**Abstract**

Gonadotropins, including human chorionic gonadotropin (hCG), have been used since and for several decades to treat infertility by ovarian stimulation. hCG is the most important protein for embryogenesis and embryo development and implantation in uterus upon fertilization of oocytes. The hCG used for in-vitro fertilization (IVF) is being extracted from urine of pregnant women, and it does inevitably contains other proteins secreted into urine. The presence of other proteins varies from batch to batch, and it can be significantly high. Due to the fact that many of the proteins identified in these formulations can trigger an allergic reaction, which, in turn, can affect the embryogenesis and prevent embryo implantation, it is very important to check the amount and type of contaminant proteins in pharmaceutical formulations. It was found that the total protein content varied from batch to batch, and a large number of contaminant urinary proteins were identified in all analyzed samples except for the recombinant product.

**Keywords:** embryogenesis, in-vitro fertilization, human chorionic gonadotropin, proteomics

### **1. Introduction**

Human chorionic gonadotropin (hCG) is one of the most widely studied markers in embryonic development. It is used as an obstetric marker, and it is often regarded as little more than a signal for maternal recognition of pregnancy. Human chorionic gonadotropin is a member of the dimeric glycoprotein hormone family that also includes FSH, LH, and TSH. The members of this hormone family share a common α subunit and have a unique β subunit to each hormone. Additionally, each hormone shows a different level of glycosylation, which determines circulating half-life and receptor binding affinity [1].

The success of embryo implantation upon IVF and embryo transfer depends on various factors related to the embryo quality and patient's endometrial receptivity. Upon implantation, it is important that the embryo reaches the endometrial cavity during the period of time in which the endometrium is receptive. It has been estimated that 50–75% of lost pregnancies are due to the embryo's implantation failure as described by Tsampalas et al. [2]. Many factors are involved in the implantation

process, which is very intricate process and the success can be influenced by many factors. The most important regulation of embryogenesis and embryo implantation in uterus is performed by hCG, and studies performed by Licht et al. [3] showed that an intrauterine injection of 500 IU of hCG/mL inhibited the expression of intrauterine insulin-like growth factor-binding protein 1 and the macrophage colony stimulating factor. It was also demonstrated that an intrauterine injection of 500 IU of hCG performed before embryo transfer significantly improved both the implantation and pregnancy rates in IVF/intracytoplasmic sperm injection cycles.

The use of gonadotropin derived from either animal or human tissues was not always without clinical danger (e.g., antibody formation from pregnant mare serum gonadotropin and Creutzfeld-Jacob disease from human pituitary gonadotropin).

The hCG is being extracted from urine of pregnant women (uhCG) for almost three decades, and it is being used for induction of mid-cycle follicular maturation and ovulation in women undergoing an IVF treatment. Originally, hCG in pharmacological preparations was derived only from the urine of pregnant women. However, due to their biological origin, these hCG products show large biological variability and significant batch-to-batch variation. Therefore, recombinant technology has been introduced for the production of recombinant hCG (rhCG) with higher purity and higher batch-to-batch reproducibility and the possibility to control their availability in different doses The availability of different amounts of active substance in recombinant products provides a good starting point to develop personalized therapy for patients depending on their individual hormonal status.

Although a recombinant product is available on the market, urinary preparations of this hormone are still manufactured and are widely used [4–9]. Often, the urinary preparations are associated with problems arising from the fact that the starting material might origin from unknown sources, have poor purity, and lead to large batch-to-batch variations in activity and the amount of other proteins.

Analysis of commercially available, uhCG, was performed earlier [8], and discussions about the possible risks of infection were published [9–15].

We have analyzed several batches of both urinary derived and recombinant hCG formulations and have compared the obtained results in terms of number of identified proteins and their function during the embryogenesis.

#### **2. Materials and methods**

#### **2.1 Analyzed hCG: source of the material**

Different batches of both uhCG and rhCG were purchased through the pharmacy of the General Hospital of Vienna and by direct purchase from pharmacies in Bosnia-Herzegovina and Serbia. Details on manufacturer and batches analyzed are shown in **Table 1**.

#### **2.2 Proteomics sample preparation**

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), triethyl bicarbonate (TEAB), and dithiothreitol were purchased from Sigma-Aldrich (Vienna, Austria). Digestion of hCG and FSH was performed using the routine approach described in earlier publications [16].

**13**

 **2.4 Data analysis**

**Table 1.**

Tyr were set as variable modifications.

*Background Proteins in Human Chorionic Gonadotropin Pharmaceutical Formulations…*

Pregnyl\_M038101 5000 IU/mL hCG Urinary-

Pregnyl\_M011526 5000 IU/mL hCG Urinary-

Pregnyl\_354043 5000 IU/mL hCG Urinary-

Predalon\_117730 5000 IU/mL hCG Urinary-

Pregnyl\_117447 5000 IU/mL hCG Urinary-

Pregnyl\_116935 1500 IU/mL hCG Urinary-

Pregnyl\_M018720 5000 IU/mL hCG Urinary-

Chorimon\_160432 5000 IU/mL hCG Urinary-

*Analyzed products, batch numbers, and manufacturers' names of analyzed samples.*

**Dosage Principal** 

**component**

**Origin Manufacturer**

MSD (N.V. organon, NL)

MSD (N.V. organon, NL)

MSD (N.V. organon, NL)

MSD (N.V. organon, NL)

MSD (N.V. organon, NL)

MSD (N.V. organon, NL)

MSD

Institut Biochimique SA, CH

(Feltham, Middlesex, UK)

derived

derived

derived

derived

derived

derived

derived

derived

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

**Commercial name and charge number**

 **2.3 Chromatographic separation and detection**

B (50%AcN, 30%MeOH, 10% TFE, 10% water, and 0.1%FA).

All separations were performed using the nanoRSLC UltiMate 3000 HPLC system coupled to the Q-Exactive Orbitrap Plus mass spectrometer (ThermoScientific, Vienna, Austria). Digested hCG and FSH were separated using trap column for sample loading and focusing (Acclaim PepMap C18, 300 μm ID × 5 mm, ThermoScientific, Vienna, Austria) and the pillar-arrayed-column (μPAC) with 2 μm interpillar distance and 2 m separation path (PharmaFluidics, Gent, Belgium) as the separation column. Both columns were operated in the column oven at 50°C. The sample was loaded onto the trap column using aqueous 0.01% HFBA at 30 μL/min and separated using the gradient generated by mixing mobile phases A (95% water, 5%AcN, and 0.1%FA) and

Ovitrelle 250 μg/0.5 mL hCG Recombinant Merck Serono

Detection was performed using both UV at 214 nm and MS using positive electrospray ionization with a nanosource and ionization needle of 20 μm ID and 10 μm tip. The 20 most intensive signals in each MS scan were selected for MS/MS (fragmentation) with HCD at normalized collision energy (NCE) set to 30.

Raw data were transformed into Mascot generic files (MGF) for database search

using MSConvert (www.proteowizzard.sourceforge.net). The database search was performed using the in-house Mascot server v.2.6 and the SwissProt database (status January 2018) using following parameters: trypsin was selected as enzyme, peptide mass precision was set to 10 ppm, carboxymethylation on Cys was selected as fixed modification and oxidation on Met, and phosphorylation on Ser, Thr, and

Pathway analysis was performed using String (www.string-db.org).

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


#### **Table 1.**

*Embryology - Theory and Practice*

hormonal status.

**2. Materials and methods**

shown in **Table 1**.

 **2.1 Analyzed hCG: source of the material**

 **2.2 Proteomics sample preparation**

process, which is very intricate process and the success can be influenced by many factors. The most important regulation of embryogenesis and embryo implantation in uterus is performed by hCG, and studies performed by Licht et al. [3] showed that an intrauterine injection of 500 IU of hCG/mL inhibited the expression of intrauterine insulin-like growth factor-binding protein 1 and the macrophage colony stimulating factor. It was also demonstrated that an intrauterine injection of 500 IU of hCG performed before embryo transfer significantly improved both the implantation and pregnancy rates in IVF/intracytoplasmic sperm injection cycles. The use of gonadotropin derived from either animal or human tissues was not always without clinical danger (e.g., antibody formation from pregnant mare serum gonadotropin and Creutzfeld-Jacob disease from human pituitary gonadotropin). The hCG is being extracted from urine of pregnant women (uhCG) for almost three decades, and it is being used for induction of mid-cycle follicular maturation and ovulation in women undergoing an IVF treatment. Originally, hCG in pharmacological preparations was derived only from the urine of pregnant women. However, due to their biological origin, these hCG products show large biological variability and significant batch-to-batch variation. Therefore, recombinant technology has been introduced for the production of recombinant hCG (rhCG) with higher purity and higher batch-to-batch reproducibility and the possibility to control their availability in different doses The availability of different amounts of active substance in recombinant products provides a good starting point to develop personalized therapy for patients depending on their individual

Although a recombinant product is available on the market, urinary preparations of this hormone are still manufactured and are widely used [4–9]. Often, the urinary preparations are associated with problems arising from the fact that the starting material might origin from unknown sources, have poor purity, and lead to

We have analyzed several batches of both urinary derived and recombinant hCG formulations and have compared the obtained results in terms of number of identi-

Different batches of both uhCG and rhCG were purchased through the pharmacy of the General Hospital of Vienna and by direct purchase from pharmacies in Bosnia-Herzegovina and Serbia. Details on manufacturer and batches analyzed are

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), triethyl bicarbonate (TEAB), and dithiothreitol were purchased from Sigma-Aldrich (Vienna, Austria). Digestion of hCG and FSH was performed using

large batch-to-batch variations in activity and the amount of other proteins. Analysis of commercially available, uhCG, was performed earlier [8], and

discussions about the possible risks of infection were published [9–15].

fied proteins and their function during the embryogenesis.

the routine approach described in earlier publications [16].

**12**

*Analyzed products, batch numbers, and manufacturers' names of analyzed samples.*

#### **2.3 Chromatographic separation and detection**

All separations were performed using the nanoRSLC UltiMate 3000 HPLC system coupled to the Q-Exactive Orbitrap Plus mass spectrometer (ThermoScientific, Vienna, Austria). Digested hCG and FSH were separated using trap column for sample loading and focusing (Acclaim PepMap C18, 300 μm ID × 5 mm, ThermoScientific, Vienna, Austria) and the pillar-arrayed-column (μPAC) with 2 μm interpillar distance and 2 m separation path (PharmaFluidics, Gent, Belgium) as the separation column. Both columns were operated in the column oven at 50°C. The sample was loaded onto the trap column using aqueous 0.01% HFBA at 30 μL/min and separated using the gradient generated by mixing mobile phases A (95% water, 5%AcN, and 0.1%FA) and B (50%AcN, 30%MeOH, 10% TFE, 10% water, and 0.1%FA).

Detection was performed using both UV at 214 nm and MS using positive electrospray ionization with a nanosource and ionization needle of 20 μm ID and 10 μm tip. The 20 most intensive signals in each MS scan were selected for MS/MS (fragmentation) with HCD at normalized collision energy (NCE) set to 30.

#### **2.4 Data analysis**

Raw data were transformed into Mascot generic files (MGF) for database search using MSConvert (www.proteowizzard.sourceforge.net). The database search was performed using the in-house Mascot server v.2.6 and the SwissProt database (status January 2018) using following parameters: trypsin was selected as enzyme, peptide mass precision was set to 10 ppm, carboxymethylation on Cys was selected as fixed modification and oxidation on Met, and phosphorylation on Ser, Thr, and Tyr were set as variable modifications.

Pathway analysis was performed using String (www.string-db.org).
