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Saeed Zavareh

Associate Professor of Cell and Molecular Biology

Biography

Dr. Saeed Zavareh is currently associate professor in the department of Cell and Molecular Biology of Damghan University in Iran. He also holds the positions as consultant and researcher of the reproductive biology unit at the Institute of Biological Sciences in Damghan University. He received his Ph.D. degree in Theriogenology from Urmia University, and his Ph.D. thesis was partly done at University of Tarbiat Modares, Iran. His primary area of research focused on developing novel methods in fertility preservation including cryopreservation of ovarian tissue, oocyte and embryo. He also interested to study the role of stem cells in reproductive biology.

Education

  • Ph.D. 2004-2009

    Theriogenology

    Urmia University, Urmia, Iran

  • DVM. 2004

    Veterinary Medicine

    Urmia University, Urmia, Iran

Teaching

  • Sex Determination and Reproduction
  • Human Reproductive Biology
  • Zoology

Selected Publications

Hashemi-Moghaddam, H., Zavareh, S., Gazi, E.M., Jamili, M. Assessment of novel core–shell Fe3O4@poly L‑DOPA nanoparticles for targeted Taxol® delivery to breast tumor in a mouse model (2018) Materials Science and Engineering C, 93, pp. 1036-1043.

DOI: 10.1016/j.msec.2018.09.005

Drug delivery systems using nanoparticles can deliver to tumor cells without affecting normal cells. In this study, a novel well dispersed magnetic nano drug was synthesized. Thus, a selective drug delivery system was designed for potential cancer treatment. A new nanocomposite, poly 3,4‑dihydroxy‑L‑phenylalanine/Fe3O4 (L‑DOPA/Fe3O4), was synthesized and used for targeted Taxol® delivery to breast tumor in inbreed Balb/c mice model with or without magnetic field. Fe and Taxol® concentrations were measured by flame atomic absorption spectrometry and high-performance liquid chromatography, respectively. Antitumor effectiveness was investigated in terms of tumor growth features. In the presence of magnetic field, Taxol® was significantly deposited in tumor tissue in Taxol-nanocomposite-treated group. In addition, the Taxol®-nanocomposite-treated group with magnetic field showed higher antitumor efficacy than the commercial Taxol and Taxol-nanocomposite without magnetic field. The magnetic nanocomposite is promising for targeted Taxol® delivery to breast tumor in a mouse model yielding high performance. © 2018 Elsevier B.V.

AUTHOR KEYWORDS: Breast cancer; Magnetic nanoparticles; Poly L‑DOPA; Taxol®
INDEX KEYWORDS: Absorption spectroscopy; Amino acids; Atomic absorption spectrometry; Controlled drug delivery; Diseases; High performance liquid chromatography; Iron oxides; Magnetic fields; Magnetite; Mammals; Nanocomposites; Nanomagnetics; Nanoparticles; Tumors, Anti-tumor efficacy; Breast Cancer; Drug delivery system; Flame atomic absorption spectrometry; Magnetic nano-particles; Magnetic nanocomposites; Tumor tissues; Well-dispersed, Targeted drug delivery, indole derivative; magnetite nanoparticle; nanocomposite; paclitaxel; polydopamine; polymer, animal; Bagg albino mouse; chemistry; drug delivery system; experimental mammary neoplasm; female; metabolism; mouse; pathology; procedures, Animals; Drug Delivery Systems; Female; Indoles; Magnetite Nanoparticles; Mammary Neoplasms, Experimental; Mice; Mice, Inbred BALB C; Nanocomposites; Paclitaxel; Polymers

Zavareh, S., Gholizadeh, Z., Lashkarbolouki, T. Evaluation of changes in the expression of Wnt/β-catenin target genes in mouse reproductive tissues during estrous cycle: An experimental study (2018) International Journal of Reproductive BioMedicine, 16 (2), pp. 69-76.

DOI: 10.29252/ijrm.16.2.69

Background: The Wingless-type (Wnt)/β-catenin signaling pathway controls cell homeostasis. Reproductive tissues are dynamic in response to steroidal hormone changes. Steroidal hormones are known to control the Wnt/β-catenin pathway, but their role in reproductive tissues remains unknown. Objective: The present study aims to investigate the expression patterns of Wnt/β- catenin target genes in mouse reproductive tissues during the normal estrous cycle. Materials and Methods: In this experimental study, 16 adult NMRI mice were grouped as proestrus, estrus, metestrus, and diestrus according to vaginal smear and histological evaluation of uterine and ovarian tissues. Uterine horns and ovarian tissues were collected. Reverse transcription quantitative polymerase chain reaction was performed to evaluate the expression of Wnt/β-catenin target genes (Myc2, Ppard, Id2, Birc5, and Ascl2) at different stages of the estrous cycle. Results: The expression levels of Id2, Ascl2, and Pprd in uterine tissue were significantly higher at the proestrus phase than at the other stages. Meanwhile, Birc5 expression in uterine tissue was significantly higher at the metestrus stage than at the other stages. Furthermore, Myc2 expression was significantly higher at the diestrus stage than at the estrus and metestrus stages. In the ovarian tissue, the highest expression of Id2, Ascl2, and Birc5 was detected at the proestrus stage, whereas the highest expression of Myc2 and Ppard was observed at the estrus stage. Conclusion: This study showed that Wnt/β-catenin target genes profiles are different among estrous cycle. It seems that different hormonal profiles during estrous cycles play a key role in the expression pattern of Wnt/β-catenin target genes in ovarian and uterine tissue. © 2018, Research and Clinical Center for Infertitlity. All rights reserved.

AUTHOR KEYWORDS: Beta catenin; Estrous cycle; Mice; Wnt signaling pathway
INDEX KEYWORDS: beta catenin; Wnt protein, adult; animal experiment; animal tissue; Article; controlled study; estrus cycle; female; gene expression; histology; mouse; nonhuman; ovary tissue; reverse transcription polymerase chain reaction; signal transduction; uterus horn; vagina cytology

Hashemi-Moghaddam, H., Zavareh, S., Karimpour, S., Madanchi, H. Evaluation of molecularly imprinted polymer based on HER2 epitope for targeted drug delivery in ovarian cancer mouse model (2017) Reactive and Functional Polymers, 121, pp. 82-90.

DOI: 10.1016/j.reactfunctpolym.2017.10.025

In this study, an artificial tumor-specific antigen was synthesized by using capabilities of molecular imprinted polymers and epitope of HER2 protein as template. Thus, a specific drug delivery system was designed for potential cancer treatment. The epitope of HER2 protein was designed and synthesized. Conformational epitopes are synthesized by residues that are sequentially discontinuous however similar together in three-dimensional space. Molecularly imprinted polymers (MIPs) were synthesized on the surface of silica nanoparticles. Dopamine was used as monomer and the conformational epitope of HER2 and doxorubicin (DOX) were the templates. The synthesized MIPs were used for targeting DOX delivery in an ovarian cancer mouse model. The antitumor effectiveness of different groups was determined in terms of parameters such as; tumor size and DOX distribution in different tissues. Results showed high efficiency of DOX-EPI-IP in suppressing tumor size. Moreover, higher DOX concentration was observed in the tumor tissue of the DOX-EPI-IP group compared with that of other groups. The HER2 epitope-based MIP is a promising candidate as a vehicle for controlled bio distribution of DOX in ovarian cancer therapy. © 2017 Elsevier B.V.

AUTHOR KEYWORDS: Doxorubicin; Epitope; HER2; Imprinted polymer; Ovarian cancer
INDEX KEYWORDS: Bioelectric phenomena; Biosynthesis; Conformations; Diseases; Mammals; Polymers; Proteins; Silica; Synthesis (chemical); Targeted drug delivery; Tissue; Tumors, Conformational epitopes; Doxorubicin; HER2; Imprinted polymers; Molecular imprinted polymers; Molecularly Imprinted Polymer; Ovarian cancers; Three dimensional space, Epitopes

Hosseinzadeh, E., Zavareh, S., Lashkarbolouki, T. Antioxidant properties of coenzyme Q10-pretreated mouse pre-antral follicles derived from vitrified ovaries (2017) Journal of Obstetrics and Gynaecology Research, 43 (1), pp. 140-148.

DOI: 10.1111/jog.13173

Aim: This study evaluated the antioxidant status of pre-antral follicles derived from vitrified ovaries pretreated with coenzyme Q10 (CoQ10). Methods: Mouse pre-antral follicles derived from fresh and vitrified warmed ovarian tissue were cultured with or without CoQ10 (50 μmol/L). Follicular growth, total antioxidant capacity (TAC), malondialdehyde (MDA) level, and superoxide dismutase (SOD), glutathione peroxidase (GPX), and catalase (CAT) activity during cultivation were assessed. Results: The growth rate of the fresh pre-antral follicles was higher compared with the vitrified groups, especially in the CoQ10-treated than non-treated groups. MDA increased while TAC decreased at 96 h of the cultivation period. TAC was higher while MDA was lower in the fresh pre-antral follicles than in the vitrified groups. These rates were higher in the CoQ10-treated than non-treated groups. The vitrified and fresh CoQ10-pretreated groups had significantly higher SOD, GPX, and CAT activity compared with the CoQ10 non-treated groups. Conclusion: CoQ10-supplemented maturation medium can increase antioxidant enzyme activity and decrease lipid peroxidation in cultured pre-antral follicles derived from fresh and vitrified mouse ovaries. © 2016 Japan Society of Obstetrics and Gynecology

AUTHOR KEYWORDS: antioxidant enzyme; coenzyme Q10; ovary; vitrification
INDEX KEYWORDS: catalase; glutathione peroxidase; malonaldehyde; superoxide dismutase; ubidecarenone; antioxidant; catalase; glutathione peroxidase; malonaldehyde; superoxide dismutase; ubidecarenone; ubiquinone, animal cell; animal tissue; Article; controlled study; culture medium; enzyme activity; female; lipid peroxidation; metabolic parameters; mouse; nonhuman; ovary follicle; ovary tissue; total antioxidant capacity; vitrification; analogs and derivatives; animal; drug effects; enzymology; metabolism; ovary follicle, Animals; Antioxidants; Catalase; Female; Glutathione Peroxidase; Malondialdehyde; Mice; Ovarian Follicle; Superoxide Dismutase; Ubiquinone; Vitrification

Kashka, R.H., Zavareh, S., Lashkarbolouki, T. Augmenting effect of vitrification on lipid peroxidation in mouse preantral follicle during cultivation: Modulation by coenzyme Q10 (2016) Systems Biology in Reproductive Medicine, 62 (6), pp. 404-414.

DOI: 10.1080/19396368.2016.1235236

Cryopreservation-induced oxidative stress (OS) may lead to lipid peroxidation, which may be responsible for decreased cell survival rate. Coenzyme Q10 (CoQ10) as a potent antioxidant may improve cell viability by neutralizing OS. In this study, oxidative lipid injury following the vitrification of preantral follicles was investigated. The effects of CoQ10 treatment on the malondialdehyde (MDA) levels, lipid peroxidation products, and activities of enzymatic and nonenzymatic antioxidants of vitrified preantral follicles were also studied. Preantral follicles were isolated from immature mouse ovaries and were vitrified. After warming, these follicles were cultured with or without CoQ10 for four days. The levels of total antioxidant capacity (TAC) and MDA, as well as the activities of superoxide dismutase (SOD), glutathione peroxidase (GPX), and catalase (CAT), were assessed at 0, 24, 48, 72, and 96 hours of culture period. The MDA level in the vitrified preantral follicles was higher than that in the fresh groups. By contrast, the MDA level was significantly lower in the groups with CoQ10 treatment than in those without this treatment during cultivation. The TAC level was higher in the fresh preantral follicles than in the vitrified groups. The rates were also higher in the CoQ10-treated groups than in those without this treatment. The activities of SOD, GPX, and CAT were also significantly higher in the fresh groups than in the vitrified groups, especially in the groups with CoQ10 treatment than in those without this treatment. Lowering the vitrification-induced lipid peroxidation of preantral follicles by CoQ10-supplemented maturation medium may be mediated by increasing SOD, GPX, and CAT activities and TAC level during cultivation. © 2016 Taylor & Francis.

AUTHOR KEYWORDS: Coenzyme Q10; lipid peroxidation; preantral follicles; vitrification
INDEX KEYWORDS: antioxidant; catalase; glutathione peroxidase; lipid peroxide; malonaldehyde; superoxide dismutase; ubidecarenone; antioxidant; catalase; glutathione peroxidase; malonaldehyde; superoxide dismutase; ubidecarenone; ubiquinone, animal cell; antioxidant activity; Article; cell culture; cell maturation; controlled study; enzyme activity; in vitro study; lipid peroxidation; mouse; nonhuman; ovary; ovary follicle; ovary follicle cell; ovary follicle development; ovary follicle fluid; priority journal; total antioxidant capacity; vitrification; warming; analogs and derivatives; animal; drug effects; enzymology; female; metabolism; ovary follicle, Animals; Antioxidants; Catalase; Female; Glutathione Peroxidase; Lipid Peroxidation; Malondialdehyde; Mice; Ovarian Follicle; Superoxide Dismutase; Ubiquinone; Vitrification

Zavareh, S., Mahdi, M., Erfanian, S., Hashemi-Moghaddam, H. Synthesis of polydopamine as a new and biocompatible coating of magnetic nanoparticles for delivery of doxorubicin in mouse breast adenocarcinoma (2016) Cancer Chemotherapy and Pharmacology, 78 (5), pp. 1073-1084.

DOI: 10.1007/s00280-016-3169-5

Purpose: Carrier-mediated drug delivery systems can be used to increase the intracellular concentration of drugs in cancerous cells, thereby improving drug biodistribution and minimizing unwanted side effects. This study aimed to investigate the effect of synthesized magnetic molecularly imprinted polydopamine for controlled doxorubicin (DOX) delivery in a breast adenocarcinoma model of BALB/c mice with an external magnetic field. Methods: The synthesized DOX-imprinted polydopamine (DOX-IP) was characterized using Fourier transform infrared spectroscopy and scanning electron microscopy. The efficacy of DOX-IP in tumor growth suppression was assessed in terms of tumor growth delay, tumor doubling time, inhibition ratio, and histopathology. High-performance liquid chromatography and flame atomic absorption spectrometry were performed to investigate the drug distribution among tissues. Results: The findings showed higher efficacy of DOX-IP with magnetic field in suppressing tumor growth than free DOX and DOX-IP without magnetic field. Significantly high DOX concentration in tumor tissue was found in the DOX-IP group with magnetic field. Conclusion: Magnetic DOX-IP demonstrates effective tumor-targeted drug delivery in a mouse model of breast cancer. © 2016, Springer-Verlag Berlin Heidelberg.

AUTHOR KEYWORDS: Breast adenocarcinoma; Doxorubicin; Imprinted polydopamine; Magnetic nanoparticle
INDEX KEYWORDS: dopamine derivative; doxorubicin; magnetic nanoparticle; polydopamine; unclassified drug; antineoplastic antibiotic; biocompatible coated material; delayed release formulation; doxorubicin; indole derivative; magnetite nanoparticle; polydopamine; polymer, animal experiment; animal model; animal tissue; Article; atomic absorption spectrometry; breast adenocarcinoma; controlled study; drug delivery system; drug distribution; drug release; drug synthesis; female; growth inhibition; high performance liquid chromatography; histopathology; in vitro study; in vivo study; infrared spectroscopy; magnetic field; mitosis index; mouse; nonhuman; priority journal; scanning electron microscopy; surface property; survival rate; tumor growth; tumor volume; adenocarcinoma; animal; Bagg albino mouse; chemistry; delayed release formulation; electromagnetism; Mammary Neoplasms, Animal; metabolism; particle size; survival analysis; synthesis; tissue distribution; treatment outcome, Adenocarcinoma; Animals; Antibiotics, Antineoplastic; Coated Materials, Biocompatible; Delayed-Action Preparations; Doxorubicin; Drug Delivery Systems; Electromagnetic Fields; Indoles; Magnetite Nanoparticles; Mammary Neoplasms, Animal; Mice; Mice, Inbred BALB C; Particle Size; Polymers; Spectrophotometry, Atomic; Spectroscopy, Fourier Transform Infrared; Survival Analysis; Tissue Distribution; Treatment Outcome

Davoudi, M., Zavareh, S., Ghorbanian, M.T., Paylakhi, S.H., Mohebbi, S.R. The effect of steroid hormones on the mRNA expression of oct4 and sox2 in uterine tissue of the ovariectomized mice model of menopause (2016) International Journal of Reproductive BioMedicine, 14 (7), pp. 471-476.

Background: The uterus is a dynamic tissue responding to hormonal changes during reproductive cycles. As such, uterine stem cells have been studied in recent years. Transcription factors oct4 and sox2 are critical for effective maintenance of pluripotent cell identity. Objective: The present research evaluated the mRNA expression of oct4 and sox2 in the uterine tissues of ovariectomized mice treated with steroid hormones. Materials and Methods: In this experimental study, adult virgin female mice were ovariectomized and treated with estradiol 17β (E2), progesterone (P4), and a combination of E2 and P4 (E2 & P4) for 5 days. Uterine tissues were removed, and immunofluorescent (IF) staining and quantitative real-time PCR of oct4 and sox2 markers were performed. Results: IF showed oct4 and sox2 expression in the uterine endometrium and myometrium among all groups. The mRNA expression of oct4 (p=0.022) and sox2 (p=0.042) in the E2-treated group significantly were decreased compared to that in the control group. By contrast, the mRNA expression of oct4 and sox2 in the P4 (p=0.641 and 0.489 respectively) and E2 & P4-treated groups (p=0.267 and 0.264 respectively) did not show significant differences compared to the control group. Conclusion: The results indicate ovarian steroid hormones change the expression of oct4 and sox2 in the mice uterine tissues, which suggest the involvement of steroid hormonal regulation in uterine stem cells. © 2016, Research and Clinical Center for Infertitlity. All rights reserved.

AUTHOR KEYWORDS: Estradiol; Mice; Progesterone; Stem cells; Uterine
INDEX KEYWORDS: estradiol; messenger RNA; octamer transcription factor 4; progesterone; steroid hormone; transcription factor Sox2, animal experiment; animal model; animal tissue; Article; controlled study; gene sequence; immunofluorescence; menopause; mouse; nonhuman; ovariectomy; protein expression; real time polymerase chain reaction; reverse transcription polymerase chain reaction; RNA extraction; sampling; signal transduction; uterine tissue

Hosseinzade, E., Zavareh, S., Lashkarboluki, T. The comparison of developed mouse vitrified preantral follicle with isolated preantral follicles from vitrified ovary (2016) Koomesh, 17 (4), pp. 981-989.

Introduction: Cryopreservation techniques are useful methods for long-term storage of cells and tissues of the reproductive system. The purpose of this study was to investigate the development and survival rate of vitrified preantral follicles compare with those of isolated preantral follicles from vitrified ovaries. Materials and methods: Preantral follicles of 14 to 16 days-old of NMRI mice ovaries were randomly divided into three groups. Group I: preantral follicles derived from fresh ovaries (control). Group II: preantral follicles derived from vitrified ovaries and group 3: vitrified preantral follicles. After thawing, preantral follicles were cultured in α-MEM medium containing 5% FBS, 100 mIU/ml hFSH, 1% ITS and 10 ng/ ml EGF. At the day 12 of culture, ovulation was induced by adding 1.5 IU/ml hCG. The preantral follicle diameter and the rates of survival, antrum formation and developmental stages of released oocytes were evaluated. Results: The results showed that the survival rate of vitrified preantral follicles was significantly higher than that of isolated preantral follicle from vitrified ovaries. The mean diameter of preantral follicles, the rates of antrum formation and MII stage oocytes in vitrified preantral follicles were significantly higher than those of isolated preantral follicles from vitrified ovaries. Conclusion: Vitrified preantral follicles were more suitable to develop to mature oocytes in comparison with isolated preantral follicles from vitrified ovaries. © 2016, Semnan University of Medical Sciences. All rights reserved.

AUTHOR KEYWORDS: Ovarian follicle; Ovary; Vitrification
INDEX KEYWORDS: animal tissue; Article; cell survival; controlled study; developmental stage; mouse; nonhuman; oocyte; ovary follicle; vitrification

Asadzadeh, R., Khosravi, S., Zavareh, S., Ghorbanian, M.T., Paylakhi, S.H., Mohebbi, S.R. Vitrification affects the expression of matrix metalloproteinases and their tissue inhibitors of mouse ovarian tissue (2016) International Journal of Reproductive BioMedicine, 14 (3), pp. 173-180.

Background: One of the most major obstacles of ovarian tissue vitrification is suboptimal developmental competence of follicles. Matrix metalloproteinases 2 (MMP-2) and 9 (MMP-9) and their tissue inhibitors TIMP-1 and TIMP-2 are involved in the remodeling of the extracellular matrix in the ovaries. Objective: This study aimed to evaluate the expression of MMP-2, MMP-9, TIMP-1, and TIMP-2 genes in the preantral follicles derived from vitrified mouse ovaries. Materials and Methods: In this experimental study, the gene expression of MMP-2, MMP-9, TIMP-1, and TIMP-2 in the isolated preantral follicles derived from fresh and vitrified ovaries of 14-16 days old female mice through real time qRT-PCR was evaluated. Developmental parameters, including survival rate, growth, antrum formation and metaphase II oocytes were also analyzed. Results: The developmental parameters of fresh preantral follicles were significantly higher than vitrified preantral follicles. The TIMP-1 and MMP-9 expression levels showed no differences between fresh and vitrified preantral follicles (p=0.22, p=0.11 respectively). By contrast, TIMP-2 expression significantly decreased (p=0.00) and MMP-2 expression increased significantly (p=0.00) in vitrified preantral follicles compared with to fresh ones. Conclusion: Changes in expression of MMP-2 and TIMP-2 after ovarian tissues vitrification is partially correlated with decrease in follicle development. © 2016, Research and Clinical Center for Infertitlity. All rights Reserved.

AUTHOR KEYWORDS: Matrix metalloproteinases; Mouse; Pre antral follicles; Vitrification
INDEX KEYWORDS: gelatinase A; gelatinase B; tissue inhibitor of metalloproteinase 1; tissue inhibitor of metalloproteinase 2, animal tissue; Article; female; gene expression; growth; metaphase; mouse; nonhuman; ovarian tissue; ovary follicle development; reverse transcription polymerase chain reaction; survival rate; vitrification

Hashemi-Moghaddam, H., Kazemi-Bagsangani, S., Jamili, M., Zavareh, S. Evaluation of magnetic nanoparticles coated by 5-fluorouracil imprinted polymer for controlled drug delivery in mouse breast cancer model (2016) International Journal of Pharmaceutics, 497 (1-2), pp. 228-238.

DOI: 10.1016/j.ijpharm.2015.11.040

Nanoparticles (NPs) have been extensively investigated to improve delivery efficiency of therapeutic and diagnostic agents. In this study, magnetic molecularly imprinted polymer (MIP) was synthesized by using polydopamine. Synthesized MIP was used for controlled 5-fluorouracil (5-FU) delivery in a spontaneous model of breast adenocarcinoma in Balb/c mice in the presence of an external magnetic field. Antitumor effectiveness of 5-FU imprinted polymer (5-FU-IP) was evaluated in terms of tumor-growth delay, tumor-doubling time, inhibition ratio, and histopathology. Results showed higher efficacy of 5-FU-IP in the presence of magnetic field upon suppressing tumor growth than free 5-FU and 5-FU-IP without magnetic field. The 5-FU and Fe distribution among tissues were evaluated by high-performance liquid chromatography and flame atomic absorption spectrometry, respectively. The obtained results, showed significantly deposition of 5-FU in the 5-FU-IP treated group with magnetic field. Thus, magnetic 5-FU-IP is promising for breast cancer therapy with high efficacy. © 2015 Elsevier B.V. All rights reserved.

AUTHOR KEYWORDS: 5-Fluorouracil; Imprinted polymer; Magnetic nanoparticles; Polydopamine
INDEX KEYWORDS: fluorouracil; iron; magnetic nanoparticle; molecularly imprinted polymer; polymer; antineoplastic antimetabolite; fluorouracil; indole derivative; magnetite nanoparticle; polydopamine, animal experiment; animal model; animal tissue; Article; atomic absorption spectrometry; breast adenocarcinoma; cancer inhibition; controlled drug release; controlled study; drug distribution; drug efficacy; high performance liquid chromatography; histopathology; infrared spectroscopy; kidney parenchyma; liver; magnetic field; mouse; nonhuman; particle size; priority journal; survival rate; animal; Bagg albino mouse; Breast Neoplasms; chemistry; disease model; drug delivery system; drug release; female; procedures; survival analysis; tissue distribution; tumor cell line; ultrastructure, Animals; Antimetabolites, Antineoplastic; Breast Neoplasms; Cell Line, Tumor; Disease Models, Animal; Drug Delivery Systems; Drug Liberation; Female; Fluorouracil; Indoles; Magnetic Fields; Magnetite Nanoparticles; Mice; Mice, Inbred BALB C; Particle Size; Polymers; Survival Analysis; Tissue Distribution

Zavareh, S., Karimi, I., Salehnia, M., Rahnama, A. Effect of in vitro maturation technique and alpha lipoic acid supplementation on oocyte maturation rate: Focus on oxidative status of oocytes (2016) International Journal of Fertility and Sterility, 9 (4), pp. 442-451.

Background: Therapeutic potential of in vitro maturation (IVM) in infertility is growing with great promise. Although significant progress is obtained in recent years, existing IVM protocols are far from favorable results. The first aim of this study was to investigate whether two step IVM manner change reactive oxygen species (ROS) and total antioxidant capacity (TAC) levels. The second aim was to find the effect of alpha lipoic acid (ALA) supplementation on oocyte maturation rate and on ROS/TAC levels during IVM. Materials and Methods: In this experimental study, mouse germinal vesicle (GV) oocytes divided into cumulus denuded oocytes (DOs) and cumulus oocyte complexes (COCs) groups. GVs were matured in vitro in the presence or absence of ALA only for 18 hours (control) or with pre-culture of forskolin plus cilostamide for an additional 18 hours. Matured oocytes obtained following 18 and 36 hours based on experimental design. In parallel, the ROS and TAC levels were measured at different time (0, 18 and 36 hours) by 2',7'-dichlorodihydrofluorescein (DCFH) probe and ferric reducing/antioxidant power (FRAP) assay, respectively. Results: Maturation rate of COCs was significantly higher than DOs in control group (P<0.05), while there was no significant difference between COCs and DOs when were pre-cultured with forskolin plus cilostamide. ROS and TAC levels was increased and decreased respectively in DOs after 18 hours while in COCs did not change at 18 hours and showed a significant increase and decrease respectively at 36 hours (P<0.05). ROS and TAC levels in the presence of ALA were significantly decreased and increased respectively after 36 hours (P<0.05) whereas, maturation rates of COCs and DOs were similar to their corresponding control groups. © 2015, Royan Institute (ACECR). All rights reserved.


AUTHOR KEYWORDS: Alpha lipoic acid; Oocytes maturation; Oxidative statuse
INDEX KEYWORDS: cilostamide; forskolin; reactive oxygen metabolite; thioctic acid, animal cell; antioxidant assay; Article; controlled study; female; in vitro oocyte maturation; mouse; nonhuman; oocyte maturation

Hatami, S., Zavareh, S., Salehnia, M., Lashkarbolouki, T., Ghorbanian, M.T., Karimi, I. Total oxidative status of mouse vitrified pre-antral follicles with pre-treatment of alpha lipoic acid (2014) Iranian biomedical journal, 18 (3), pp. 181-188.

BACKGROUND: Cryopreservation of pre-antral follicles is a hopeful technique to preserve female fertility. The aim of the present study was to evaluate reactive oxygen species (ROS) and total antioxidant capacity (TAC) levels of mouse vitrified pre-antral follicles in the presence of alpha lipoic acid (ALA).
METHODS: Isolated pre-antral follicles (140-150 µm in diameter) were divided into vitrified-warmed and fresh groups. Each group was subjected to in vitro maturation with or without ALA for 12 days, followed by adding human chronic gonadotropin to induce ovulation. In vitro fertilization was performed to evaluate their developmental competence. In parallel, the amount of ROS and TAC were assessed after 0, 24, 48, 72, and 96 h of culture by 2',7'-dichlorofluorescin assay and ferric reducing/antioxidant power assay, respectively.
RESULTS: The respective rates of survival, antrum formation, and metaphase II oocytes were significantly higher in ALA-supplemented groups compared to the groups not treated with ALA. TAC and ROS levels were significantly decreased and increased, respectively during the culture period up to 96 h in the absence of ALA in both vitrified and non-vitrified samples. However, with pretreatment of ALA, TAC levels were increased significantly and remained constant up to 96 h in vitrified-warmed pre-antral follicles, while ROS levels completely returned to the level of starting point after 96 h of culture in the presence of ALA.
CONCLUSION: Pretreatment of ALA positively influences development of pre-antral follicles in vitrified and non-vitrified samples through increasing follicular TAC level and decreasing ROS levels.


INDEX KEYWORDS: antioxidant; reactive oxygen metabolite; thioctic acid, animal; cell culture; cell differentiation; drug effects; female; human; metabolism; mouse; oocyte; ovary follicle; oxidation reduction reaction; vitrification, Animals; Antioxidants; Cell Differentiation; Cells, Cultured; Female; Humans; Mice; Oocytes; Ovarian Follicle; Oxidation-Reduction; Reactive Oxygen Species; Thioctic Acid; Vitrification

Hatami, S., Zavareh, S., Salehnia, M., Lashkarbolouki, T., Karimi, I. Comparison of oxidative status of mouse pre-antral follicles derived from vitrified whole ovarian tissue and vitrified pre-antral follicles in the presence of alpha lipoic acid (2014) Journal of Obstetrics and Gynaecology Research, 40 (6), pp. 1680-1688.

DOI: 10.1111/jog.12394

Aim The main goal of this study was to compare developmental competence and oxidative status of vitrified-warmed pre-antral follicles (VPF) with pre-antral follicles derived from vitrified-warmed ovarian tissue (VOF) in the presence of alpha lipoic acid (ALA). Materials and Methods Ovarian tissue and isolated pre-antral follicles were exposed to equilibration solution and then vitrification solution. After thawing of LN2 snap-frozen samples, pre-antral follicles were cultured with or without ALA for 12 days that followed by hCG-induced ovulation. MII oocytes were in vitro fertilized and embryo cleavage assessed. Reactive oxygen species (ROS) and total antioxidant capacity (TAC) levels of cultured pre-antral follicles were measured. Results The rates of survival, antral-like cavity formation, MII oocytes, fertilization, 2-cell embryo and blastocyst development were higher in VPF compared to VOF. These rates were higher in ALA-supplemented groups in comparison to their respective groups. An increase and a decrease in ROS production and TAC levels were observed up to the 96 h during cultivation period, respectively. ROS level was lower in cultured VPF compared to VOF. In ALA-treated groups, ROS level decreased to reach comparable values of starting point and TAC levels increased after 24 h of culture and then remained constant. Conclusion Developmental outcomes showed vitrification of pre-antral follicles is more appropriate method than that of whole ovarian tissue. Moreover, it seems that inclusion of ALA improved in vitro development of pre-antral follicles in both vitrified and non-vitrified samples. © 2014 The Authors. Journal of Obstetrics and Gynaecology Research © 2014 Japan Society of Obstetrics and Gynecology.


AUTHOR KEYWORDS: alpha lipoic acid; ovarian tissue; oxidative status; vitrification
INDEX KEYWORDS: reactive oxygen metabolite; thioctic acid; antioxidant; reactive oxygen metabolite; thioctic acid, animal cell; article; blastocyst; cell survival; comparative study; controlled study; embryo; embryo cleavage; embryo development; female; fertilization in vitro; mouse; nonhuman; oocyte; ovary follicle; oxidative stress; thawing; vitrification; vitrified warmed ovarian tissue; vitrified warmed preantral follicle; animal; cryopreservation; drug effects; fertilization; metabolism; ovary follicle, Animals; Antioxidants; Cryopreservation; Embryonic Development; Female; Fertilization; Mice; Ovarian Follicle; Reactive Oxygen Species; Thioctic Acid; Vitrification

Hatami, S., Zavareh, S., Salehnia, M., Lashkarbolouki, T., Ghorbanian, M.T., Karimi, I. Total oxidative status of mouse vitrified pre-antral follicles with pre-treatment of alpha lipoic acid (2014) Iranian Biomedical Journal, 18 (3), pp. 180-187.

DOI: 10.6091/ibj.1258.2014

Background: Cryopreservation of pre-antral follicles is a hopeful technique to preserve female fertility. The aim of the present study was to evaluate reactive oxygen species (ROS) and total antioxidant capacity (TAC) levels of mouse vitrified pre-antral follicles in the presence of alpha lipoic acid (ALA). Methods: Isolated pre-antral follicles (140-150 μm in diameter) were divided into vitrified-warmed and fresh groups. Each group was subjected to in vitro maturation with or without ALA for 12 days, followed by adding human chronic gonadotropin to induce ovulation. In vitro fertilization was performed to evaluate their developmental competence. In parallel, the amount of ROS and TAC were assessed after 0, 24, 48, 72, and 96 h of culture by 2',7'-dichlorofluorescin assay and ferric reducing/antioxidant power assay, respectively. Results: The respective rates of survival, antrum formation, and metaphase II oocytes were significantly higher in ALA-supplemented groups compared to the groups not treated with ALA. TAC and ROS levels were significantly decreased and increased, respectively during the culture period up to 96 h in the absence of ALA in both vitrified and non-vitrified samples. However, with pretreatment of ALA, TAC levels were increased significantly and remained constant up to 96 h in vitrified-warmed pre-antral follicles, while ROS levels completely returned to the level of starting point after 96 h of culture in the presence of ALA. Conclusion: Pretreatment of ALA positively influences development of pre-antral follicles in vitrified and nonvitrified samples through increasing follicular TAC level and decreasing ROS levels.

AUTHOR KEYWORDS: Alpha lipoic acid (ALA); Pre-antral follicle; Reactive oxygen species (ROS); Total antioxidant capacity (TAC); Vitrification
INDEX KEYWORDS: chorionic gonadotropin; dichlorodihydrofluorescein diacetate; reactive oxygen metabolite; thioctic acid, animal cell; antioxidant activity; antioxidant assay; article; blastocyte; cell culture; cell isolation; cell maturation; cell structure; cell survival; controlled study; enzyme assay; female; fertilization in vitro; in vitro study; metaphase; nonhuman; oocyte development; ovary follicle cell; ovulation; ovulation induction; oxidation; rat; vitrification

Hatami, S., Zavareh, S., Salehnia, M., Lashkarbolouki, T., Ghorbanian, M.T., Karimi, I. The impact of alpha lipoic acid on developmental competence of mouse vitrified pre-antral follicles in comparison to those isolated from vitrified ovaries (2014) Iranian Journal of Reproductive Medicine, 12 (1), pp. 57-64.

Background: Cryopreservation of ovarian tissues and pre-antral follicles is a promising prospect for preservation of women fertility. Objective: The aim of this study was to evaluate the in vitro developmental competence of mouse vitrified pre-antral follicles in comparison to isolated pre-antral follicles derived from vitrified ovaries in the presence of alpha lipoic acid (ALA). Materials and Methods: Pre-antral follicles derived from fresh, vitrified-warmed ovarian tissues and vitrified–warmed pre-antral follicles were cultured individually with or without ALA, followed by adding hCG to induce ovulation. The follicle growth, oocyte maturation, and embryo development were assessed. Results: The diameter and development of follicles, oocyte maturation and embryo development rates were significantly higher in ALA supplemented groups compared to the respective ALA-free conditions groups. Aforementioned parameters were significantly higher in vitrified-warmed follicles in comparison to follicles derived from vitrified-warmed ovaries. Conclusion: These findings support a superior performance of pre-antral follicles when vitrified rather than when isolated from vitrified ovaries with regard to increasing the rates of developmental parameters. Moreover, ALA improves the in vitro maturation of pre-antral follicles in vitrified and non-vitrified samples. © 2014, Research and Clinical Center for Infertitlity. All rights reserved.

AUTHOR KEYWORDS: Alpha lipoic acid; Ovary; Preantral follicles; Vitrification
INDEX KEYWORDS: chorionic gonadotropin; thioctic acid, animal cell; animal tissue; Article; controlled study; embryo; embryo development; female; fertilization in vitro; in vitro study; intermethod comparison; male; mouse; nonhuman; oocyte maturation; ovary follicle; ovary follicle development; ovulation; preantral follicle; tissue culture; vitrification

Rahnama, A., Zavareh, S., Ghorbanian, M.T., Karimi, I. The effects of cAMP-elevating agents and alpha lipoic acid on in vitro maturation of mouse germinal vesicle oocytes (2013) Journal of Reproduction and Infertility, 14 (4), pp. 173-183.

Background: In spite of extensive efforts to improve in vitro oocyte maturation, the obtained results are not very efficient. This study was conducted to assess impacts of cAMP elevating agents and alpha lipoic acid (ALA) on in vitro oocyte maturation and fertilization. Methods: Mouse germinal vesicle (GV) oocytes were categorized into cumulus denuded oocytes (DOs; n=896) and cumulus oocyte complexes (COCs; n=1077) groups. GV oocytes were matured in vitro with or without ALA; (I) without the meiotic inhibitors; (II) supplemented with cilostamide; (III) supplemented with forskolin and (IV) supplemented with Forskolin plus cilostamide. The obtained metaphase II (MII) oocytes were subjected to in vitro fertilization. Independent-samples t-testand ANOVA were used for data analysis. A p-value less than 0.05 was considered to be statistically significant. Results: The COCs maturation, fertilization and two cell embryo rates were higher than those of DOs in the control group, while no significant difference was observed between relevant COCs and DOs when they were cultured with cilostamide meiotic inhibitors in two step manner. Combined treatment of cilostamide and forskolin significantly elevated the developmental rates in both COCs and DOs as compared to other groups. The developmental rates of COCs and DOs in the presence of ALA were similar to their respective groups without ALA. Conclusion: cAMP elevating agents were more effective on DOs than COCs with regard to rates of maturation and fertilization. However, ALA did not affect the developmental rates of both COCs and DOs in in vitro maturation in one or two step manner.

AUTHOR KEYWORDS: ALA; cAMP-elevating agents; Cumulus cell; In vitro maturation; Mouse; Oocyte
INDEX KEYWORDS: cilostamide; cyclic AMP; forskolin; thioctic acid, animal cell; animal experiment; article; controlled study; cumulus cell; drug effect; embryo; embryo culture; female; germinal vesicle; in vitro oocyte maturation; male; mouse; nonhuman; oocyte maturation; spermatozoon; spermatozoon density

Salehnia, M., Töhönen, V., Zavareh, S., Inzunza, J. Does cryopreservation of ovarian tissue affect the distribution and function of germinal vesicle oocytes mitochondria? (2013) BioMed Research International, 2013, art. no. 489032, .

DOI: 10.1155/2013/489032

The aim of this study was to evaluate mitochondrial alteration and ATP content of germinal vesicle (GV) oocytes isolated from fresh and vitrified ovaries. After superovulation, the ovaries from adult mice were collected and divided into control and vitrified groups. GV oocytes were isolated mechanically from each group. Half were cultured for 24 hours and their maturation was assessed. Metaphase II oocytes were collected and submitted to in vitro fertilization and their fertilization rates and development to the blastocyst stage were evaluated. In the remaining GV oocytes, ATP levels were quantified, and mitochondrial distribution, mitochondrial membrane potential, and intracellular free calcium were detected with rhodamine 123, JC-1 and Flou-4 AM staining, using laser-scanning confocal microscopy. Maturation and fertilization rates of GV oocytes and the developmental rates of subsequent embryos were significantly lower in vitrified samples (P < 0.05). The ATP content and Ca2+ levels differed significantly in fresh and vitrified GV oocytes (P < 0.05). Most mitochondria were seen as large and homogenous aggregates (66.6%) in fresh GV oocytes compared to vitrified oocytes (50%). No significant differences in mitochondrial membrane potential were found between the groups. The lower maturation and fertilization rates of GV oocytes from vitrified ovaries may be due to changes in their mitochondrial function and distribution. © 2013 Mojdeh Salehnia et al.


INDEX KEYWORDS: adenosine triphosphate; calcium; flou 4 am; fluorescent dye; JC 1; rhodamine 123; unclassified drug; adenosine triphosphate; adenosine triphosphate, animal cell; animal experiment; animal tissue; article; blastocyst; calcium cell level; cell culture; cell function; cell isolation; cell maturation; cellular distribution; confocal laser microscopy; controlled study; cryopreservation; developmental stage; embryo; embryo development; female; fertilization; fertilization in vitro; germinal vesicle; in vitro study; in vivo study; metaphase; mitochondrial membrane potential; mitochondrion; mouse; nonhuman; oocyte; ovary; ovary tissue; staining; superovulation; vitrification; animal; cell survival; cytology; metabolism; mitochondrion; physiology; vitrification; metabolism; oocyte; ovary, Adenosine Triphosphate; Animals; Blastocyst; Cell Survival; Cryopreservation; Female; Fertilization; Fertilization in Vitro; Membrane Potential, Mitochondrial; Mice; Mitochondria; Oocytes; Ovary; Vitrification, Adenosine Triphosphate; Animals; Blastocyst; Cell Survival; Cryopreservation; Female; Fertilization; Fertilization in Vitro; Membrane Potential, Mitochondrial; Mice; Mitochondria; Oocytes; Ovary; Vitrification

Salehnia, M., Zavareh, S. The effects of progesterone on oocyte maturation and embryo development (2013) International Journal of Fertility and Sterility, 7 (2), pp. 74-81.

Oocyte maturation and embryo development are controlled by intra-ovarian factors such as steroid hormones. Progesterone (P4) exists in the follicular fluid that contributes to normal mammalian ovarian function and has several critical functions during embryo development and implantation, including endometrial receptivity, embryonic survival during gestation and transformation of the endometrial stromal cells to decidual cells. It is well known that the physiological effects of P4 during the pre-implantation stages of some mammal's embryos are mediated by P4 receptors and their gene expression is determined. The effects of P4 on oocytes and embryo development have been assessed by some investigations, with contradictory results. P4, a dominant steroid in follicular fluid at approximately 18 hours after the luteinizing hormone (LH) surge may have a critical role in maturation of oocytes at the germinal stage. However, it has been shown that different concentrations of P4 could not improve in vitro maturation rates of germinal vesicles (GV) in cumulus oocyte complexes (COCs) and cumulus denuded oocytes (CDOs). Culture media supplemented with P4 significantly improved mouse embryo development. In addition, an in vivo experimental design has shown high blastocyst survival and implantation rates in P4-treated mice. In this review we explain some of the findings that pertain to the effects of P4 on oocyte maturation and embryo development both in vitro and in vivo.

AUTHOR KEYWORDS: Embryo; In vitro maturation; Oocyte; Progesterone
INDEX KEYWORDS: cholesterol; granulocyte macrophage colony stimulating factor; luteinizing hormone; pregnenolone; progesterone; steroid hormone, blastocyst; culture medium; embryo development; fertilization; gene expression; hormone substitution; human; nonhuman; oocyte maturation; ovary function; ovary polycystic disease; review; Western blotting

Alijan-Pour, J., Abrari, K., Bluki, T.L., Ghorbanian, M.T., Goudarzi, I., Salmani, M.E., Mirshekar, M. Acute ethanol administration affects memory reactivation: A look at the neuronal density and apoptosis in the rat hippocampus (2012) Pharmacology Biochemistry and Behavior, 102 (2), pp. 321-328.

DOI: 10.1016/j.pbb.2012.04.008

This study is an attempt to examine whether administration of ethanol after memory reactivation will modulate expression of memory in rats or not. We further examined whether this administration alters the number of tunnel positive cells in hippocampus. Adult male Wistar rats were trained in a fear conditioning system using two 1 s , 0.6 mA shock with an interval of 180 s. 24 h later the rats were returned to the chamber for reactivation, and then they were injected with ethanol (0.5, 1, 1.5 mg/kg) or saline, ip. Again, one, seven and fourteen days after reactivation, the rats were returned to the context for 5 min. The freezing time (absence of all movements except respiration) was scored in seconds. In the second experiment, after test 1, the animals were anesthetized and a transcardial perfuse with phosphate buffer and paraformaldehyde 4% was conducted. After post-fixation of brains 5-μm sections were stained with cresyl violet. Finally, paraffin-embedded sections of 10 μm were cut out throughout the tissue and each sample was processed with TUNEL. The number of apoptotic cells in a 130 μm-long segment of the hippocampal CA1 and CA3 fields and dentate gyrus was counted. The data demonstrate that ethanol exposure impairs post retrieval processes. Rats receiving ethanol (1.5 mg/kg) showed lower freezing levels during the first test. Moreover, ethanol decreases the density of CA1, CA3 and DG cells and increases the density of apoptotic cells in all regions of hippocampus. Therefore, ethanol exposure impairs reconsolidation of contextual fear conditioning probably via decreasing the density of CA1, CA3 and DG cells. © 2012 Elsevier Inc. All rights reserved.

AUTHOR KEYWORDS: Contextual fear conditioning; Ethanol; Hippocampus; Reconsolidation
INDEX KEYWORDS: alcohol; paraformaldehyde; phosphate buffered saline, acute drug administration; animal cell; animal experiment; animal tissue; apoptosis; article; cell density; controlled study; dentate gyrus; drug effect; freezing; hippocampal CA1 region; hippocampal CA3 region; hippocampus; male; memory; nerve cell; nick end labeling; nonhuman; priority journal; rat; tissue fixation; tissue section, Animals; Apoptosis; Behavior, Animal; Ethanol; Hippocampus; Male; Memory; Neurons; Rats; Rats, Wistar, Animalia; Rattus; Rattus norvegicus

Talebi, A., Zavareh, S., Kashani, M.H., Lashgarbluki, T., Karimi, I. The effect of alpha lipoic acid on the developmental competence of mouse isolated preantral follicles (2012) Journal of Assisted Reproduction and Genetics, 29 (2), pp. 175-183.

DOI: 10.1007/s10815-011-9706-6

Purpose: This study was designed to investigate the effect of alpha-lipoic acid (ALA) on reactive oxygen species (ROS) production, total antioxidant capacity (TAC) and developmental competence of cultured pre-antral follicles derived from mouse ovarian tissue. Methods: Pre-antral follicles were isolated from immature mouse ovaries and were cultured in α- minimal essential medium supplemented with different concentrations (0, 50, 100, 250 and 500 uM) of ALA. Follicular growth, oocyte maturation and embryo development were evaluated. Separately, ROS and TAC were measured after 0, 24, 48, 72 and 96 h of culture with spectrofluorometery and ferric reducing/antioxidant power (FRAP) assay, respectively. Results: In the presence of 100 uM ALA, developmental rates of follicles, oocytes and embryos were significantly higher than other groups (p < 0.05). At 96 h after culture, a decrease in ROS and an increase in TAC were observed in ALA group compared to control group (p < 0.05). Conclusion: ALA (100 uM) improves the in vitro development of follicles. This effect may be mediated by decreasing ROS concentration and increasing follicular TAC level during the culture period. © 2011 Springer Science+Business Media, LLC.


AUTHOR KEYWORDS: Alpha-lipoic acid; Preantral follicles; Reactive oxygen species; Total antioxidant capacity
INDEX KEYWORDS: antioxidant; reactive oxygen metabolite; thioctic acid, animal experiment; article; competence; controlled study; embryo development; female; fertilization in vitro; immaturity; mouse; nonhuman; oocyte maturation; ovary follicle development; priority journal; spectrofluorometry; survival rate, Animals; Cell Culture Techniques; Female; Fetus; Mice; Oocytes; Ovarian Follicle; Reactive Oxygen Species; Thioctic Acid

Saberivand, A., Karimi, I., Becker, L.A., Moghaddam, A., Azizi-Mahmoodjigh, S., Yousefi, M., Zavareh, S. The effects of Cannabis sativa L. seed (hempseed) in the ovariectomized rat model of menopause (2010) Methods and Findings in Experimental and Clinical Pharmacology, 32 (7), pp. 467-473.

DOI: 10.1358/mf.2010.32.7.1487085

Cannabis sativa L. has been used for the treatment of various gynecological diseases in traditional medicine. The potential of this plant to protect against complications of menopause has been raised but rarely studied. Twenty female rats were divided into five groups: shamoperated (sham), ovariectomized (OVX) and three other ovariectomized groups: HST1%, HST2% and HST10% which received 1%, 2% and 10% hempseed, respectively, in their diet for 3 weeks. The effects of hempseed on plasma lipid and lipoprotein profiles, estradiol and calcium levels were evaluated. Rats were tested for behavioral changes using the forced swimming test. The results showed that ovariectomy, independent of the type of diet, caused elevation of plasma calcium, total cholesterol and HDL-cholesterol levels, while hempseed modified this effect. Plasma estradiol levels were significantly lower in the OVX group compared to other groups. The swimming times for the OVX and sham groups were significantly shorter than that of the HSD10% group. All hempseed-treated groups were less anxious and showed significant declines in fecal boli compared to the sham group. The exploratory diving percent decreased in the HST10% group compared with other groups. These results suggest that hempseed may improve post-ovariectomy complications in rats. © 2010 Prous Science, S.A.U. or its licensors.

INDEX KEYWORDS: calcium; cannabis; cholesterol; estradiol; fatty acid; high density lipoprotein cholesterol; lipid; lipoprotein; calcium; estradiol; lipid; vegetable oil, article; behavior change; calcium blood level; cholesterol blood level; drug effect; estradiol blood level; forced swimming test; menopause; ovariectomy; plant seed; sham procedure; traditional medicine; animal; animal behavior; animal model; blood; female; human; menopause; rat; reproduction; Wistar rat, Animals; Behavior, Animal; Calcium; Cannabis; Estradiol; Female; Humans; Lipids; Menopause; Models, Animal; Ovariectomy; Plant Oils; Rats; Rats, Wistar; Reproductive Physiological Phenomena; Seeds

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