Synthesis and characterization of PCL-DA:PEG-DA based polymeric blends grafted with SMA hydrogel as bio-degradable intrauterine contraceptive implant.

Presently available long-acting reversible female contraceptive implants are said to be an effective way of preventing unintended pregnancy. Unacceptable side effects attributed by these contraceptive implants act as a major drawback for the practitioners. These problems pave the way for the development of a new form of long-acting non-hormonal female contraceptive implant, especially in the developing countries. PCL-DA: PEG-DA polymeric scaffold is grafted with Styrene Maleic Anhydride (SMA) based hydrogel, and their physicochemical, thermal and biological parameters are being explored for developing a bio-degradable form of the non-hormonal intrauterine contraceptive implant. With the fixed ratio of PEG-DA: PCL-DA polymer, SMA hydrogel was added at four different concentrations to determine the optimum concentration of SMA hydrogel for the development of a promising long-acting biodegradable intrauterine contraceptive implant. Structural elucidation of the polymers was confirmed using 1H and 13C NMR spectroscopic analyses. The physiochemical characterization report suggests that SMA hydrogel interacts with the PCL-DA: PEG-DA polymeric scaffold through intermolecular hydrogen bonding interaction. The in-vitro spermicidal activity of the polymeric scaffold increases when the concentration of SMA based hydrogel in the polymer samples is increased without showing any significant toxicological effects. From the study results, it may be concluded that SMA hydrogel grafted PCL-DA: PEG-DA scaffold can be developed as intra-uterine biodegradable non-hormonal female contraceptive implant due to its excellent bio-compatibility and spermicidal activity.

Biocompatible polyvinyl alcohol and RISUG® blend polymeric films with spermicidal potential.

Majority of the commercially available vaginal contraceptives encompasses cervicovaginal membrane disrupting detergent molecules as pharmacologically active ingredients. Development of a tissue-compatible vaginal contraceptive agent is necessary to circumvent the existing demand for female contraception in the reproductive healthcare sector. With this objective, the present study delineates the use of RISUG® based non-hormonal female contraceptive films. RISUG® was blended with polyvinyl alcohol (PVOH) to formulate biodegradable intra-vaginal contraceptive films. The formulated films were characterized for their thermal, physiochemical and biological features. The results showed that both RISUG® and PVOH were miscible and interacted at the intermolecular level. Variations in the concentration of RISUG® resulted in the changes in physicochemical, thermal and rheological characteristics of the formulated blends. In vitro toxicological assay of the polymeric formulations did not show any significant toxicity. However, the blend films retained spermicidal potential of RISUG®. Furthermore, in vivo toxicological evaluation of the polymeric blend in the rat model revealed about their biocompatibility with no significant organ toxicity, hematological and biochemical alterations. These results together confirm the potential applicability of the PVOH:RISUG® blend films as a vaginal contraceptive.

RISUG® based improved intrauterine contraceptive device (IIUCD) could impart protective effects against development of endometrial cancer.

Intrauterine Contraceptive Devices with multifaceted application potential is a need of an hour. Although, copper-based IUDs exert an effective contraceptive as well as anticancer effects in a long-term basis, but also results in multiple complications. In this regard, RISUG® a polymer based contraceptive device has been introduced as a suitable alternative. However, its potential to impart protective effects against development of endometrial cancer still remains unexplored. This article presents the hypothesis on this unexplored domain and provides scientific facts to support the hypothesis. The mechanism of anticancerous activity is hypothesized that RISUG® involves its lipid membrane destabilizing activity. This activity is modulated by both, the cellular microenvironment and lipid bilayer composition. Acidic environment along with the significantly higher fluidic nature of lipid bilayer of the cancerous cells make them more prone to lipid solubilisation effect of RISUG®. We here present an in-depth insight into the factors that would favour faster solubilisation of cancer cell membrane, thereby exerting an anticancer effect.

Impact of styrene maleic anhydride (SMA) based hydrogel on rat fallopian tube as contraceptive implant with selective antimicrobial property.

Development of non-hormonal female contraception is a need to combat against increasing population growth. The presently available short term or long term female contraceptives and sterilization methods have their own restrictions and side effects. With this objective, herein, we describe an innovative insight about the use of hydrogel formulation consisting of Styrene Maleic Anhydride (SMA) dissolved in Dimethyl Sulfoxide (DMSO) as non-hormonal fallopian tube contraceptive implant. Firstly, in vitro behavior of SMA hydrogel was evaluated by in vitro swelling and rheological properties to comprehend the polymeric hydrogel property post implantation inside the fallopian tube. Simulated Uterine Fluid (SUF) was used to simulate female reproductive tract environment in this study. Mechanical strength of the hydrogel when subjected to dynamic environment post implantation in the fallopian tube was estimated by the G' values demonstrated. SMA hydrogel expressed selective antimicrobial activity against opportunistic pathogens (Escherichia coli, Pseudomonas aeruginosa and Staphylococcus aureus) while having limited consequence over the growth of Lactobacillus spp. After confirmation of cytocompatibility against primary rat endometrial cell lines, the polymeric hydrogel was implanted inside the uterine horns of Sprague-Dawley rats. In vivo biocompatibility of the hydrogel was confirmed by histological and immunohistochemical evaluation of uterine tissue sections. Hematology, blood biochemistry and organ toxicity (kidney, liver, spleen, lungs and heart) also revealed biocompatibility of SMA hydrogel. The results of the current study indicated that the SMA copolymer dissolved in DMSO to form hydrogel has excellent biocompatibility for application as female contraceptive gel which can be implanted in the fallopian tube.

Probing suitable therapeutic nanoparticles for controlled drug delivery and diagnostic reproductive health biomarker development.

Nanomaterial mediated drug delivery represents a highly promising technique while its selectivity for reproductive healthcare application still remains a challenge. Since the delicate structure and functional role of reproductive tissue and gametes require the use of biocompatible nanomedicine/devices that do not affect fertility or the development of resulting offspring, this paper reports an intercomparative study of human spermatozoa interaction with three different nanoparticles (NPs) namely; iron oxide (Fe3O4), multiwalled carbon nanotubes (MWCNT) and graphene platelet nanopowder (GPN) to probe their suitability for drug delivery carrier and biomarker development purposes. ATR-FTIR results revealed that the sperm cell interaction with GPN had maximum amide I absorption for cell proteins and CO stretching of the peptide backbone at the band around 1657 cm(-1) followed by iron oxide NPs whereas MWCNT had no absorption. These results showed that GPN followed by iron oxide NPs got maximally entrapped by cell membrane protein with maximum disruption but MWCNT exhibited less entrapment but significantly higher internalization which was further validated by morphological analysis of these cell NP interaction by SEM, HRTEM and fluorescence microscopy. The uptake kinetics and penetration mechanism of NPs were examined with isothermal titration calorimetry (ITC). Interestingly, ITC results confirmed ATR-FTIR and morphological observations that the binding of GPN and Fe3O4 NPs with cell was exothermic and their bindings were favored by both negative enthalpy and positive entropy whereas in the case of MWCNT it was endothermic supported by unfavorable positive enthalpy and a favorable entropy change. Hence, it was evident that MWCNT had better internalization efficiency without disrupting the sperm lipid membrane compared to Fe3O4 and GPN NPs. Therefore, this work proposes CNT as promising means.

An emerging interface between life science and nanotechnology: present status and prospects of reproductive healthcare aided by nano-biotechnology.

Among the various applications of nano-biotechnology, healthcare is considered one of the most significant domains. For that possibility to synthesize various kind of nanoparticles (NPs) and the ever-increasing ability to control their size as well as structure, to improve surface characteristics and binding NPs with other desired curing agents has played an important role. In this paper, a brief sketch of various kinds of nanomaterials and their biomedical applications is given. Despite claims of bio-nanotechnology about to touch all areas of medical science, information pertaining to the role of nanotechnology for the betterment of reproductive healthcare is indeed limited. Therefore, the various achievements of nano-biotechnology for healthcare in general have been illustrated while giving special insight into the role of nano-biotechnology for the future of reproductive healthcare betterment as well as current achievements of nanoscience and nanotechnology in this arena.

Poly(styrene-co-maleic acid)-based pH-sensitive liposomes mediate cytosolic delivery of drugs for enhanced cancer chemotherapy.

pH-responsive polymers render liposomes pH-sensitive and facilitate the intracellular release of encapsulated payload by fusing with endovascular membranes under mildly acidic conditions found inside cellular endosomes. The present study reports the use of high-molecular weight poly(styrene-co-maleic acid) (SMA), which exhibits conformational transition from a charged extended structure to an uncharged globule below its pK(1) value, to confer pH-sensitive property to liposomes. The changes in the co-polymer chain conformation resulted in destabilization of the liposomes at mildly acidic pH due to vesicle fusion and/or channel formation within the membrane bilayer, and ultimately led to the release of the encapsulated cargo. The vesicles preserved their pH-sensitivity and stability in serum unlike other polymer-based liposomes and exhibited no hemolytic activity at physiological pH. The lysis of RBCs at endosomal pH due to SMA-based liposome-induced alterations in the bilayer organization leading to spherocyte formation indicated the potential of these vesicles to mediate cytosolic delivery of bio-active molecules through endosome destabilization. The SMA-loaded liposomes exhibiting excellent cytocompatibility, efficiently delivered chemotherapeutic agent 5-Fluorouracil (5-FU) within colon cancer cells HT-29 in comparison to neat liposomes. This caused increased cellular-availability of the drug, which resulted in enhanced apoptosis and highlighted the clinical potential of SMA-based vesicles.

Probing molecular interactions of poly(styrene-co-maleic acid) with lipid matrix models to interpret the therapeutic potential of the co-polymer.

To understand and maximize the therapeutic potential of poly(styrene-co-maleic acid) (SMA), a synthetic, pharmacologically-active co-polymer, its effect on conformation, phase behavior and stability of lipid matrix models of cell membranes were investigated. The modes of interaction between SMA and lipid molecules were also studied. While, attenuated total reflection-Fourier-transform infrared (ATR-FTIR) and static (31)P nuclear magnetic resonance (NMR) experiments detected SMA-induced conformational changes in the headgroup region, differential scanning calorimetry (DSC) studies revealed thermotropic phase behavior changes of the membranes. (1)H NMR results indicated weak immobilization of SMA within the bilayers. Molecular interpretation of the results indicated the role of hydrogen-bond formation and hydrophobic forces between SMA and zwitterionic phospholipid bilayers. The extent of membrane fluidization and generation of isotropic phases were affected by the surface charge of the liposomes, and hence suggested the role of electrostatic interactions between SMA and charged lipid headgroups. SMA was thus found to directly affect the structural integrity of model membranes.

An approach to noninvasive delivery, biodistribution, and fertility control potential evaluation of the Cuproferrogel iron oxide-copper-styrene maleic anhydride-dimethyl sulphoxide in the female.

Under guidance of an external pulsed magnetic field the Cuproferrogel iron oxide-copper-styrene maleic anhydride-dimethyl sulphoxide delivered into the rat/rabbit oviduct resulted in oocytes with granulated cytoplasm, zona enlargement, membrane disintegration, and finally loss of viability in 72 hours. Also, the percentage biodistribution of magnetic and electrically conductive particles observed under safe level advocates the use of Cuproferrogel as a potential female fertility control molecule.

Smart RISUG: a potential new contraceptive and its magnetic field-mediated sperm interaction.

The rationale and technique underlying a novel concept of noninvasive fertility control by a new Cuproferrogel contraceptive drug, iron oxide-copper-styrene maleic anhydride-dimethyl sulphoxide (Fe3O4-Cu-SMA-DMSO) composite named 'Smart RISUG' (smart reversible inhibition of sperm under guidance) in presence of pulsed magnetic field (PMF; 1 mT to 800 mT) is explained. It was synthesized by dispersing iron oxide particles and copper particles into SMA-DMSO (male contraceptive RISUG) and characterized for particle distribution, particle size measurement and transmittance peaks, etc. Interaction of the RISUG particles as well as Smart RISUG particles with Albino rat sperm cell was studied in presence as well as absence of PMF. To find an explanation to increased reaching of the Smart RISUG drug into sperm under influence of magnetic field, the transport properties were characterized by high resolution transmission electron microscopy and atomic force microscopy. Smart RISUG could be mobilized into sperm cell membrane at the PMF, 760 mT in about 50 seconds. Adoption of novel drug Smart RISUG involving new technique may open the pathway for non surgical control of drug distribution, detection and restoration of the normal fertility after removal of the contraceptive from the male/female reproductive tube in presence of electromagnetic field.

RISUG: a potential candidate for the entry inhibitor group of antiretroviral drugs.

Entry inhibitors are a group of antiretroviral drug which prevents HIV from entering human immune cells. They include both fusion and attachment inhibitors. A hypothesis is put forward in which a new male contraceptive drug with proven antimicrobial property is proposed as a possible candidate for the entry inhibitor group of antiretroviral drugs. The proposed mechanism of action involves (i) interaction with gp120 and thereby preventing binding to CD4 and (ii) competitive binding with the viral glycoprotein and inhibit the glycoprotein - cell surface glyocosaminoglycan Heparan Sulfate (HS) interaction. A new drug RISUG (Reversible Inhibition of Sperm Under Guidance) presently undergoing Phase III clinical trials throughout India for its contraceptive effect in male has also antimicrobial actions. RISUG is a chemical complex of styrene maleic anhydride (SMA(AN)) and dimethyl sulfoxide. On injection into the vas deferens, it reacts with the components of intravas fluid, the spermatic fluid and gets converted to styrene maleic acid (SMA(AC)) and breakdown products like mandelic acid. An anti HIV activity of RISUG is likely due to its electrical charge and mandelic acid generation. For experimental validation HIV in vitro assays can be performed which will involve infectivity assays, luciferase assay and soluble gp120 assays. A positive result from the studies will validate the hypothesis.

Polyelectrolyte polymer properties in relation to male contraceptive RISUG action.

RISUG a polyelectrolytic hydrogel (styrene maleic anhydride and dimethyl sulfoxide) has proven to be efficacious as a contraceptive for a long term when injected into the lumen of vas deferens. Currently it is in advanced phase III clinical trials in India. Present investigation analyzes the swelling characteristics of RISUG hydrogel in different pH buffers and various biological fluids to understand its retention in the vas deferens as reported in previous studies. Significant variation in degree of swelling and equilibrium swelling ratio with transformation of Fickian to non-Fickian mode of diffusion was observed with increased pH. This might be due to ionization of carboxylic groups at high pH resulting in increased electrostatic repulsive force and high osmotic pressure inside the hydrogel network affecting its physical cross-linking and increases the free volume. Conversely, at low pH the dissociation of carboxylic group is limited making the hydrogel more stable. Interaction with various biomolecules present in various biological fluids was also studied. SEM, AFM and FTIR were used to analyze the topological and structural parameters of the polymer in different mediums. Loosening of structure and increasing porosity with significant adsorption of various biomolecules was observed. AFM revealed a significant change in overall roughness of polymer surface on interaction with different biological fluids. These observations suggest that the swelling and increased roughness will lead to increased resistance to sperm movement in the vas deferens.

Study of the micro-structural properties of RISUG--a newly developed male contraceptive.

A new male contraceptive given the name RISUG (an acronym for reversible inhibition of sperm under guidance) and presently undergoing advanced clinical trials has been developed. When injected into the lumen of the vas deferens, its polyelectrolytic nature induces a surface charge imbalance on sperm membrane system leading to the leakage of enzymes essential for fertilization. Contact mode atomic force microscopy (AFM) has been used to analyze quantitatively the micro-structural properties of RISUG and its precipitate in various systems. Hydrolysis of the contraceptive gel resulted in the formation of pores of varying dimensions. RISUG being a highly charged molecule, as evident from zeta potential measurements, has a tendency to form a complex with ionic biomolecules present in the seminal plasma. This is supported by the experimental observations using AFM. This RISUG-biomolecule complex possibly acts as an ionic trap for spermatozoa passing through the vas deferens. Micro-structural properties of RISUG including amplitude (root mean square, peak-to-valley distance, skewness and kurtosis) and spatial roughness have been studied to understand its response to various physiological conditions. Significant alterations in the surface charge distribution of the sperm cell is observed on exposure to RISUG.

Quantitative analysis of surface micro-roughness alterations in human spermatozoa using atomic force microscopy.

A new male contraceptive given the name RISUG (an acronym for Reversible Inhibition of Sperm Under Guidance) has been developed by our research group. RISUG is a bioactive polymer and is injected into the lumen of the vas deferens using a no-scalpel approach. The polyelectrolytic nature of this contraceptive induces a surface charge imbalance on sperm membrane system leading to its destabilization. Complete disintegration of the plasma membrane with subsequent rupture and dispersion of the acrosomal contents is observed on RISUG treatment. In the present study, micro-structural properties of human spermatozoa exposed to RISUG in vitro have been quantitatively analysed using atomic force microscopy. The parameters used to quantify these morphological changes include amplitude (peak-valley height difference, arithmetic roughness, root mean square roughness) and spatial roughness. Factor loadings (Varimax rotation) have been used to determine the parameters displaying maximum variation. Further, sperm cells have been classified in various principal-component planes using principal-component analysis. The periodic structural features of the atomic force microscopy images have also been obtained using power spectral analysis.

Biophysical mechanism-mediated time-dependent effect on sperm of human and monkey vas implanted polyelectrolyte contraceptive.

AIM: To determine the short and long-term morphological effects on sperm as induced by intra-vas alteration of pH and electrical charge. METHODS: Desired biophysical influences were obtained by injection of reversible inhibition of sperm under guidance (RISUG) into the lumen of the vas deferens of human subjects and the monkey. RISUG is a polyelectrolyte hydrogel complex of styrene maleic anhydride (SMA) and dimethyl sulfoxide (DMSO) which generates an electrostatic charge and also lowers in a near space of pH domain. The morphology of sperm was examined by light microscopy, scanning and transmission electron microscopy. Human study enabled semen collection by masturbation as early as 3 h after injection and studies extended up to 6 months. In the monkey, on vas excision after RISUG implantation, sperm characteristics were examined in serial sections. RESULTS: Semenology in clinical studies and histological data of the monkey showed a time-sequenced sperm plasma membrane, tail mitochondria and nuclear decondensation alterations in sperm structural components, which beared marked similarity to changes in the sperm head and tail during capacitation and entry into the ovum. CONCLUSION: The findings provide a means of causing such changes in the sperm that inhibit the fertilizing ability before the nucleus is affected. Therefore achieving non-obstructive vas-based contraception, without genotoxic or teratogenic effects caused by infertile sperm passing into the semen, is feasible.

Topological alterations in human spermatozoa associated with the polyelectrolytic effect of RISUG.

A new method of male contraception has been developed which results in long-term infertility and has the potential advantage of being reversible. The contraceptive, given the name RISUG (an acronym for Reversible Inhibition of Sperm Under Guidance) is a polyelectrolytic compound and when injected into the lumen of the vas deferens, induces a surface charge imbalance on the sperm membrane system leading to its destabilization. In the present study, morphological and topological alterations in human spermatozoa induced by RISUG have been investigated using atomic force microscopy (AFM). Complete disintegration of the plasma membrane with subsequent rupture and dispersion of the acrosomal contents is observed on treatment with RISUG in vitro. Considerable damage to the midpiece region with significant clustering of the mitochondria and its fusion with the head region is also observed. These observations are in agreement with the significant increase in the volume of RISUG-treated sperm-head region. Topological alterations in the flagellar and midpiece region of RISUG-treated spermatozoa have also been studied.

Atomic force microscopy: a powerful tool for high-resolution imaging of spermatozoa.

Atomic force microscopy (AFM) has emerged as the only technique capable of real-time imaging of the surface of a living cell at nano-resolution. Since AFM provides the advantage of directly observing living biological cells in their native environment, this technique has found many applications in pharmacology, biotechnology, microbiology, structural and molecular biology, genetics and other biology-related fields. AFM has also proved to be a valuable tool for reproductive biologists. An exhaustive review on the various applications of AFM to sperm cells is presented. AFM has been extensively applied for determining the structural and topological features of spermatozoa. Unstained, unfixed spermatozoa in their natural physiological surroundings can be imaged by this technique which provides valuable information about the morphological and pathological defects in sperm cells as three-dimensional images with precise topographical details. Sperm head defects and the acrosome at the tip of the head responsible for fertilization, can be examined and correlated with the lack of functional integrity of the cell. Considerable amount of work is reported on the structural details of the highly condensed chromatin in sperm head using AFM. Detailed information on 3D topographical images of spermatozoa acquired by AFM is expected to provide a better understanding of various reproductive pathways which, in turn, can facilitate improved infertility management and/or contraceptive development.

RISUG (reversible inhibition of sperm under guidance)--an antimicrobial as male vas deferens implant for HIV free semen.

HIV transmission from the male to the female is a major health problem. A hypothesis proposing an intra vas deferens implant of an antimicrobial compound to prevent the infection spread is presented. Mechanisms of action for the inhibition could include inactivating HIV in sperms passing through the vas deferens; drug release from the implant to destroy HIV entering into semen from genital structures distal to the vas deferens; and sperm acrosome released hyaluronidase mediated reabsorption of HIV. A subcomponent of the implant flowing along sperm pathway may have a role in reducing the entry of HIV from a positive female into penile tissue. A new drug RISUG (reversible inhibition of sperm under guidance) presently undergoing clinical trials for its contraceptive effect in the male (because it disrupts the sperm acrosome by an electrical charge and pH lowering effects) has also antimicrobial action. The drug being a combination of styrene maleic anhydride (SMA) and dimethyl sulfoxide (DMSO) on being injected into the lumen of the vas deferens produces styrene maleic acid thereby lowering pH; induces electrochemical action leading to a stable electrical charge generation; releases mandelic acid; and induces acrosome reaction in sperms with consequent release of hyaluronidase and sperm inactivation. Moreover, one time administration into the lumen of the vas gives long term action. All these phenomena very well match with the needs for HIV clearance of semen and hence RISUG is here proposed as a possible candidate material for the HIV inhibiting vas deferens implant when delivered in below contraceptive threshold dosage. For experimental validation, after obtaining data on the semen HIV load under control conditions in the HIV positive males inducted into the study, 30 mg of SMA in 120 microl of DMSO (contraceptive dose being 60 mg SMA+120 microl DMSO) is to be injected into vasa deferens bilaterally. Thereafter at intervals of one month the viral load needs to be determined in semen obtained either by masturbation or in lubricant free condom at intercourse - the method of collection remaining the same throughout for a particular subject. A significant reduction in the semen viral load following RISUG administration will validate the hypothesis. Speculated reduced female to male HIV transmission is more difficult to test. Nonspecific indications will come from a population study of the incidence of RISUG treated men becoming HIV positive as compared to that in the general population.

Effect of a new injectable male contraceptive on the seminal plasma amino acids studied by proton NMR spectroscopy.

Effect of RISUG, a newly developed male contraceptive, on various amino acids of seminal plasma ejaculates was studied by proton magnetic resonance spectroscopy at 400 MHz. Levels of amino acids were compared with the seminal plasma of obstructive azoospermia and controls. Glutamic acid, glutamine, and arginine were found to be high in concentration in human seminal plasma. The concentration of aromatic amino acids such as tyrosine, histidine, and phenylalanine in RISUG-injected subjects showed no significant difference compared to controls (p > 0.1); however, there was a statistically significant decrease in the concentration of these amino acids in obstructive azoospermia. The concentration of some prominent amino acids that showed overlapping resonances, such as isoleucine+leucine+valine (p < 0.01), alanine+isoleucine+lysine (p < 0.01), arginine+lysine+leucine (p < 0.01), and glutamic acid+glutamine (p < 0.01), showed a statistically significant decrease in RISUG-injected subjects compared to controls. Overlap of these amino acid resonances were noticed even at 600 MHz. In general, the total amino acids concentration in RISUG-injected subjects was found to be higher than in azoospermic subjects, confirming the occurrence of 'partial' obstructive azoospermia in subjects injected with this contraceptive.

Prediction of electrical impedance parameters for the simulated leg segment of an aircraft pilot under G-stress.

BACKGROUND: Blood accumulation in the lower extremities of fighter pilots has been known to induce loss of peripheral vision and consciousness. G suits and anti-G straining maneuvers applied to lower limbs do not work according to the actual status of blood accumulation. Therefore, the problem of blood accumulation in the legs requires further investigation. METHOD: The leg segment of an aircraft pilot has been modeled as a cylinder containing its anatomical structure of skin-fat layer, muscles, bones, and blood volume in the form of inner cylinders. Finite element analysis (FEA) was applied to the leg model with suitable boundary conditions. Predicted leg-segment electrical impedance (LEI) without blood accumulation has been compared with the measured experimental value using electrical impedance plethysmography (EIP). The effects of 7% blood pooling were analyzed by electrical impedance of the leg segment and its constituents by increasing the diameter of blood volume cylinder. RESULTS: The LEI has been predicted to be 46.7 omega, which corresponds well with experimental value of 46.0 omega and predicted value, being 1.58% higher. Predicted LEI was reduced to 44.2 omega during simulation of blood accumulation in FE model. The relative contributions of admittance for leg constituents fat, muscle, bones and blood volume are also found to be 2.96, 75.58, 0.33, and 11.13%, respectively. CONCLUSION: A computer simulation technique has been developed to predict electrical impedance parameters of the leg segment and its constituents with and without blood pooling. These results will be useful in further research and clinical applications for aircraft pilots under acceleration (G) stress.