps using SDS-gel separation and MALDI-ToF analysis of the final product [17]. Therefore, it was a great surprise to identify a number of proteins originating from the starting

A discussion on contaminant proteins in hCG formulations has already been published [4, 5, 7, 17–19], and several publications address the possibility of the presence of harmful substances in commercial formulation [13–15, 20–22] but no final decision was made and the urinary-derived hCG formulations are still widely used although it has been shown that recombinant hCG can be used with the same

Due to the lack of published data and reports on hCG formulations and the lack

of information about these products, we have analyzed commercially available formulations that are routinely prescribed for patients undergoing IVF treatment. The major active component, hCG, was identified in all analyzed samples and, in addition, human serum albumin (HSA), luteotropin subunit beta (LSHB), and glycoprotein hormones alpha chain (CGA). The presence of HSA in all samples can be explained by its secretion in urine and by the need of growing CHO cells, for recombinant hCG production, in culturing medium supplemented with HSA, which might contain a number of other proteins that were not removed when HSA purification was performed. The pathway and interaction analysis, shown in **Figure 3**, explains the dependence of co-expression between the hCG, LHB, and CGA and explains why these proteins can also be identified in recombinant products. Obviously, the production of hCG also induces the expression of other proteins involved in ovarian steroidogenesis, hormone activity, and regulation of hormone levels, which are identified with high confidence with MS/MS analysis. However, in this case, the identification of LHB is a false positive one, and we cannot claim that this protein is really present in the recombinant sample. The reason is that hCG and LHB have a common amino acid sequence between amino acids on positons 22

product (urine) in different batches of the final product.

**16**

**Figure 3.**

*products.*

*Pathway describing the interdependence of hCG and LHB both of which were identified in recombinant* 

#### **4.3 Embryogenesis and hCG, influence of contaminants**

Often, hCG is also known as "the hormone of pregnancy". It plays an important part for establishing and maintaining pregnancy, and it is extensively used in IVF procedures. A serial measurement of hCG every 48 hours is being used to confirm the early pregnancy and to distinguish between normally progressing pregnancies from ectopic pregnancies or spontaneous abortions [10]. It was shown that the miscarriage rate is higher when assisted reproductive techniques (ARTs) are applied than with naturally occurring pregnancy [11]. Girard et al. evaluated the association between early β-hCG concentration increases, blastocyst morphology, and pregnancy evolution in a single-blastocyst transfer program. In most IVF laboratories, blastocyst transfers are associated with higher success rates of implantation as compared with the cleavage stage embryo as shown by Oron et al. [12] and Girard et al. [10]. Most laboratories report that blastocysts are selected for transfer according to their morphological characteristics. That means that physicians decide on implantation based on, that is, expansion degree, hatching status of the blastocysts, inner cell mass, or trophectoderm characteristics.

It is taught that hCG is involved in embryo implantation by modulating the activity of collagenases and plasminogen activators in an in-vitro system, as described by Yagel et al. [13]. Furthermore, it is an interesting and noteworthy feature that most of the mediators that have earlier been considered essential for the implantation process (i.e., EGF and IL-1/IL-6) are also involved in the regulation of hCG biosynthesis by the placental syncytiotrophoblasts [13–15] probably by modulating trophoblast differentiation. Therefore, Licht et al. postulated that hCG may play a central part in the hypothetical embryo-maternal cross talk and tested the hypothesis by simulating the effect of a very early pregnancy on the decidualized endometrium. The results suggested that, no immunoreactive hCG was found in the peripheral circulation (hCG<5 mIU/mL) and the treatment did not alter progesterone secretion by the corpus luteum, thus suggesting that the effects observed were direct.

As described in previous paragraph, urinary-derived hCG contains high number of contaminant proteins. Considering that, particularly, EGF is present in high concentrations in almost all tested commercial preparations of urinary-derived hCG, and additional experiments are needed to find out whether the effects observed are due to hCG or to a contamination from the formulation.

Furthermore, contaminant proteins identified in hCG formulations can be the source of severe allergic reactions and can also trigger immune response that must not necessarily been manifested externally; however, it can negatively affect embryo implantation and embryogenesis. Koh et al. [16] and Phipps et.al [17] described the cases of IgE-mediated immunoreaction after intramuscular administration of urinary-derived gonadotropins. This type of allergic reactions occurs shortly after administration of urinary-derived gonadotropin, and it occurs with symptoms like shortness of breath, wheezing, flushing, general weakness, and dizziness. In all reported cases, the IgE-mediated allergic reaction could be confirmed after intradermal skin testing with the same uhCG lot. Testing with rhCG triggered no reaction, thus confirming the theory that some urinary proteins can trigger an immune response and might be responsible for anaphylactic reactions. Immune reaction could also influence the development of the embryo and hinder normal embryogenesis and embryo implantation in endometrium.

Kajihara et al. [18] proposed that some urinary-derived hCG (uhCG) impurities like EDN or ribonuclease 2 could influence inflammatory and immunological processes through regulation of apoptosis in endometrial cells and, therefore, directly influence embryo nesting and development during IVF. Considering that some patients undergo lengthy and intensive treatment with uhCG, an effect cannot be excluded.

**19**

**Author details**

**5. Conclusion**

human urine.

effective.

identified on products' leaflets.

Vienna, Austria

Tanja Panić-Janković

provided the original work is properly cited.

1

in general and embryo implantation in uterus.

and Goran Mitulović

© 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,

1,2\*

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

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

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

*Background Proteins in Human Chorionic Gonadotropin Pharmaceutical Formulations…*

immunosuppressive effect on T-cell-mediated allogenic rejection.

Uromodulin, as the most abundant protein in human urine, is also detected in various uhCG formulations as the most abundant one. Uromodulin has been described as a powerful stimulator of the immune system through its ability to bind on the surface of almost all blood cells and to encourage the cellular production of cytokines, increase lymphocyte proliferation and phagocytosis [19, 20]. Using a mouse model, de Silva Antunes et al. suggested that uromodulin, but also some other urinary proteins, that is, major urinary proteins [13, 14, 17] or kidney androgen-regulated protein act as a possible allergens in the T-cell-mediated allergic reaction [21]. Otherwise, Phinuster et al. [22] suggested protective role of uromodulin in amniotic fluid in the fetus defense against anti-allogenic antibodies and

Formulations of hCG, both urinary and recombinant, contain contaminant proteins that originate from either starting material or has been introduced during the manufacturing process. The information about these contaminants cannot be

We could show that urinary-derived products contain a significant number of human proteins that obviously originate from the starting raw material—the

We think that this issue must be approached and discussed since the recombinant proteins show no such contamination and their use has been proven safe and

Due to an important role played by hCG in embryo development in healthy subjects and embryo implantation during the IVF procedure, it is of great importance to thoroughly check the quality of the formulations and apply production steps for the best possible removal of all contaminant proteins from the final product and prevent possible adverse and allergic reactions, which might affect embryogenesis

*DOI: http://dx.doi.org/10.5772/intechopen.82652*

*Background Proteins in Human Chorionic Gonadotropin Pharmaceutical Formulations… DOI: http://dx.doi.org/10.5772/intechopen.82652*

Uromodulin, as the most abundant protein in human urine, is also detected in various uhCG formulations as the most abundant one. Uromodulin has been described as a powerful stimulator of the immune system through its ability to bind on the surface of almost all blood cells and to encourage the cellular production of cytokines, increase lymphocyte proliferation and phagocytosis [19, 20]. Using a mouse model, de Silva Antunes et al. suggested that uromodulin, but also some other urinary proteins, that is, major urinary proteins [13, 14, 17] or kidney androgen-regulated protein act as a possible allergens in the T-cell-mediated allergic reaction [21]. Otherwise, Phinuster et al. [22] suggested protective role of uromodulin in amniotic fluid in the fetus defense against anti-allogenic antibodies and immunosuppressive effect on T-cell-mediated allogenic rejection.
