Publications by authors named "Bita Ebrahimi"

30 Publications

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Paternal trans-fatty acid and vitamin E diet affects rat offspring's semen quality and PPARs expression.

Andrologia 2021 Apr 27:e14082. Epub 2021 Apr 27.

Department of Genetics, Reproductive Biomedicine Research Centre, Royan Institute for Reproductive Biomedicine, ACECR, Tehran, Iran.

Trans-fatty acids (TFAs) consumption has created concerns regarding male/female reproductive system. However, the effects of TFA in paternal diet on offspring's reproduction have not been addressed. The purpose of this study is to investigate the effects of rat paternal TFAs and vitamin E consumption on offspring's sperm quality and expression pattern of peroxisome proliferator-activated receptors (PPARs) in testis tissues. Forty adult male rats were randomly divided into four groups: Control diet (C); Control diet plus TFA (CTH); diet supplemented with vitamin E (E) and a diet containing vitamin E and TFA (ETH). Mother rats had normal diet during gestation period. Three offspring from each group were chosen randomly and their testicular samples were collected, and sperm parameters were measured by CASA. Our results indicate that feeding fathers with TFA can negatively affect offspring's sperm concentration and motility, while consumption of vitamin E can improve these parameters (p < .05). The paternal diet containing TFA down-regulated the expression of PPARβ and PPARγ genes, whereas vitamin E-containing diet up-regulated the transcription of PPAR genes. In conclusion, TFA intake in paternal diet may have negative effects on reproductive system of the offspring while vitamin E may not diminish these effects.
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http://dx.doi.org/10.1111/and.14082DOI Listing
April 2021

The effect of agar substrate on growth and development of cryopreserved-thawed human ovarian cortical follicles in organ culture.

Eur J Obstet Gynecol Reprod Biol 2021 Mar 29;258:139-145. Epub 2020 Dec 29.

Department of Embryology, Reproductive Biomedicine Research Center, Royan Institute for Reproductive Biomedicine, ACECR, Tehran, Iran. Electronic address:

Objective: To preserve human ovarian tissue structure and improve follicular growth and survival during in-situ culture, various biomaterials are used. In this study we aimed to compare agar as a cultivation substrate with matrigel-coated insert in order to achieve an optimum system for in-situ human follicle culture.

Study Design: Frozen-thawed human ovarian cortical tissues were cultured on either matrigel-coated inserts or agar-soaked substrates. The proportion of morphologically viable and degenerated follicles at different developmental stages, secreted hormonal levels, and apoptotic and proliferation gene expressions were compared between the cultured groups after 7-days of culture.

Results: The follicular growth was not significantly different between the two cultured groups, although showing higher percentage of growing follicles in agar cultured group. The secreted hormonal levels didn't have any difference between two cultured groups. Although the apoptotic gene expressions didn't show any difference between the cultured groups, the apoptotic index was lower in agar cultured group. In addition, Ki67 gene expression, a proliferative marker, showed a significantly higher expression in agar cultured group.

Conclusion: Based on the results, agar is as suitable as matrigel-coated inserts for the survival and growth of follicles during culture. Therefore, agar can be an inexpensive alternative substrate for culturing frozen-thawed human ovarian cortical strips.
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http://dx.doi.org/10.1016/j.ejogrb.2020.12.048DOI Listing
March 2021

The combination of basic fibroblast growth factor and kit ligand promotes the proliferation, activity and steroidogenesis of granulosa cells during human ovarian cortical culture.

Cryobiology 2020 10 29;96:30-36. Epub 2020 Aug 29.

Department of Embryology, Reproductive Biomedicine Research Center, Royan Institute for Reproductive Biomedicine, ACECR, Tehran, Iran. Electronic address:

Different factors, such as basic fibroblast growth factor (bFGF) and kit ligand (KL), are used in ovarian cortical culture to promote activation of primordial follicles. In the present study, the effects of bFGF and KL, alone and in combination, were evaluated on human follicular activation and growth during in-situ cortical culture. Slow frozen-thawed human ovarian cortical tissues (n = 6) were cultured in 4 different groups: 1) control (base medium), 2) KL (base medium; BM + 100 ng/ml KL), 3) bFGF (BM + 100 ng/ml bFGF) and 4) bFGF + KL (BM + 100 ng/ml KL + 100 ng/ml bFGF) for a week. The proportion of morphologically normal and degenerated follicles at different developmental stages, secreted hormonal levels and specific gene expressions were compared. Although the proportion of growing follicles was higher than primordial counterpart in all cultured groups, no significant differences were observed among the cultured groups. In all cultured groups, anti-Müllerian hormone (AMH), progesterone and estradiol hormones levels increased after 7 days of culture; however, this increase was only significant for estradiol in the bFGF + KL group. The expression of Ki67 gene indicated an increase in ovarian cell proliferation in the three experimental groups compared to the control group, however this increment was only significant for the bFGF + KL group. It can be concluded that KL and bFGF factors individually have no beneficial effects on in-situ follicular growth, but their combination positively influences steroidogenesis of granulosa cells without significantly increasing the number of growing follicles.
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http://dx.doi.org/10.1016/j.cryobiol.2020.08.011DOI Listing
October 2020

Noninvasive sexing of human preimplantation embryos using RT-PCR in the spent culture media: A proof-of-concept study.

Eur J Obstet Gynecol Reprod Biol 2020 Sep 12;252:89-93. Epub 2020 Jun 12.

Department of Genetics, Reproductive Biomedicine Research Center, Royan Institute for Reproductive Biomedicine, ACECR, Tehran, Iran.

Preimplantation genetic testing (PGT) routinely requires biopsy which is an invasive approach. The aim of this study was to examine a noninvasive approach for sexing of preimplantation embryos using polymerase chain reaction (PCR)/reverse transcriptase-PCR (RT-PCR) based on the presence of SRY DNA/RNA in the spent culture medium. Two groups were evaluated: in group 1, 40 embryos of routine PGT volunteers were cultured individually after biopsy and in group 2, 31 embryos were cultured individually until Day-5 post-fertilization. Each group was further divided into three subgroups: RNA extraction (RE), nucleic acid (NA) and DNase treated (DT). In the NA and DT subgroups, cDNA synthesis was performed directly on culture medium with or without DNase treatment in DT and NA subgroups, respectively. The results of sexing based on the PCR/RT-PCR in the culture medium, were compared with the results of sexing by fluorescence in situ hybridization (FISH) technique. In group 1, all samples were correctly diagnosed. In group 2, five female samples were misdiagnosed. Test's sensitivity, specificity and accuracy were 100 %, 94.44 % and 96.88 %, in RE, 100 %, 81.82 % and 93.55 % in DT and 100 %, 71.43 % and 85.71 % in NA, respectively. Preimplantation sexing without embryo biopsy, in the spent embryo culture media using RNA amplification based methods including RE and DT seem to be more reliable while nucleic acid based method, NA, led to the highest misdiagnoses probably due to DNA contamination. Since all male samples were correctly diagnosed in all subgroups of this preliminary study, preimplantation noninvasive sexing on culture medium seems feasible, however all sources of nucleic acid contamination must be carefully avoided.
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http://dx.doi.org/10.1016/j.ejogrb.2020.06.023DOI Listing
September 2020

A novel approach for human sperm cryopreservation with AFPIII.

Reprod Biol 2020 Jun 10;20(2):169-174. Epub 2020 Apr 10.

Department of Andrology, Reproductive Biomedicine Research Center, Royan Institute for Reproductive Biomedicine, ACECR, Tehran, Iran. Electronic address:

Sperm cryopreservation causes different stresses including thermal shock, osmotic damage, and ice crystal formation, thereby reducing sperm quality. Few studies have evaluated the application of AFPs in cryopreservation. The effects of antifreeze protein III (AFP III) on human sperm cryopreservation is not fully understood therefore, we conducted this study to investigate the effects of AFPIII treatment on human sperm parameters following cryopreservation. First, for 20 semen samples the effects of various concentrations of AFPIII (0, 0.01, 0.1, 1, 5, 10 μg/ml) were evaluated. Sperm parameters, such as motility and viability were assessed in order to identify an optimal dose. Next, liquefied 20 semen samples were divided into three aliquots and diluted in glycerol-egg-yolk-citrate (GEYC) cryopreserved without AFPIII (control), with optimal dose of AFPIII, as well as fresh groups. After thawing, samples were evaluated for plasma membrane integrity (PMI), DNA fragmentation index (DFI), reactive oxygen species (ROS), and total antioxidant capacity (TAC) levels. Spermatozoa treatment with 0.01, 0.1 and 1 μg/ml AFPIII increased the sperm motility and viability compared to the control group, but the highest concentrations were ineffective. In conclusion, the results showed that the addition of AFPIII to GEYC at 1 μg/ml improved motility, PMI, viability and TAC, and decreased ROS and DNA fragmentation of cryopreserved human semen compared to the control group.
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http://dx.doi.org/10.1016/j.repbio.2020.03.006DOI Listing
June 2020

Chitosan Hydrogel Supports Integrity of Ovarian Follicles during In Vitro Culture: A Preliminary of A Novel Biomaterial for Three Dimensional Culture of Ovarian Follicles.

Cell J 2020 Jan 29;21(4):479-493. Epub 2019 Jul 29.

Department of Embryology, Reproductive Biomedicine Research Center, Royan Institute for Reproductive Biomedicine, ACECR, Tehran, Iran.

Objective: Testing novel biomaterials for the three dimensional (3D) culture of ovarian follicles may ultimately lead to a culture model which can support the integrity of follicles during culture (IVC). The present study reports the first application of a chitosan (CS) hydrogel in culturing mouse preantral follicles.

Materials And Methods: In this interventional experiment study, CS hydrogels with the concentrations of 0.5, 1, and 1.5% were first tested for fourier transform infrared spectroscopy (FT-IR), Compressive Strength, viscosity, degradation, swelling ratio, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) cytotoxicity and live/dead assay. Thereafter, mouse ovarian follicles were encapsulated in optimum concentration of CS (1%) and compared with those in alginate hydrogel. The follicular morphology, quality of matured oocyte and steroid secretion in both CS and alginate were assessed by enzyme-linked immunosorbent assay (ELISA). The expression of folliculogenesis, endocrine, and apoptotic related genes was also evaluated by quantitative real-time polymerase chain reaction (qRT-PCR) and compared with day that in 0.

Results: The rates of survival, and diameter of the follicles, secretion of estradiol, normal appearance of meiotic spindle and chromosome alignment were all higher in CS group compared with those in alginate group (P≤0.05). The expression of and in CS group was significantly higher than that of the alginate group (P≤0.05).

Conclusion: The results showed that CS is a permissive hydrogel and has a beneficial effect on encapsulation of ovarian follicle and its further development during 3D culture.
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http://dx.doi.org/10.22074/cellj.2020.6393DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6722450PMC
January 2020

Evaluating two ovarian decellularization methods in three species.

Mater Sci Eng C Mater Biol Appl 2019 Sep 30;102:670-682. Epub 2019 Apr 30.

Department of Embryology, Reproductive Biomedicine Research Center, Royan Institute for Reproductive Biomedicine, ACECR, Tehran, Iran. Electronic address:

Since there is dearth of practical ways to obtain mature follicles from cryopreserved or native ovarian tissues, especially in patients suffering from ovarian dysfunction, tissue engineering may help in restoring ovarian function and/or fertility. In the present study, the effects of sodium dodecyl sulfate (SDS) and sodium hydroxide (NaOH) on the decellularization of ovarian tissues were studied in order to ascertain their suitability in creating suitable bioscaffolds. Cells were removed from the ovarian tissues of mouse, sheep and human. The samples were distributed among three groups, viz., control (not treated), SDS and NaOH treated. Qualitative histological evaluations, quantitative assessments (nuclear contents, collagen and glycosaminoglycan), immunohistochemistry staining (for laminin, fibronectin and Collagen I), cell viability and scanning electron microscopic (SEM) assays were performed for all experimental groups. Finally, suspensions of mouse ovarian cells were injected into human NaOH treated scaffolds and subsequently auto-transplanted to ovariectomized mice. H&E and IHC staining (GDF-9) were performed on human recellularized NaOH treated scaffolds 1 month after auto-transplantation. Although histological studies and quantitative evaluations confirmed the successful decellularization and presence of key factors in ovarian scaffolds under both treatment methods, NaOH showed more interesting outcomes. Cell metabolic activity in sheep and human ovaries treated with NaOH was statistically (p < 0.05) higher than for SDS treated samples after 72 h. Moreover, spherical associations with cuboidal cells in human NaOH treated scaffolds were observed and this follicular reconstruction was also confirmed by GDF-9. NaOH was found to be more suitable than SDS for the decellularization of ovarian tissues and it supports follicular reconstruction better than SDS. This is a valuable finding in tissue engineering research and can help in the creation of appropriate ovarian bioscaffolds.
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http://dx.doi.org/10.1016/j.msec.2019.04.092DOI Listing
September 2019

An improved method for vitrification of in vitro matured ovine oocytes; beneficial effects of Ethylene Glycol Tetraacetic acid, an intracellular calcium chelator.

Cryobiology 2018 10 3;84:82-90. Epub 2018 Jul 3.

Department of Reproductive Biotechnology, Reproductive Biomedicine Research Center, Royan Institute for Biotechnology, ACECR, Isfahan, Iran.

Vitrification affects fertilization ability and developmental competence of mammalian oocytes. This effect may be more closely associated with an intracellular calcium rise induced by cryoprotectants. The present study aimed to assess whether addition of Ethylene Glycol Tetraacetic acid (EGTA) to vitrification solution could improve quality and developmental competence of in vitro matured ovine oocytes. Vitrified groups were designed according to the presence or absence of EGTA and/or calcium in base media, including: mPB1+ (modified PBS with Ca), mPB1 (modified PBS without Ca), mPB1/EGTA (mPB1 containing EGTA), mPB1/EGTA (mPB1 containing EGTA). In vitro development, numerical chromosome abnormalities, hardening of zona pellucida, mitochondrial distribution and function of viable oocytes were evaluated and compared between groups. Quality of blastocysts was assessed by differential and TUNEL staining. Also, mRNA expression levels of six candidate genes (KIF11, KIF2C, CENP-E, KIF20A, KIF4A and KIF2A), were quantitatively evaluated by RT-PCR. Our results showed that calcium-free vitrification and EGTA supplementation can significantly increase the percentage of normal haploid oocytes and maintain normal distribution and function of mitochondria in vitrified ovine oocytes, consequently improving developmental rate after in vitro fertilization. qRT-PCR analysis showed no significant difference in mRNA expression levels of kinesin genes between vitrified and fresh oocytes. Also, the presence of calcium in vitrification solution significantly increased zona hardening. In conclusion, we have shown for the first time that supplementation of vitrification solution with EGTA, as a calcium chelator, improved the ability of vitrified ovine oocytes to preserve mitochondrial distribution and function, as well as normal chromosome segregation.
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http://dx.doi.org/10.1016/j.cryobiol.2018.07.001DOI Listing
October 2018

Developmental competence of in vitro matured ovine oocytes vitrified in solutions with different concentrations of trehalose.

Reprod Domest Anim 2018 Oct 25;53(5):1159-1167. Epub 2018 Jun 25.

Department of Reproductive Biotechnology, Reproductive Biomedicine Research Center, Royan Institute for Biotechnology, ACECR, Tehran, Iran.

This study aimed to determine the optimum concentration of trehalose in solutions used for vitrification of in vitro matured (IVM) ovine oocytes. IVM oocytes were randomly divided into four experimental (vitrified) and one control (fresh) groups. Experimental groups were treated with different concentrations (0.0, 0.25, 0.5 and 1.0 M) of trehalose. After warming, some viable oocytes were exposed to 0.25% pronase to test zona pellucida hardening, whereas the others were fertilized and cultured in vitro for 8 days to evaluate their developmental competence. Blastocysts quality was assessed by differential staining and TUNEL test. Survival and developmental rates of oocytes vitrified in the presence of 0.5 M trehalose were significantly higher than those of the other vitrified groups. Furthermore, there was a significant difference between fresh and vitrified groups in total blastocyst rate. Analysis of blastocysts quality also revealed a significant difference between the group treated with 0.5 M trehalose and other groups in terms of apoptotic index. Furthermore,zona pellucida digestion time period was longer in trehalose-free (0.0 M) group compared to other groups. In conclusion, we found that IVM ovine oocytes vitrified in solutions containing 0.5 M trehalose are fertilization-competent and are able to produce good-quality blastocysts with an apoptotic index comparable to that of the fresh oocytes. Therefore, 0.5 M may be considered the optimum concentration of trehalose to be used in solutions prepared for vitrification of oocytes.
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http://dx.doi.org/10.1111/rda.13221DOI Listing
October 2018

Utilizing Fibrin-Alginate and Matrigel-Alginate for Mouse Follicle Development in Three-Dimensional Culture Systems.

Biopreserv Biobank 2018 Apr 24;16(2):120-127. Epub 2018 Jan 24.

1 Department of Embryology, Reproductive Biomedicine Research Center, Royan Institute for Reproductive Biomedicine , ACECR, Tehran, Iran .

In vitro culture of ovarian follicles is a new technique in reproductive technology, which helps in understanding the process of folliculogenesis. The in vitro culture of follicles could be carried out using three-dimensional (3D) natural scaffolds that mimic the ovarian tissue stroma. Selection of the right matrix and culture media in these scaffolds could increase the survival and maturation of the follicles. In this work, the applicability of matrigel-alginate (MA) and fibrin-alginate (FA) 3D scaffolds for folliculogenesis was assessed. The ovaries of 13-day-old Naval Medical Research Institute (NMRI) mice were isolated and distributed into control and vitrification groups. Preantral follicles (mean diameter: 120-140 μm) were mechanically isolated from control and vitrified-warmed ovaries, encapsulated in MA or FA scaffold and cultured for 12 days. Follicle survival, growth, maturation, and quantitative expression of oocyte maturation genes (Gdf9, Bmp15, Fgf8, KitL, Kit, and Amh) and proteins (GDF9 and BMP15) were assessed. Survival rate of culture preantral follicles in control groups was found to be significantly higher than vitrified follicles. Antrum formation was similar in all groups. Follicle diameters were significantly increased in all groups during culture period. A decreasing pattern of gene expression was seen for all genes in all groups. This trend was verified through evaluation of protein expression, during which there was strong staining in antral follicles from all groups in the last day of in vitro culture. The better survival and maturation rate of follicles in the MA compared to FA scaffold indicates that the MA matrix, being rich in extracellular matrix components, could mimic the ovarian condition better and presents a good environment for follicle development.
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http://dx.doi.org/10.1089/bio.2017.0087DOI Listing
April 2018

Slow freezing versus vitrification technique for human ovarian tissue cryopreservation: An evaluation of histological changes, WNT signaling pathway and apoptotic genes expression.

Cryobiology 2017 12 4;79:29-36. Epub 2017 Oct 4.

Department of Embryology, Reproductive Biomedicine Research Center, Royan Institute for Reproductive Biomedicine, ACECR, Tehran, Iran; Department of Anatomy, Faculty of Medical Science, Tarbiat Modares University, Tehran, Iran. Electronic address:

This study compared slow freezing and vitrification of ovarian tissue by evaluation of histological changes, WNT signaling pathway and apoptotic genes expression. Ovarian tissue was obtained from women aging 27-38 years old. Ovarian cortex from each patient was divided into three pieces and randomly grouped as slow freezing, vitrification and control groups for investigation of WNT signaling gene expression and β-CATENIN presence as well as histological studies. The stromal structure of all ovaries were preserved. The number of secondary follicles decreased in vitrified group (P < 0.05). WNT-3, β-CATENIN, FZD-2 and GSK-3β expressions were significantly higher in slow frozen and vitrified groups, compared to control group (P < 0.05). On the contrary, AXIN1 expression in slow frozen samples were significantly lower than that of the vitrified and control group. The expression of apoptotic genes, excluding CASP3, was significantly decreased in slow-frozen samples (P < 0.05). Conversely, BAX:BCL-2 percentage significantly increased in vitrification versus slow freezing and control(P < 0.05). Follicles in slow frozen samples displayed nuclear and cytoplasmic β-CATENIN staining, while control and vitrification groups only showed β-CATENIN protein in the cytoplasm. The presented data show that slow freezing results in a better preservation regardless of the type of follicle. Therefore, it is concluded that slow freezing is still an ideal method for ovary cryopreservation.
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http://dx.doi.org/10.1016/j.cryobiol.2017.09.007DOI Listing
December 2017

Fertility Preservation in Cancer Patients: and Options.

Cell J 2017 Jul-Sep;19(2):173-183. Epub 2017 Feb 22.

Department of Embryology, Reproductive Biomedicine Research Center, Royan Institute for Reproductive Biomedicine, ACECR, Tehran, Iran.

Oocyte, embryo and ovarian tissue cryopreservation are being increasingly proposed for fertility preservation among cancer patients undergoing therapy to enable them to have babies after the cancer is cured. Embryo cryopreservation is not appropriate for single girls without any sperm partner and also because oocyte retrieval is an extended procedure, it is impossible in cases requiring immediate cancer cure. Thus ovarian tissue cryopreservation has been suggested for fertility preservation especial in cancer patients. The main goal of ovarian cryopreservation is re-implanting the tissue into the body to restore fertility and the hormonal cycle. Different cryopreservation protocols have been examined and established for vitrification of biological samples. We have used Cryopin to plunge ovarian tissue into the liquid nitrogen and promising results have been observed. Ovarian tissue re-implantation after cancer cure has one problem- the possibility of recurrence of malignancy in the reimplanted tissue is high. Xenografting-implantation of the preserved tissue in another species- also has its drawbacks such as molecular signaling from the recipient. follicle culturing is a safer method to obtain mature oocytes for fertilization and the various studies that have been carried out in this area are reviewed in this paper.
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http://dx.doi.org/10.22074/cellj.2016.4880DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5412777PMC
February 2017

Oocyte maturation, embryo development and gene expression following two different methods of bovine cumulus-oocyte complexes vitrification.

Vet Res Commun 2017 Mar 10;41(1):49-56. Epub 2016 Dec 10.

Shiraz Rostami Human Assisted Reproductive Center, Shiraz, Iran.

Objective: To examine the maturational competence, embryo development and expression of genes involved in oocyte maturation and cumulus expansion (GDF9, BMP15, HAS2, TNFAIP6, FGF17 and FSHr) following two standard methods of bovine COCs vitrification.

Methods: Bovine cumulus-oocyte complexes (COCs) were aspirated from slaughtered ovaries and then distributed into three groups: non-vitrified COCs (control), vitrification 1 group (V1); vitrification was performed by 15% ethylene glycol (EG) and 15% DMSO in holding media (TCM-199 with 20% FCS); and vitrification 2 group (V2); vitrification was performed by 40% EG in holding media. After vitrification, COCs were warmed in two steps and cultured and then evaluated for nuclear maturation, embryo development and gene expressions.

Results: The mean (±SD) percentages of nuclear maturation and blastocyst/cleaved were higher in control group (79.5 ± 8.0 and 31.0 ± 5.1%) than the V1 (34.8 ± 9.1 and 4.4 ± 5.1%) and V2 (47.8 ± 11.7 and 7.1 ± 5.8%) groups (P < 0.05), respectively. Further, COCs in V2 group showed higher mean (±SD) percentages of cleavage compared to V1 group (31.8 ± 1.0 vs 21.7 ± 2.8%; P < 0.05). GDF9 and BMP15 expression levels were higher in COCs in the control than of the vitrification groups (P < 0.05). In addition, expression level of GDF9 and BMP15 was higher in V2 group than in V1group (P < 0.05). The expression of HAS2 and FGF17 in V1 group was lower (P < 0.05) than that of the V2 groups.

Conclusions: Expression of oocyte maturation genes was affected by vitrification procedure and conditions. Using EG alone for vitrification of bovine immature COCs, resulted in higher expression of GDF9, BMP15 and production of more in vitro matured and cleaved oocytes.
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http://dx.doi.org/10.1007/s11259-016-9671-8DOI Listing
March 2017

Dietary Vitamin E Is More Effective than Omega-3 and Omega-6 Fatty Acid for Improving The Kinematic Characteristics of Rat Sperm.

Cell J 2016 Jul-Sep;18(2):262-70. Epub 2016 May 30.

Department of Andrology, Reproductive Biomedicine Research Center, Royan Institute for Reproductive Biomedicine, ACECR, Tehran, Ira.

Objective: Although key roles for dietary vitamin E (VITE) and fatty acid (FA) in fertility have been confirmed, limited data are available on the effects of VITE alone, or a constant level of VITE supplemented by dietary omega-6 and omega-3 FAs in combination on male reproduction. Consequently in this paper, the effects of VITE, sunflower oil, fish oil and their combination on rat sperm were investigated.

Materials And Methods: We divided 50 mature male Wistar rats into 5 groups (n=10) in a experimental completely randomized design for eight weeks: i. Control (CTR): standard diet; ii. Vitamin E diet (VITE): 2 times greater than recommendations; iii. Sunflower oil group (n-6) [gavaged with 0.5 ml/day/rat sunflower oil+VITE diet]; iv. Fish oil group (n-3): [gavaged with 0.5 ml/day/rat fish oil+VITE diet] and v. n-3+n-6 group [gavaged with 0.3 ml fish oil/day/rat+0.2 ml sunflower oil/day/rat+VITE diet]. The sperm parameters were measured by computer assisted semen analyzer (CASA). All data were analyzed with SPSS software.

Results: Feed intake decreased in groups which were administered sunflower oil compared with the other groups (P<0.05). The groups which received only VITE or fish oil+VITE had a significantly higher concentration of sperm compared with the n-6+n-3 and CTR group (P<0.05). VITE and n-3 showed significant improved progressive motility compared to the CTR group, whereas the n-6 and n-6+n-3 groups were in the middle (P<0.05). The highest sperm kinematic parameters were observed in the VITE only group. There was no strong correlation between sperm parameters and blood lipid profiles.

Conclusion: Dietary VITE and fish oil+VITE can improve sperm quality. Our findings can be a focus for improvements in sperm quantity and motility in fertile animals using only dietary VITE.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4988426PMC
http://dx.doi.org/10.22074/cellj.2016.4322DOI Listing
August 2016

Comparison of apoptosis pathway following the use of two protocols for vitrification of immature mouse testicular tissue.

Theriogenology 2016 Nov 13;86(8):2073-82. Epub 2016 Jul 13.

Razi Herbal Medicines Research Center, Lorestan University of Medical Sciences, Khorramabad, Iran.

Our objective was to evaluate the apoptosis incidence in immature mouse testicular tissue after two different protocols of vitrification and short-term culture. Testes of 7-day-old Naval Medical Research Institute mice were isolated and distributed into control and vitrification groups. In vitrification 1 group, testes were vitrified using a combination of ethylene glycol and DMSO in three steps, and in vitrification 2 group, testes were vitrified using a combination of ethylene glycol and sucrose in five steps. Then, fresh and vitrified-warmed testis fragments were cultured for 20 hours. Morphology, cell viability, apoptosis incidence, and apoptosis gene expression (BAX, BCL2, Caspase 3, Fas, Fas ligand, p53) were evaluated at 0, 3, and 20 hours of culture by light microscopy, flow cytometry, and real-time polymerase chain reaction, respectively. Significant decrease of early apoptosis (annexin V+/PI- cells in vitrification 1 and 2 groups at 0 hours of culture, 37.34 ± 0.91 and 30.72 ± 2.2, and at 20 hours of culture, 1.46 ± 0.28 and 0.76 ± 0.11, respectively), increase of late apoptosis (annexin V+/PI+ cells in vitrification 1 group at 0 hours of culture, 14.46 ± 0.86, and at 20 hours of culture, 37.18 ± 2.34), and BAX/BCL-2 ratio (in vitrification 1 and 2 groups at 0 hours of culture, 7.31 ± 0.31 and 6.83 ± 1.38, and at 20 hours of culture, 24.08 ± 4.32 and 9.35 ± 1.91, respectively) were observed in vitrification groups during culture period. Caspase 3 expression was significantly decreased in all groups after 3 hours of culture (in control, vitrification 1, and vitrification 2 groups at 0 hours of culture, 1.00 ± 0.0, 1.56 ± 0.09, and 0.79 ± 0.06, and at 20 hours of culture, 0.37 ± 0.0, 0.96 ± 0.10, and 0.12 ± 0.03, respectively). Expression of p53 was significantly lower in vitrification 1 (0.32 ± 0.02) and control (0.50 ± 0.03) groups in 20 hours of culture as compared with vitrification 2 (0.88 ± 0.14) group. Fas (in vitrification 1 and 2 groups at 0 hours of culture, 2.29 ± 0.23 and 1.14 ± 0.15, and at 20 hours of culture, 12.43 ± 0.46 and 6.7 ± 0.48, respectively) and Fas Ligand (in vitrification 1 and 2 groups at 0 hours of culture, 1.2 ± 0.28 and 5.24 ± 0.32, and at 20 hours of culture, 21.75 ± 2.00 and 25.82 ± 2.15, respectively) expressions significantly increased in vitrification groups after 20 hours of culture. Although both vitrification protocols cause cell death via apoptotic and necrotic pathway, it seems that vitrification 1 protocol induces cell death more via apoptotic pathway than via necrosis. The apoptosis incidence after vitrification may have occurred independent of p53.
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http://dx.doi.org/10.1016/j.theriogenology.2016.06.027DOI Listing
November 2016

An Introduction to The Royan Human Ovarian Tissue Bank.

Int J Fertil Steril 2016 Jul-Sep;10(2):261-3. Epub 2016 Jun 1.

Department of Embryology, Reproductive Biomedicine Research Center, Royan Institute for Reproductive Biomedicine, ACECR, Tehran, Iran; Department of Anatomy, Faculty of Medical Sciences, Tarbiat Modares University, Tehran, Iran.

From December 2000 until 2010, the researchers at Royan Institute conducted a wide range of investigations on ovarian tissue cryopreservation with the intent to provide fertility pres- ervation to cancer patients that were considered to be candidates for these services. In 2010, Royan Institute established the Royan Human Ovarian Tissue Bank as a subgroup of the Embryology Department. Since its inception, approximately 180 patients between the ages of 747 years have undergone consultations. Ovarian samples were cryopreserved from 47 patients (age: 7-35 years) diagnosed with cervical adenocarcinoma (n=9); breast carcinoma (n=7), Ewing's sarcoma (n=7), opposite side ovarian tumor (n=7), endometrial adenocarci- noma (n=4), malignant colon tumors (n=3), as well as Hodgkin's lymphoma, major thalas- semia and acute lymphoblastic leukemia (n=1-2 patients for each disease). Additionally, two patients requested ovarian tissue transplantation after completion of their treatments.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4948080PMC
http://dx.doi.org/10.22074/ijfs.2016.4918DOI Listing
July 2016

Intramuscular Autotransplantation of Vitrified Rat Ovary Encapsulated with Hyaluronic Acid Hydrogel.

Biopreserv Biobank 2016 Apr 1;14(2):114-21. Epub 2016 Feb 1.

2 Department of Embryology at Reproductive Biomedicine Research Center, Royan Institute for Reproductive Biomedicine , ACECR, Tehran, Iran .

Acceleration of revival of ovarian function and maintaining of follicular reserve is mandatory after transplantation of cryopreserved ovarian tissue. In this study, hyaluronic acid hydrogel was used as a scaffold to improve restoration of ovarian estrous cycle and follicular preservation. Mature (∼ 8 weeks old) female Wistar rats with normal estrous cycles were divided in two groups: A: autotransplanted vitrified ovarian tissue without hyaluronic acid (HA), and B: autotransplanted vitrified ovarian tissue encapsulated with HA. Bilateral ovariectomy was performed in the diestrus stage; then ovaries were vitrified, warmed, and autotransplanted intramuscularly. Daily vaginal monitoring was performed until re-initiation of first full estrous cycle. Thereafter, follicular preservation, fibrosis, and apoptosis incidence were assessed histologically and immunohistochemically. The serum follicle stimulating hormone (FSH) levels were also accessed and compared for normal and ovariectomized rats. Re-initiation of first full cycle, atretic follicles, apoptotic index, and area of fibrosis in group A were approximately similar to group B. However, the total numbers of intact follicles were significantly lower in group B than group A. Moreover, the level of FSH in both experimental groups and normal rats was similar and in group B reduced significantly compared to the ovariectomized rats. Hyaluronic acid hydrogel did not show any negative effect on restoration of estrous cycle, but could not support follicular preservation after autotransplantation.
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http://dx.doi.org/10.1089/bio.2015.0021DOI Listing
April 2016

Oocyte maturation and expression pattern of follicular genes during in-vitro culture of vitrified mouse pre-antral follicles.

Gene Expr Patterns 2016 Jan 15;20(1):63-70. Epub 2015 Dec 15.

Department of Stem Cells and Developmental Biology, Cell Science Research Center, Royan Institute for Stem Cell Biology and Technology, ACECR, P.O.Box: 19395-4644, Tehran, Iran.

Our aim was to evaluate the oocyte maturation rate and follicular genes expression pattern during in-vitro culture of vitrified mouse pre-antral follicles. Middle sized pre-antral follicles were isolated mechanically from the ovaries of pre-pubertal mice and distributed in vitrification and control groups. In the vitrification group, follicles were washed in equilibration and vitrification solutions and then were immersed in liquid nitrogen after loading on cryotop tips. After warming in descending concentrations of sucrose solutions, fresh and vitrified-warmed follicles were cultured for 13 days. Follicles survival rate and follicular genes expression were assessed during in vitro culture. Finally, at the end of the culture period oocytes maturation rate were compared in both groups. In the vitrification group, follicles survival rate was lower significantly comparing to the control group (P < 0.05), whereas oocytes maturation rate were similar. Although at the beginning of the culture period, expression of some genes such as Gdf9, Bmp15, Tgfβ1 and BmprII were higher in the vitrification group (P < 0.05), during the rest of the culture period expression pattern of all follicular genes were similar in both groups. In conclusion, survival rate of cryotop vitrified pre-antral follicles reduced during culture period while oocytes maturation and follicular genes expression did not show any noticeable alteration.
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http://dx.doi.org/10.1016/j.gep.2015.12.001DOI Listing
January 2016

Three dimensional in vitro culture of preantral follicles following slow-freezing and vitrification of mouse ovarian tissue.

Cryobiology 2015 Dec 14;71(3):529-36. Epub 2015 Nov 14.

Department of Embryology, Reproductive Biomedicine Research Center, Royan Institute for Reproductive Biomedicine, ACECR, Tehran, Iran.

To evaluate the effects slow-freezing and vitrification on three dimensional in vitro culture of preantral follicles, ovaries of 12-14 days old female NMRI mice were isolated and randomly assigned to fresh control, slow-freezing and vitrification groups. Slow-freezing was performed using programmable freezer. Vitrification was carried out in a medium consisting of ethylene glycol (EG) and dimethyl sulphoxide (Me2SO) by needle immersion method. middle sized preantral follicles were mechanically isolated and cultured for 12 days in 0.7% sodium alginate gel. The follicles development and quantitative expression of oocyte specific genes (Bmp15, Gdf9, Fgf8) and the growth related genes (Igf1, Kit, Kit-l) were assessed after 1, 8 and 12 days of culture. Both cryopreserved groups showed reduction of follicular survival rates compared to the control group on days 8 and 12 of culture (P < 0.05). Antrum formation rates reduced in slow-freezing after 12 days of culture (P < 0.05). Evaluation of gene expression showed reduction of Bmp15, Gdf9, Fgf8, Kit and Kit-l during 12 days of culture (P < 0.05). Kit and Kit-l expression in slow-freezing group significantly reduced on day 8 of culture (p < 0.05). Igf1 expression was lower in slow-freezing group on 1st day of culture than vitrification and control groups (P < 0.05). Finally, intergroup comparison showed same expression pattern of genes after 12 days of culture. Thus, cryopreservation of mouse ovaries by both methods can preserve most developmental parameters and expression of maturation genes. However, vitrification is a better method for cryopreservation of mouse ovaries due to greater antrum formation and expression of growth related markers.
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http://dx.doi.org/10.1016/j.cryobiol.2015.11.001DOI Listing
December 2015

Mouse preantral follicle development in two-dimensional and three-dimensional culture systems after ovarian tissue vitrification.

Eur J Obstet Gynecol Reprod Biol 2015 Nov 30;194:206-11. Epub 2015 Sep 30.

Department of Genetics, Reproductive Biomedicine Research Center, Royan Institute for Reproductive Biomedicine, ACECR, Tehran, Iran.

Objectives: This work on follicle culture and evaluation of expression of oocyte maturation genes helps to better understand the complicated processes of folliculogenesis and to develop new approaches for infertility treatment.

Study Design: Ovaries of 12-day-old female NMRI mice were divided into control and vitrification groups. After vitrification and warming procedures, ovarian tissue morphologies were histologically evaluated and compared to those of the control group. In the second stage, preantral follicles were mechanically isolated from non-vitrified and vitrified ovaries and cultured for 12 days in two-dimensional (2D) and three-dimensional (3D) systems. Finally, the survival and growth rate of follicles and quantitative expression of oocyte maturation genes (Gdf9, Bmp15 and Bmp6) were studied.

Result: Morphological integrity of ovarian tissue in vitrification group was well preserved. Survival rates of cultured preantral follicles in control group during 2D and 3D systems were somewhat similar, but were significantly different between 2D and 3D systems in vitrification group. Although the growth rate of follicles was similar in the 3D system in both groups, substantially higher growth rate was observed for the control group in the 2D system. Expressions of oocyte maturation genes were, to some extent, similar between control and vitrification groups. There was a remarkable reduction in expression pattern of genes in 3D compared to 2D system in both experimental groups, during the 12th day of culture period.

Conclusions: 3D in-vitro culture system could be appeared more appropriate than 2D culture system for preservation of follicles in terms of spatial morphology, growth rate and expression reduction of maturation genes.
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http://dx.doi.org/10.1016/j.ejogrb.2015.09.028DOI Listing
November 2015

Maternal-Effect Gene Expression in Cultured Preantral Follicles Derived from Vitrified-Warmed Mouse Ovary.

Cell J 2015 11;17(2):332-8. Epub 2015 Jul 11.

Department of Embryology at Reproductive Biomedicine Research Center, Royan Institute for Reproductive Biomedicine, ACECR, Tehran, Iran.

Objective: This study was conducted to assess survival of follicles, their oocyte maturation and fertilization potential as well as expression of early embryo developmental genes in in vitro cultured pre-antral follicles derived from vitrified-warmed mouse ovary.

Materials And Methods: In this experimental study, ovaries of 12-day old Naval Medical Research Institute (NMRI) female mice were placed into non-vitrified and vitrifiedwarmed groups. Isolated preantral follicles from experimental groups were cultured in vitro for 12 days. On the 12(th) day of culture, oocyte maturation was induced and then matured oocytes were in vitro fertilized. The rates of oocyte maturation and two-cell stage embryo formation were assessed. Relative expression of Mater and Zar1 was evaluated on days 1, 6, 10 and 12 of culture. Data analysis was performed by t test and two-way ANOVA (P<0.05).

Results: Our data showed no significant difference between the control and vitrification groups in the rate of follicular survival, oocyte maturation and two-cell stage embryo formation. The level of gene expression was higher on the 6(th)and 10(th)days of culture for Mater and Zar1 in vitrified-warmed group compared with non-vitrified group, however, there was no significant difference between the two groups.

Conclusion: It seems that the applied vitrification method did not reveal any negative effect on maturation and developmental competence of oocytes surrounded in preantral follicles and therefore could preserve follicular reserves efficiently.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4503847PMC
http://dx.doi.org/10.22074/cellj.2016.3742DOI Listing
July 2015

Vitrification of mouse preantral follicles versus slow freezing: Morphological and apoptosis evaluation.

Anim Sci J 2015 Jan 7;86(1):37-44. Epub 2014 Jul 7.

Department of Anatomy, Faculty of Medical Science, Tarbiat Modares University, Tehran, Iran.

The aim of this study was evaluation of survivability, maturation rate and apoptotic gene expression of preantral follicles after vitrification and slow freezing technique. Normal mouse preantral follicles were randomly divided into three experimental groups. In the control group, follicles were cultured immediately; in the vitrification and slow freezing groups, follicles were cultured after vitrification-warming and slow freezing-thawing procedures. Follicular viability was assessed by using 0.4% trypan blue, and molecular evaluation of messenger RNA levels of apoptosis-related genes was performed by the semi-quantitative RT-PCR method after 3 h of culture. Oocyte maturation rates were also evaluated on day 14 of culture. Survival and maturation rate in the slow freezing group were significantly lower than those in control and vitrification groups (P ≤ 0.05). Although there was no difference in Survivin expression among the three experimental groups, Bcl-2 expression was significantly lower in the slow freezing group compared to the other groups (P ≤ 0.05). The expression of Bax, P53, Fas and Bax/Bcl-2 ratio in the slow freezing group was significantly higher than control and vitrification groups (P ≤ 0.05). Preantral follicle vitrification seems to be better than slow freezing as seen in the survival, maturation and expression rates of apoptotic gene variants.
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http://dx.doi.org/10.1111/asj.12244DOI Listing
January 2015

A comparative study of saffron aqueous extract and its active ingredient, crocin on the in vitro maturation, in vitro fertilization, and in vitro culture of mouse oocytes.

Taiwan J Obstet Gynecol 2014 Mar;53(1):21-5

Department of Embryology, Reproductive Biomedicine Research Center, Royan Institute for Reproductive Biomedicine, ACECR, Tehran, Iran; Department of Embryology, Faculty of Biological Sciences, Shahid Beheshti University, Tehran, Iran.

Objective: Reactive oxygen species have effects on gamete quality and gamete interaction; they influence spermatozoa, oocytes, embryos, and their environment. In this study, we evaluated the antioxidant effect of different concentrations of saffron (Crocus sativus L.) aqueous extract (SAE) and its ingredient, crocin, on the improvement of in vitro maturation (IVM) and subsequent in vitro fertilization (IVF) and embryo development of mouse oocytes.

Materials And Methods: Cumulus oocyte complexes were collected from ovaries, and germinal vesicle oocytes were cultured in the presence of SAE and crocin. SAE was added at dosages of 5 μg/mL, 10 μg/mL, and 40 μg/mL; dosages of crocin were 50 μg/mL, 100 μg/mL, and 400 μg/mL. All dosages were added to maturation medium and a group without SAE or crocin was considered as the control group. Following IVM, metaphase II oocytes were fertilized and cultured in vitro in order to observe embryo development.

Results: Both SAE and crocin improved the rate of IVM, IVF, and in vitro culture. Addition of 40 μg/mL SAE to maturation medium significantly increased the rate of IVM, IVF, and in vitro culture (p < 0.05). Furthermore 100 μg/mL crocin significantly increased the IVM rate compared to the control group (p < 0.05).

Conclusion: Use of SAE during IVM can affect on IVM, IVF, and early embryo development in a dose-dependent manner. SAE appears to have a stronger effect than pure crocin.
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http://dx.doi.org/10.1016/j.tjog.2012.11.004DOI Listing
March 2014

Effect of ovarian tissue vitrification method on mice preantral follicular development and gene expression.

Theriogenology 2014 Jan 1;81(2):302-8. Epub 2013 Oct 1.

Department of Embryology at Reproductive Biomedicine Research Center, Royan Institute for Reproductive Biomedicine, ACECR, Tehran, Iran.

Vitrification is considered a viable method for cryopreservation of ovarian tissue and selection of methods that minimize follicular damage is important. The objective of the present study was to evaluate the effects of two vitrification methods on ovarian tissue morphology, preantral follicles survival rate during in vitro culture, and relative expression of genes associated with oocyte maturation and cumulus expansion. Ovaries from 12-day-old mice were vitrified in media containing ethylene glycol, dimethyl sulphoxide, and sucrose. Before plunging in liquid nitrogen, ovaries were first loaded into an acupuncture needle (needle immersion vitrification [NIV]) or placed on a cold steel surface for 10 to 20 seconds (solid surface vitrification [SSV]). The integrity of the ovarian tissue was well-preserved after vitrification and was similar controls. Follicle viability in the SSV group was lower (P < 0.05) than in the control group after 6 days of culture and the NIV group after 10 day of culture. Follicle viability after 12 day of culture was 92.8%, 82.1%, and 58.4% in control, NIV, and SSV groups, respectively. Bmp15, Gdf9, BmprII, Alk6, Alk5, Has2, and Ptgs2 gene expression patterns were similar among groups. However, the level of gene expression in the vitrification groups during Days 6 to 10 were higher compared with the control group. In conclusion, ovarian tissue morphologic integrity was well-preserved, regardless of the vitrification method. Vitrification using the needle immersion method resulted in greater follicular survival after 12 day of culture than the SSV method. Gene expression patterns during culture did not seem to explain the reduced survival rate observed in the solid surface group.
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http://dx.doi.org/10.1016/j.theriogenology.2013.09.029DOI Listing
January 2014

Effect of Different Thawing Rates on Post-Thaw Viability, Kinematic Parameters and Chromatin Structure of Buffalo (Bubalus bubalis) Spermatozoa.

Cell J 2013 20;14(4):306-13. Epub 2013 Feb 20.

1. Department of Clinical Science, Faculty of Veterinary Medicine, Urmia Branch, Islamic Azad University, Urmia, Iran.

Objective: The aim of the present study was to evaluate three thawing rates on the post thaw motility, viability and chromatin structure of buffalo semen frozen in 0.5-ml straws.

Materials And Methods: In this experimental study semen was collected with artificial vagina (42℃) from four buffalo bulls.Split pooled ejaculates (n=4) were extended at 37℃ with a Bioxcell® extender. Semen was cooled to 4℃ within 2 hours, equilibrated at 4℃ for 4 hours, then filled in 0.5 ml French straws, and frozen in programmable cell freezer before plunging into liquid nitrogen. Straws were thawed at water bath temperatures of 37, 50 or 70℃ for 30, 15 and 6 seconds, respectively. Semen was incubated at 37℃ for 2 hours and evaluated for post thaw motility, viability, acrosomal and DNA integrity of spermatozoa. Analysis of variance (ANOVA) was used for comparisons of means. When the ANOVA test showed statistical differences, the mean of the treatments were compared using Duncan's multiple range tests.

Results: The initial postthaw motility (0 hour) averaged 62.7 ± 7.2%, 73.1 ± 9.77%, and 74.9 ± 8.58% for the three thaw rates, respectively. Kinematic parameters such as average path velocity, linearity and beat/cross frequency in the thaw rate of 70℃ for 6 seconds were superior to other rates studied (p<0.05). After 2 hours of incubation, proportions of progressive motility and Kinematic parameters decreased in all groups (p>0.05). A positive correlation was detected between sperm motility and thawing rate after two hours incubation times. The percentage of viable spermatozoa and spermatozoa with an intact acrosome and plasma membrane integrity were not different between the groups of samples thawed at different temperatures (p>0.05). The percentage of spermatozoa with chromatin dispersion forthe thaw rate of 70℃ for 6 seconds was significantly higher than for the to other rates studied (p< 0.05). In contrast with motility and viability, the DNA integrity of post thaw spermatozoa remained unaffected during 2 hours incubation.

Conclusion: The post thaw motility and kinematic parameters of buffalo spermatozoa were significantly improved immediately after thawing by increasing the thawing rate from 37℃ in 30 seconds to70℃ in 6 seconds. However, this relative advantage had disappeared after incubation in a water bath at 37℃ for two hours.A thaw rate of 70℃ for 6 seconds was associated with higher chromatin dispersion than the other thaw rates studied. Sperm thawing over at 50 degrees could be safely used to improve motility recovery after sperm cryopreservation in buffalo bulls.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3593936PMC
April 2013

Development of 4-cell mouse embryos after re-vitrification.

Cryobiology 2012 Feb 23;64(1):23-6. Epub 2011 Nov 23.

Department of Embryology, Reproductive Biomedicine Center, Royan Institute for Reproductive Biomedicine, ACECR, Tehran, Iran.

This paper reports studies on the effects of re-vitrification by the CPS (Closed Pulled Straw) method on the development of 4-cell stage mouse embryos. The procedure involved culturing 2-cell mouse embryos in G-1 medium until the 4-cell stage followed by the division of the normal 4-cell stage embryos into a control group (non-vitrified) and two experimental subgroups (vitrified and re-vitrified). Embryos in the vitrified subgroup were cryopreserved by the CPS vitrification method. In the second experimental subgroup (re-vitrified), embryos that were already vitrified were warmed and cryopreserved again by the same method. There was no significant reduction in the rate of blastocyst formation after vitrification and re-vitrification. However, re-vitrification reduced the total cell number, ICM (inner cell mass) percent and blastocyst diameter (P<0.05). These results showed that vitrification and re-vitrification by the CPS method did not negatively affect the development of vitrified-warmed 4-cell mouse embryos, whereas re-vitrification significantly reduced both the cell number and diameter of blastocysts.
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http://dx.doi.org/10.1016/j.cryobiol.2011.11.003DOI Listing
February 2012

Effects of retinoic acid on maturation of immature mouse oocytes in the presence and absence of a granulosa cell co-culture system.

J Assist Reprod Genet 2011 Jun 17;28(6):553-8. Epub 2011 Jun 17.

Department of Embryology, Royan Institute for Reproductive Biomedicine Research, ACECR, P. O. Box: 166565-9711, Tehran, Iran.

Purpose: Evaluation of the all-trans retinoic acid (t-RA) effects on in vitro maturation (IVM) and in vitro fertilization (IVF) of immature mouse oocytes in the presence and absence of granulosa cell monolayer.

Methods: Denuded oocytes isolated from mice ovaries and matured in IVM medium alone (Control I), IVM medium in the presence of granulosa cells (Control II), IVM medium with t-RA (Experimental I) and IVM medium simultaneously with t-RA and granulosa cells (Experimental II). After 24 h, matured oocytes were fertilized in T6 medium and their development was followed until the blastocyst stage. Metaphase II oocytes ploidy were evaluated by chromosome counting.

Results: The t-RA group compared to the control groups showed no obvious abnormalities. Additionally maturation and embryo development rates significantly increased in the t-RA treated granulosa cell co-culture system.

Conclusions: In conclusion, association of t-RA with granulosa cell co-culture during in vitro maturation increases meiosis resumption, formation of metaphase II oocytes, as well as 2-cell and blastocyst stage embryos.
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http://dx.doi.org/10.1007/s10815-011-9579-8DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3158250PMC
June 2011

Ultrastructural changes of sheep cumulus-oocyte complexes following different methods of vitrification.

Zygote 2012 May 17;20(2):103-15. Epub 2011 Feb 17.

Department of Anatomy, Faculty of Medical Science, Tarbiat Modares University, P.O. Box: 14115-111, Tehran, Iran.

To determine the ultrastructural changes of sheep cumulus-oocyte complexes (COCs) following different methods of vitrification, good quality isolated COCs (GV stage) were randomly divided into the non-vitrified control, conventional straw, cryotop and solid surface vitrification groups. In both conventional and cryotop methods, vitrified COCs were respectively loaded by conventional straws and cryotops, and then plunged directly into liquid nitrogen (LN2); whereas in the solid surface group, vitrified COCs were first loaded by cryotops and then cooled before plunging into LN2. Post-warming survivability and ultrastructural changes of healthy COCs in the cryotop group especially in comparison with the conventional group revealed better viability rate and good preservation of the ooplasm organization. However in all vitrification groups except the cryotop group, mitochondria were clumped. Solely in the conventional straw group, the mitochondria showed different densities and were extremely distended. Moreover in the latter group, plenty of large irregular connected vesicles in the ooplasm were observed and in some parts their membrane ruptured. Also, in the conventional and solid surface vitrification groups, cumulus cells projections became retracted from the zona pellucida in some parts. In conclusion, the cryotop vitrification method as compared with other methods seems to have a good post-warming survivability and shows less deleterious effects on the ultrastructure of healthy vitrified-warmed sheep COCs.
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http://dx.doi.org/10.1017/S0967199410000638DOI Listing
May 2012

In vitro maturation, apoptotic gene expression and incidence of numerical chromosomal abnormalities following cryotop vitrification of sheep cumulus-oocyte complexes.

J Assist Reprod Genet 2010 May 9;27(5):239-46. Epub 2010 Mar 9.

Department of Anatomy, Faculty of Medical Science, Tarbiat Modares University, Tehran, Iran.

Purpose: The purpose of this study was to evaluate the effects of cryotop vitrification of sheep cumulus-oocyte complexes (COCs) on oocyte maturation, apoptotic gene expression and incidence of chromosomal abnormalities.

Methods: Freshly isolated (control group) and vitrified-warmed COCs (cryotop group) were matured in vitro. The expression of pro- and anti-apoptotic genes was investigated by real-time PCR. The incidence of numerical chromosomal abnormalities was evaluated by cytogenetic analysis.

Results: The mean percentage of oocytes in the cryotop and control groups that reached metaphase II was 49.25 +/- 3.01% and 51.94 +/- 2.7% respectively. The expression rates of pro- and anti-apoptotic genes were similar in both groups, whereas the incidence of numerical chromosomal abnormalities was higher in the cryotop group compared to the control group (42.5% vs. 20%, p < 0.05).

Conclusion: Although cryotop vitrification of COCs did not affect the incidence of oocyte maturation or apoptotic gene expression, significant deficiencies in the maintenance of oocyte chromosomal organization were seen.
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http://dx.doi.org/10.1007/s10815-010-9401-zDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2881203PMC
May 2010

IVM and gene expression of sheep cumulus-oocyte complexes following different methods of vitrification.

Reprod Biomed Online 2010 Jan 30;20(1):26-34. Epub 2009 Oct 30.

Department of Anatomy, School of Medical Science, Tarbiat Modarres University, PO Box 14115-111, Tehran, Iran.

To determine the optimal vitrification conditions for sheep cumulus-oocyte complexes (COC), good-quality isolated COC were randomly divided into non-vitrified control, conventional straw, cryotop and solid-surface vitrification groups. In the conventional and cryotop methods, the vitrified COC were respectively loaded by conventional straw and cryotop, whereas in the solid-surface group, the vitrified COC were loaded by cryotop and then cooled before plunging in liquid nitrogen. The results indicated that the mean percentage of viability of vitrified-warmed COC was higher in both cryotop groups than that of the conventional group (83.84+/-2.85 and 78.56+/-1.72 versus 63.43+/-1.48%, P<0.05). In the cryotop group, although the mean percentage of oocyte maturation was similar to that in the control group (48.81+/-3.09 versus 51.94+/-3.01%), it was significantly higher than the other vitrification groups (48.81+/-3.09 versus 36.60+/-1.69 and 6.09+/-2.51%, respectively, P<0.05). However, the expression of maturation genes (GDF9, BMP15) was retarded after vitrification. Among the vitrification groups, the cryotop group had better expression. BMPRII was also expressed highly in the control, whereas ALK5 was similar in all groups. In conclusion, direct cryotop, when compared with other vitrification methods, seemed to be safe and could increase the viability, post-warming maturation and maturation-gene expression rates of sheep COC.
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http://dx.doi.org/10.1016/j.rbmo.2009.10.020DOI Listing
January 2010