Journal of Endocrinology and Reproduction

K. M. Rangari ¹, T. G. Shrivastav ¹

Affiliations
1 Dept of Reproductive Biomedicine, National Institute of Health and Family Welfare, Munirka, New Delhi-110067, IN

Abstract

The neem leaf extract (NLE) has been reported to exhibit spermicidal and oocidal activities under in vitro cultured conditions but the effect of NLE on various sperm function parameters have not been studied. Therefore, in the present study we have assessed effect of different concentrations of NLE on various sperm functions characteristics to develop a water-based vaginal contraceptive. The human sperm (10 million sperm/mL), were exposed to different concentrations of NLE (3, 9, 15, and 30mg/mL) for 20 seconds at 37°°C. Thereafter, sperm function characteristics such as motility, viability, hypo-osmotic swelling (HOS), nuclear decondensation (NDC), acrosome intactness (AI) and deoxyribonucleic acid (DNA) stability were analyzed. A dose-dependent inhibition of sperm motility was observed, while 15mg/mL dose immobilized 100% of spermatozoa. Whereas other sperm functional parameters like viability, DNA intactness, HOS, NDC and AI decreased from 94% to 9%, 71% to 29%, 77% to 25%, 62% to 29% and 53% to 25%, respectively. The data obtained in the present study reveal that treatment of NLE causes reduction in functional parameters to infertility range. Therefore, a preparation containing 15mg of NLE in 150 μl of 0.9% NaCl was tested in rat as a vaginal contraceptive, and the data revealed 100% efficacy in mating studies after 21 days of vaginal application. The findings indicate the potential use of NLE for developing water-based vaginal contraceptive.

Keywords

Azadirachta indica, Neem Leaf Extract (NLE), Sperm Function Tests, DNA Stability, Vaginal Contraceptive.

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T. G. Shrivastav ¹, S. Nara ¹, V. Tripathi ¹, Z. Sayeed ¹, K. Rangari ¹, S. K. Chaube ¹, H. Singh ²

Affiliations
1 Department of Reproductive Biomedicine, National Institute of Health and Family Welfare, Munirka, New Delhi-110067, IN
2 Centre for Biomedical Engineering, Indian Institute of Technology, Hauz Khaus, New Delhi, IN

Abstract

Preparation of horseradish peroxidase (HRP) carbamide that is HRP linked to urea (HRP-carbamide/HRP-U) is demonstrated and its potential application in the development of enzyme immunoassays (EIAs) for haptens is described. In this new strategy, the lysine residues of HRP were acylated and then acylated HRP was activated to create highly reactive functional groups by periodate oxidation of its carbohydrate moiety and, subsequently, forming a peptide bond with one of the amino groups of urea. The resulting HRP-carbamide was then coupled to carboxylic derivatives of cortisol-21-hemisuccinate (F-21-HS), 17α-OH progesterone-3-O-carboxymethyl-oxime (17α-OHP-3-O-CMO) and nandrolone-3-O-carboxymethyl-oxime (N-3-O-CMO) to prepare enzyme conjugates adopting N-hydroxysuccinimide-carbodiimide method. The F-21-HS-U-HRP, 17α-OHP-3-O-CMO-U-HRP, and N-3-O-CMO-U-HRP enzyme conjugates thus prepared were used for the development of an enzyme linked immunosorbent assays (ELISAs) for the estimation of cortisol, 17α-OHP and nandrolone. The sensitivity of cortisol, 17α-OHP and nandrolone assays were 0.4 ng/ml, 0.05 ng/ml, and 0.12 ng/ml, respectively, and the analytical recovery ranged from 93.3% to 100%, 94.3% to 98.7%, and 93.5 % to 104%, respectively. In the present study, the strategy adopted for preparing HRP-carbamide and its subsequent use in preparing enzyme conjugate has been shown to result in an increase in thermostability of enzyme conjugate, amino group's availability in enzyme for conjugation with increase in bridge length between analyte and enzyme to reduce steric hindrance.

Keywords

HRP-Carbamide, Immunoassay, 17α-OHP, Cortisol, Nandrolone, Urea.

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Kiran Rangari ¹, T. G. Shrivastav ¹

Affiliations
1 Department of Reproductive Biomedicine, National Institute of Health and Family Welfare, New Delhi -110067, IN

Abstract

Male infertility is a multi-factorial disorder. The functional ability of spermatozoa is primarily determined by their motility. Many factors are responsible for reduction in sperm motility but immunological and / or hormonal factors are vital. At present, the impact of immunological factor along with hormonal imbalance in reducing sperm motility is not known. In the present study, steroid hormones (testosterone, estradiol, progesterone and cortisol) and anti-sperm antibodies (ASAB) were measured in serum and seminal plasma of males with normal or reduced sperm motility. The ASAB was not detected in serum samples of volunteers with normal sperm motility but detected in 22.7% of serum samples of volunteers with low sperm motility. On the other hand ASAB was detected in seminal plasma of volunteers with normal sperm motility (31.8%) and low sperm motility (36.3%). Significantly low levels of testosterone and cortisol (p<0.001 and p<0.05, respectively), and significantly high levels of estradiol and progesterone (p<0.005 and p<0.001, respectively) were observed in the seminal plasma of samples with low sperm motility when compared with samples with normal sperm motility. A correlation was observed between presence of ASAB and altered steroid levels such as low cortisol, and high progesterone and estradiol in seminal plasma of volunteers with low sperm motility. There was no difference in serum steroid levels. These results suggest a relationship between testicular steroid hormone levels with autoimmunity to sperm antibodies.

Keywords

Anti-Sperm Antibodies, Male Infertility, Sex Steroids, Sperm Motility.

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T. G. Shrivastav ¹

Affiliations
1 Department of Reproductive Biomedicine, National Institute of Health and Family Welfare, Munirka, New Delhi 110067, IN

Abstract

Charcoal stripped serum, generally used for the preparation of standards for enzyme immunoassay, some time gives positive bias values. To eliminate positive biases in testosterone immunoassay, natural steroids that don't have any cross-reaction with testosterone antibody, have been supplemented to charcoal stripped serum. This supplemented serum was used for the preparation of standards for the development of direct testosterone enzyme Immunoassay.

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