INTRODUCTION:

The World Health Organization (WHO) has universally accepted infertility as a public health issue. Several scientists are taking the initiative to develop new therapeutic approaches for effective infertility treatment [1]. Infertility is a most critical disorder in the reproductive system, described as a couple's inability to access a medical pregnancy after 12 months of regular, unprotected sexual intercourse or loss of an individual's ability or reproductive capacity with his/her spouse. Several factors, including but not limited to the quantity and quality of the ovarian reserve, are responsible for infertility [2,3].

Such factors include age, delayed pregnancy, poor diet, non-exercise or exercise, obesity, psychological stress, alcohol consumption, smoking, drug use, exposure to toxins and chemicals in the atmosphere, genital tract infections and sexually transmitted diseases [4]. There are no accurate figures of the global infertility rate, but more than 186 million people suffer from infertility, most of which belong to developing countries [2]. The global incidence of female infertility is growing and ranges between 10 and 20%. Due to the importance of women's health, several studies have been carried out to improve the health of women [5]. Treatment with fertility drugs is the primary approach for women with ovulatory disorders that want to conceive. Treatments chosen for a patient can differ and depend on the infertility cause defined [6].

Different drugs used based on various causes of infertility include:estrogen receptor modulator like clomiphene citrate [7], aromatase inhibitors such as letrozole and anastrozole [8], gonadotropins including menotropins [9] and human chorionic gonadotropins [10], Gonadotropin-releasing hormone analogs like gonadorelin acetate, nafarelinacetate, leuprolide acetate, triptorelinpamoate, goserelin acetate, ganirelix acetate and cetrorelix acetate [11,6].

Follicle stimulating hormone (FSH) is another class of hormonal drug used widely for the treatment of female infertility. FSH is used when PCOS and hypogonadotropic hypogonadal anovulation induces infertility, where the rate of FSH is suboptimal due to arrest and anovulation in follicular growth [9]. Urinary FSH is extracted and purified from urine obtained from post-menopausal women. Purified FSH is injected intramuscularly or injected subcutaneously in order to improve the fertility [12]. The physicochemical properties of FSH are given in Table 1 [13,14].

Table 1: Physicochemical properties of FSH (Follitropin)

Name	Human follicle stimulating hormone
Description	White powder
Molecular Formula	C₉₇₅H₁₅₁₃N₂₆₇O₃₀₄S₂₆
Molecular Weight	30 kDa
Melting Point	55 °C
Isoelectric point	5.5
t_1/2	3-4 hours
Protein average weight	22672.9 Da
Dose	Min. 75 IU/Day for 1-2 week; SC/IM Max. 300 IU/Day for 1-2 week; SC/IM

In the present study, various analytical tools have been used in order to evaluate the integrity of urinary isolated FSH. The availability and application of suitable technologies for improved assessment of the light absorption characteristics, structural integrity, purity and consistency of FSH is the main objective of this study. Appropriate analytical tools to allow a physicochemical evaluation of urinary FSH is important for achieving this objective.

MATERIALS AND METHODS:

Purified urinary human follicle stimulating hormone (FSH) was purchased from ProSpec-TanyTechnoGene Ltd., Ness-Ziona, Israel. Human FSH Elisa kit was supplied by Demeditec Diagnostics GmbH, Kiel, Germany. Pyrogen free sterile water was obtained from Sun Pharmaceuticals India Ltd., Mumbai.Sodium dodecyl sulphate and Commassie blue dye were purchased from Sigma Aldrich, St Louis, MO, USA. All other chemicals and solvents were of analytical grade.

Description/ Appearance of FSH:

FSH was noted visually to verify that it complies with the vendor's specifications as per the certificate of analysis (CoA).

Determination of UV absorption spectrum of FSH:

The UV absorption spectrum of FSH was determined by using UV-Visible spectrophotometer (UV-1601PC, Shimadzu, Japan). FSH solution (5µg/mL) in phosphate buffer saline pH 7.4 was scanned in UV range of 200 to 400nm against the blank.

Determination of circular dichroism spectra of FSH:

Circular dichroism (CD) spectra of pure FSH sample in PBS pH 7.4 were recorded by scanning the sample in CD spectrophotometer (J-815, JASCO, Tokyo, Japan). About 350µL (10µg) of pure FSH samples were kept in a rectangular quartz cuvette of path length 1mm. The samples were scanned in the wavelength range of 280-190 nm in triplicate at 25°C using 1 nm bandwidth. The baseline correction was done by using the PBS pH 7.4 [15,16].

Determination of molecular weight by SDS-PAGE:

The molecular weight of FSH was examined by SDS-PAGE method. The minimum detectable amount (≈ 15 µg in 30µL) was dissolved in sample buffer (2X) and processed with 5X loading buffer. The protein sample was allowed to run on a gradient gel of polyacrylamide (12% w/v) for 3 hours with 1X running buffer. The gel stack was stained with commassie blue dye after migration of the protein sample and further de-stained overnight to remove excess dye. The gel stack was observed under BioRad imager and compared with the standard molecular weight markers of range 10 to 170 KD (PageRuler^TM, ThermoFisher Scientific) to trace the protein molecular weight.

Determination of FTIR spectra of FSH:

FSH sample (500µg/mL) was prepared in Milli Q water and scanned in the IR range of 4,500 to 400 cm^-1 in IR spectrophotometer (Spectrum Two^TM, Perkin Elmer Inc., USA). Milli Q water was taken as blank and subtracted from the spectra. Subtracted data was baseline corrected and smoothed, and then plotted.

FSH standard ELISA test procedure:

FSH solid phase ELISA works on the sandwich principle. It consists of microtiter well plate which is pre-coated with a monoclonal antibody that specifically binds to the unique antigenic domain of FSH molecule. The patient serum sample was incubated with an anti-FSH monoclonal antibody conjugated with horseradish peroxidase enzyme in the well plate. The free conjugate was washed with aqua dest. The bound enzyme is proportionate to FSH concentration in the sample which in-turn develops the colour when substrate solution is added. The intensity of colour developed is proportional to the concentration of FSH in the sample. The same principal and test was used for estimation of FSH in in vitro samples.

Assay Procedure:

The assay test procedure was performed as per the manufacturer’s protocol. The ready to use FSH control (0mIU/mL) and standards (5-100mIU/mL) were dispensed into wells (25µL) and incubated for 30 min with anti-FSH monoclonal antibody conjugated with horseradish peroxidase enzyme (100µL). The wells were rinsed thoroughly with aqua dest (400µL per well) and 100µL of the substrate solution (Tetramethylbenzidine) was added so as to incubate for 10 min. The enzymatic reaction was terminated with 50µL stop solution in each well (1N acidic solution) and the absorbance of each well was measured at 450nm in a micro plate reader (ELx800, BioTek Instruments Inc., Vermont, USA). Assay was performed for two replicates and the mean value was calculated. Mean absorbance value (Y-axis) vs concentration (X-axis) was plotted. Regression coefficient (R²) and slope was calculated from the plot.

RESULTS AND DISCUSSION:

Description/ Appearance of FSH:

Human follicle stimulating hormone (FSH) obtained from ProSpec-Tany Techno Gene Ltd., Ness-Ziona, Israel, is a sterile, filtered, lyophilized, white colored, amorphous powder. It is in compliance with the CoA with respect to the organoleptic as well as physicochemical properties. FSH is hygroscopic powder with molecular weight 30kD and isoelectric point 5.5 [13,14]. It is also known as Follitropin subunit beta or follicle stimulating hormone beta subunit or FSH-beta or FSH-B etc.

Determination of UV absorption spectrum of FSH

Fig.1 showed UV absorption spectrum of FSH from 200 to 400nm. Due to the presence of histidine, aromatic amino acid, cysteine and methionine, the spectrum has a band around 230nm. The peak around 200nm may be due to an artifact produced by stray light, or could be the major π- π* transition of the peptide bond, suggesting a significant contribution of random structures. The aromatic absorption band of FSH is appeared between 250 to 280nm. The absorption maxima of FSH at the concentration of 5µg/mL in phosphate buffer saline pH 7.4 was found to be 212.4 nm, which is in close agreement with [17].

Fig. 1. UV absorption spectrum of FSH

Determination of circular dichroism (CD) spectra of FSH:

CD determines the difference in the absorption of circularly polarized light on the left and right hands caused by structural asymmetry. CD spectroscopy is a powerful tool to investigate the physical stability of proteins due to its sensitivity to structural modifications. Secondary protein structures in the far UV region (around 180-250nm) can be explored by CD spectroscopy. More ordered structure follows stronger (positive or negative) CD signals. Several secondary structures have distinct CD spectra, and since helices have stronger CD signals than β- structures, no direct comparisons can be made between distinct proteins to conclude on the degree of ordered structures. Studies are generally conducted by identifying the CD spectrum as a function of external variables such as pH, temperature, stabilizer, surfactant or denaturant concentration and storage time [16]. The well-defined patterns of pure proteins with antiparallel β-pleated sheets or β-helices appeared as negative bands between 210-218nm and positive bands at 190-195nm [18]. Fig.2 shows CD spectra of pure FSH sample in PBS pH 7.4 which was recorded at 25^oC. The results revealed that pure FSH showed positive antiparallel β-pleated sheets at 195nm and negative antiparallel β-pleated sheets at 212nm. Such pattern of CD spectrum indicated the well-defined conformation of the FSH secondary structures. The results of CD spectroscopy of pure FSH were found in accordance with previous reports [16,17].

Fig. 2. Circular dichroism spectrum of FSH

Determination of molecular weight by SDS-PAGE:

SDS-PAGE analysis of pure FSH clearly indicated that the β-subunit migration takes place under the electrophoretic conditions. SDS-PAGE analysis of FSH was performed in triplicate. Each time SDS-PAGE showed bands with apparent molecular weight ≈ 30kD (Fig.3.). This band pattern appeared is in accordance with the CoA. Absence of any other band clearly revealed that the FSH sample is free from impurities and stable at electrophoretic conditions.

Fig. 3. SDS-PAGE gel electrophoresis of standard FSH

Determination of FTIR spectrum of FSH

The FTIR spectrum of standard FSH (Fig.4) showed the characteristic peak at 1640-1680 cm^-1, which corresponds to carbonyl group (-C=O) of amide. The peak of secondary amide group (-CONH-) corresponding to 1450-1540 cm^-1was also present in the spectrum. Also, amine (-NH-) group appeared at peak 3200 cm^-1. The methylene groups are appeared at 2800-2900 cm^-1. These spectral data analysis clearly indicated the precise protein structure of FSH.

Fig. 4. FTIR spectrum of standard FSH

FSH standard ELISA test procedure:

FSH standard ELISA was performed as per the manufacturer’s protocol. The results (Table 2 and Fig. 5) obtained from standard test procedure represent the liner relationship between concentrations of FSH standards (5 to 100mIU/mL) and the absorbance, performed by ELISA plate reader at 450nm. The linearity equation for the assay is represented as y = 0.017x + 0.055, having a slope value of 0.017 and intercept value of 0.055. The regression coefficient (R²) obtained for the linear equation was found to be 0.9998. These obtained results suggest that absorbance values of FSH standards are in accordance with their respective concentrations without any major deviations. Thus, this linearity equation can be used for the estimation of FSH in unknown serum samples as well as in vitro samples.

Fig. 5. Calibration curve for standard FSH

Table 2: Concentration versus absorbance data for FSH by ELISA technique

Conc. (mIU/mL)	Abs
0	0.045±0.04
5	0.15±0.01
10	0.225±0.01
20	0.405±0.01
50	0.900±0.04
100	1.765±0.08

CONCLUSIONS:

The present study concluded that various analytical techniques such as UV absorption, circular dichroism, molecular weight determination by SDS-PAGE, FT-IR as well as standard ELISA test were found to be useful to assess the light absorption characteristics, structural integrity, purity and consistency of FSH

CONFLICT OF INTEREST:

The authors declare no conflict of interest, financial or otherwise.

REFERENCES:

1. Boivin J, Bunting L, Collins JA, Nygren KG. International estimates of infertility prevalence and treatment-seeking: potential need and demand for infertility medical care. Hum Reprod. 2007; 22(6): 1506-12.

2. Vander BM, Wyns C. Fertility and infertility: Definition and epidemiology. ClinBiochem. 2018; 62: 2-10.

3. Bala J, Agrawal Y, Seth S, Goyal V, Kumar P. Correlation between anti-mullerian and follicle-stimulating hormone in female infertility. Int J Health Allied Sci. 2014; 3: 232-36.

4. Petragalia F, Serour GI, ChapronC.The changing prevalence of infertility. Int J Gynecol Obstet. 2013; 123: S4-S8.

5. Direkvand-Moghadam A, Sayehmiri K, Delpisheh A. The global trend of infertility: an original review and meta-analysis. Int J Epidemiol Res. 2014; 1 (1): 35-43.

6. Che S, Eaton A, Moultry AM. A pharmacotherapeutic review of treatment options for infertility in women. Formulary. 2005; 40: 329-41.

7. Lieu CL and Yoshida T. Infertility. In: Dipiro J.T., Talbert R.L., Hayes, P.E., et al, eds. Pharmacotherapy: A Pathophysiologic Approach. 2002; 5th ed. New York: McGraw Hill. Pp.1431–1443.

8. Polyzos NP, Tzioras S, Badawy AM. Aromatase inhibitors for female infertility: a systematic review of the literature. Reprod Biomed Online. 2009; 19(4): 456-71.

9. Smith C, Grimm M, Schwegel M. Treatment of infertility in women. J Am Pharm Assoc. 2012; 52: e27–e42.

10. Weiss JM, Ludwig M, Ortmann O, Diedrich K. GnRH antagonists in the treatment of infertility. Ann Med. 2003; 35: 512-22.

11. Hayden C. GnRH analogues: applications in assisted reproductive techniques. Eur J Endocrinol. 2008; 159: S17–S25.

12. Bassett R, Ceccarelli D, Crisci C, di Tria AM, Mancinelli M, Martelli F, Mendola D, Pezzotti A. Comparative analytical characterization of two commercial human follicle-stimulating hormones extracted from human urine. Curr Med Res ResOpin. 2005; 21(6): 899-905.

13. Palagiano A, Nesti E, Pace L. FSH: urinary and recombinant. Eur J ObstetGynecolReprod Biol. 2004; 115: S30-3.

14. Ulloa-Aguirre A, Damián-Matsumura P, Jiménez M, Zambrano E, Díaz-Sánchez V. Biological characterization of the isoforms of urinary human follicle-stimulating hormone contained in a purified commercial preparation. Hum Reprod. 1992; 7(10): 1371-8.

15. Schmidt A, MacColl R, Lindau-Shepard B, Buckler DR, Dias JA. Hormone-induced conformational change of the purified soluble hormone binding domain of follitropin receptor complexed with single chain follitropin. J Biol Chem. 2001; 276(26): 23373-81.

16. Sjögren HU, Bagger HL. Stabilization of FSH. European patentEP 2598160B1, 2011.

17. Groen BH, Bloemendal M, Mulders JWM, Hadden JM, Chapman D, Van Stokkum IHM, et al. Spectroscopic characterization of recombinant follicle stimulating hormone. SpectrochimActa Part A MolBiomolSpectrosc. 1996; 52(10): 1331–46.

18. Greenfield NJ. Using circular dichroism spectra to estimate protein secondary structure. Nat Protoc. 2006; 1(6): 2876-90.

Received on 16.11.2019 Modified on 08.01.2020

Research J. Pharm. and Tech 2020; 13(9):4315-4319.

DOI: 10.5958/0974-360X.2020.00762.3