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Cell Journal [Yakhteh]. 2019; 20 (4): 513-520
in English | IMEMR | ID: emr-199620


Objective: In vitro transplantation [IVT] of spermatogonial stem cells [SSCs] is one of the most recent methods in transplantation in recent decades. In this study, IVT and SSCs homing on seminiferous tubules of host testis in organ culture have been studied

Materials and Methods: In this experimental study, human SSCs were isolated and their identities were confirmed by tracking their promyelocytic leukemia zinc finger [PLZF] protein. These cells were transplanted to adult azoospermia mouse testes using two methods, namely, IVT and in vivo transplantation as transplantation groups, and testes without transplantation of cells were assigned in the control group. Then histomorphometric, immunohistochemical and molecular studies were done after 2 weeks

Results: After two weeks, histomorphometric studies revealed that the number of subsided spermatogonial cells [SCs] and the percentage of tubules with subsided SCs in IVT and in vivo groups were significantly more than those in the control group [P<0.05]. Immunohistochemical studies in the transplantation groups confirmed that the PLZF protein was expressed in the cells subsided on the seminiferous tubule. Quantitative reverse-transcription polymerase chain reaction [qRT-PCR] demonstrated that the PLZF gene expression was only positive in the transplantation groups, but it was not significantly different between the IVT group and the in vivo group [P>0.05]

Conclusion: Testicular tissue culture conditions after SSC transplantation can help these cells subside on the seminiferous tubule basement membrane

Cell Journal [Yakhteh]. 2018; 19 (4): 614-619
in English | IMEMR | ID: emr-189852


Objective: re-vitrification of embryos immediately after thawing or after a culture period could be used to preserve the extra embryos after embryo transfer. This study aims to clarify the effect of re-vitrification on Bax and Bcl-2 gene expressions of compact and early blastocyst stage embryos

Materials and Methods: this experimental study was performed on mouse embryos. We collected 8-cell stage embryos [n=400] from female mature mice, 60-62 hoursafter injection of human chorionic gonadotropin [hCG]. The embryos were divided into 5 groups: fresh [n=80], vitrified at the 8-cell stage [n=80], vitrified at the blastocyst stage [n=80], vitrified at the 8-cell stage, and re-vitrified at the compact [n=80] and early blastocyst stages [n=80]. Embryos were vitrified by cryolock. We analyzed the developmental rates of the vitrified-warmed embryos with the chi-square test. Quantitative polymerase chain reaction [qPCR] data were analyzed with SPSS version 16 using one-way analysis of variance [ANOVA] followed by Tukey's post hoc test. P<0.05 were considered statistically significant

Results: the expanded blastocyst formation rate showed a significant difference in re-vitrified embryos compared with fresh embryos [P<0.05]. However, this result was similar between the two re-vitrified groups. Our data showed a significant difference in expression of the Bax and Bcl-2 genes between re-vitrified and fresh embryos [P<0.05]. Expressions of the Bax and Bcl-2 genes showed no significant difference between the two re-vitrified groups

Conclusion: based on our study, re-vitrification could affect developmental rate and expressions of the Bax and Bcl2 genes

Cell Journal [Yakhteh]. 2018; 19 (4): 634-639
in English | IMEMR | ID: emr-189855


Objective: low intensity ultrasound [continues and pulsed] is a form of energy. Spermatogonial stem cells [SSCs] are at the base of male fertility. This study investigated the effects of low intensity ultrasound stimulation [LIUS] and low intensity pulsed ultrasound stimulation [LIUPS] on the expression of germ cell-specific and pluripotency genes in SSCs in vitro

Materials and Methods: in this experimental study, isolated SSCs from neonatal male mice were cultured in Dulbecco's Modified Eagle's Medium [DMEM] with 10% fetal bovine serum [FBS]. In addition, to confirm identification of SSCs, PLZF protein was detected positively in SSCs derived colonies. SSCs were stimulated by LIUS and LIUPS for 5 days, followed by assessment of expression of integrin-alpha6 [Itga6] and beta1 [Itgbeta1], as two germ cell-specific genes, and Oct- 4, as a pluripotency gene, on day 21st by quantitive reverse transcriptase-polymerase chain reaction [qRT-PCR]. To investigate the proliferation rate and colonization of SSCs in different groups, counting whole number of the cells and colonies as well as analysis of the respective diameters were performed on days 7[th], 14[th] and 21[st]. Data was analyzed by ANOVA test

Results: LIUS and LIUPS treatment of mouse SSCs increased expression of Itga6 and Itgbeta1 genes in the experimental groups, compared to the control group [P<0.05], whereas there was no significant difference between the groups, regarding the expression of Oct-4 gene. These treatments maintained survival rate, while they increased proliferation rate and colonization of SSCs during the first week of culture. However, within the second week, proliferation rate and colonization were decreased in the experimental groups

Conclusion: these results suggested that LIUS and LIUPS treatment had good effect on SSCs proliferation and colonization, based on the gene-specific marker expression during 21 days culture in vitro

IJRM-International Journal of Reproductive Biomedicine. 2018; 16 (10): 641-648
in English | IMEMR | ID: emr-206766


Background: Polycystic ovary syndrome [PCO] is one of the most common reasons for infertility. Calligonum as a plant possess some of the important antioxidants that can decrease oxidative stress

Objective: The effects of treatment with Calligonum as an antioxidant on ovary tissue of a PCO mouse model

Materials and Methods: Thirty female NMRI mice were divided into three groups [n=10/each]: control, PCO, and Calligonum. We induced PCO model with single dose of Estradiol valerate [40 mg/kg]. Then Calligonum [20 mg/kg] was intraperitoneally injected weekly for two months. The level of oxidative stress and total antioxidant capacity was assessed in the ovarian tissue by flow cytometry and fluorescence recovery after photobleaching, respectively, and the histological study was conducted by the morphometric method and embryo development with in vitro fertilization

Results: The obtained results showed that estradiol valerate was able to increase oxidative stress within the ovary and causes ovarian cysts after two months. The cyst formation was decreased in Calligonum group compared to PCO group [p=0.001]. The percentage of pre-antral and antral follicles significantly decreased in Calligonum group compared to PCO group [p=0.001]. The oxidative stress decreased in Calligonum group significantly compared to PCO group [p=0.001]. Calligonum can significantly increase the total antioxidant capacity of ovarian tissue [p=0.001] as well as the percentage of in vitro fertilization compared to the PCO group

Conclusion: Calligonum could decrease ovary cyst in PCO model, and improve in vitro fertilization rate. Also, Calligonum extract as an antioxidant could decrease oxidative stress in PCO model

Modares Journal of Medical Sciences, Pathobiology. 2016; 18 (4): 61-70
in Fa | IMEMR | ID: emr-185185


Objective: Vitrification is a convenient, effective method for freezing and storing embryos. Under certain situations, such as an unsuitable endometrial environment, extra embryos can be re-vitrified for future use. There is inadequate data on the effects of revitrification on embryos, so we have evaluated the effects of re-vitrification on the development rate and expression of apoptotic and implantation genes

Methods: Female NMRI mice, ages six-eight weeks were super-ovulated with 7.5 IU PMSG and 7.5 IU hCG. Females were mated with males from the same strain and inspected for the presence of vaginal plugs the following morning. Females with the presence of vaginal plugs were considered to be pregnant and killed 62 h post hCG injection. Eight-cell embryos were flushed from their oviducts and subsequently divided into three experimental groups: fresh, vitrified-warmed 8-cell embryos, and re-vitrifiedwarmed blastocyst embryos. RNA was extracted and we used real-time PCR to evaluate expressions of Bax, Bcl-2, and ErbB4. Data was analyzed by the chi-square and ANOVA tests

Results: A significant difference existed in blastocyst formation rate, degeneration rate, and expressions of Bax, Bcl-2, and ErbB4 in re-vitrified embryos compared to fresh embryos

Conclusion: The vitrification and warming process did not affect the developmental rate and expressions of Bax, Bcl-2, and ErbB4 in the eight-cell stage embryos. However, we observed a change in development rate and expression rates of Bax, Bcl-2, and ErbB4 after re-vitrification in the early blastocyst stage

IJRM-Iranian Journal of Reproductive Medicine. 2016; 14 (8): 511-518
in English | IMEMR | ID: emr-183810


Background: nowadays, oocytes and embryos vitrification has become a routine technique. Based on clinical judgment, re-vitrification maybe required. But little is known about re-vitrification impact on genes expression

Objective: the impact of re-vitrification on apoptotic and implanting genes, Bax, Bcl-2 and ErbB4, at compaction stage embryos were evaluated in this study

Materials and Methods: in this experimental study, 8 cell embryos [n=240] were collected from female mature mice, 60-62 hr post HCG injection. The embryos were divided randomly to 3 groups included: fresh [n=80], vitrified at 8 cell stage [n=80], vitrified at 8 cell stage thawed and re-vitrified at compaction stage [n=80]. Embryos were vitrified by using cryolock, [open system] described by Kuwayama. Q-PCR was used to examine the expression of Bax, Bcl2 ErbB4 genes in derived blastocysts

Results: our result showed that expanded blastocyst rate was similar between vitrified and re-vitrified groups, while re-vitrified embryos showed significant decrease in expanded blastocyst rate comparing with fresh embryos [p=0.03]. In addition, significant difference was observed on apoptotic gene expression when comparing re-vitrified and fresh embryos [p=0.004], however expression of Bax and Bcl-2 [apoptotic] genes didn't demonstrate a significant difference between re-vitrified and vitrified groups. The expression rate of ErbB4, an implantation gene was decreased in re-vitrified embryos comparing with fresh embryos [p=0.003], but it was similar between re-vitrified and vitrified embryos

Conclusion: re-vitrification can alter the expression of Bax, Bcl-2 and ErbB4 genes and developmental rate of mouse embryos in compaction stage

Anatomical Sciences Journal. 2015; 12 (1): 23-28
in English | IMEMR | ID: emr-179363


Introduction: Nowadays, spermatogonial stem cells [SSCs] cultivation has been used by many researchers as an effective tool for infertility treatments. Oxidative conditions can be effective on cell proliferation and differentiation of these cells. So, the aim of this study was to establish oxidative stress model for antioxidant activity of some drugs investigation during SSCs in vitro culture

Methods: Neonatal NMRI male mice [3-5 day] were used for isolation of SSCs. The cell suspension was prepared by twice enzymatic digestion. The cell suspension contents were spermatogonial and sertoli cells and treated by different doses of H2O2 logarithmic concentrations from 0-100 and muM after 24 hours. To access the optimal dose, extra doses from 10-100 and muM was evaluated. After 2 hours of H2O2 treatment, viability was determined by Trypan blue assay. The data were analyzed using SPSS software and One-way ANOVA test

Results: Our data showed that spermatogonial stem cells colonies appeared after 4 days of isolation. These cells expressed OCT4 and PLZF proteins. Many of spermatogonial stem cells were removed after using higher doses of H2O2. The results showed that 30 and muM concentration of H2O2 could induce oxidative stress in spermatogonial stem cell during in vitro culture

Conclusion: According to this study, 30 and muM concentration of H2O2 can cause cell death lower than 50% of total number of cells and can increase oxidative stress in cultivation of SSCs. This model is a suitable tool for studying some new antioxidant drugs

Anatomical Sciences Journal. 2015; 12 (3): 141-144
in English | IMEMR | ID: emr-179382


There are often duplicated ureter with types of congenital anomalies and associated clinical complications. The unilateral duplicated ureter was observed as an incidence result in usual dissection. During routine dissection of the body of an adult male with middle-aged 40-50 year old, the duality of unilateral incompletely ureter was seen in above. Two branches of ureter are coalesce in 7 cm distally hilum of kidney and form a unit ureter. Right kidney and ureter were entirely normal. This variation was accruing because ureter bud split incompletely during embryogenesis. Kidney with bifid ureter forms a clear diagnosis of renal failure. These anatomical differences may exhibit pathological obstruction urinary tract and should be considered in endourological method. Awareness of relationship between renal arteries and collecting systems for applications is necessary and vital in endourological procedures and renal surgeries

IJRM-Iranian Journal of Reproductive Medicine. 2014; 12 (9): 623-632
in English | IMEMR | ID: emr-149664


It has been hypothesized that blastocyst integrin expression changes can affect the spontaneous miscarriage in polycystic ovarian syndromes [PCOS]. In this study, the profile of integrin genes and proteins was investigated on blastocyst of the PCOS experimental mouse model. 30 NMRI female mice were equally divided into 3 groups: control, experimental [PCOS that was injected estradiol valerate [40 mg/kg]]. After 8 weeks, each group was hyper stimulated by PMSG and HCG. Vaginal plaque was checked, and mice were investigated 5 days after the test. Progesterone and estradiol levels were determined; alpha4, alpha v, beta 1 and beta 3 integrin genes and protein of blastocysts were examined by real time PCR method and immunohistochemistry, respectively. Estradiol level was significantly increased [p

Animals, Laboratory , Integrin alpha4 , Integrin alphaV , Integrin beta3 , Integrin beta1 , Blastocyst , Mice , Integrins , Embryo Implantation
AJMB-Avicenna Journal of Medical Biotechnology. 2014; 6 (4): 218-227
in English | IMEMR | ID: emr-149835


Mesenchymal Stem Cells [MSCs] are multipotent cells that can be collected from different sources. Under specific conditions, MSCs can be differentiated to tissue specific cells in vitro. Human Umbilical Cord Mesenchymal Stem Cells [hUCMSCs] can easily be harvested and cultured in in vitro conditions. Production of germ cells from mesenchymal stem cells is a very interesting and promising area in the field of reproductive medicine. In the present study, the possible trans-differentiation of hUCMSCs into Primordial like Germ Cell [PGC] was performed in vitro under specific condition. Human umbilical cord mesenchymal stem cells were cultured and expanded in DMEM medium containing 10% FBS. The cultured cells were studied for differentiation ability to adipocytes and osteocytes. Furthermore, MSCs related markers were identified by flow cytometry method. For PGC differentiation, hUCMS cells were cultured in differentiation medium containing Bone Morphogenetic Protein 4 [BMP4] and it was followed by retinoic acid [RA]. Real time PCR and immunocytochemistry analysis were performed to evaluate the expression of PGC specific genes and proteins, respectively. Our results showed that hUCMSCs cultured in the presence of BMP4 and RA are able to trans differentiate in to PGC like cells in vitro. Real time PCR and immunocytochemistry results showed that differentiated cells expressed PGC specific markers after 14 days of culture. Based on these results, it was concluded that hUCMSC may be considered as a promising alternative cell source in reproductive medicine. More studies including laboratory and also animal models are needed to evaluate the functionality of differentiated PGCs before introducing them to clinical applications

Humans , Umbilical Cord , Germ Cells , In Vitro Techniques , Bone Morphogenetic Protein 4 , Tretinoin
Modares Journal of Medical Sciences. 2014; 17 (3): 93-103
in Fa | IMEMR | ID: emr-167812


Oxidative stress as a consequence of aging can induce infertility in males. In this study, we have investigated the effects of aging on sperm parameters, intra-spermatic water soluble antioxidants, reactive oxygen species [ROS], and in vitro blastocyst formation. We chose 5 older NMRI male mice [10-12 months] and 5 younger NMRI male mice [2-3 months]. Sperm parameters, ROS, soluble antioxidants level and in vitro fertilization rate were assessed in both groups. The results were analyzed by the independent sample and chi square tests. A correlation test was performed between ROS generation and soluble antioxidant levels. Our data showed a significant decrease [P

Animals, Laboratory , Infertility/etiology , Paternal Age , Spermatozoa , Reactive Oxygen Species , Fertilization in Vitro , Oxidative Stress , Mice
Modares Journal of Medical Sciences, Pathobiology. 2014; 17 (1): 17-27
in Fa | IMEMR | ID: emr-160394


It is hypothesized that stem cells have the capability to form tumors after transplantation. Spermatogonial stem cells have proliferation potency and colonization ability related to express pluripotency genes such as c-Myc. The primary aim of this study is to investigate tumorigenicity ability of these cells after in vitro cultivation and inoculation in athymic animals. Spermatogonial stem cells from 3-5 day-old neonatal mice testes [NMRI] were cultured following two-step enzymatic digestion. After one month of culturing the spermatogonial stem cells, the obtained colonies were identified by Oct4 and PLZF markers. Expressions of Nanog, Oct4 and c-Myc pluripotency genes were subsequently studied. We subcutaneously inoculated 5 x 106 cells into athymic mice and assessed tumor formation after 8 weeks. Mouse embryonic stem cells [CCE line] were used as the positive control. Generated tumors were measured by a caliper. The colonies expressed Oct4 and PLZF proteins. Ratio of pluripotency gene expressions in these cells compared to embryonic stem cells significantly decreased [P

IJRM-Iranian Journal of Reproductive Medicine. 2014; 12 (10): 687-694
in English | IMEMR | ID: emr-148984


Endometrial integrin expression changes might be a reason for implantation failure in polycystic ovarian syndromes [PCOS]. Assessment of integrin genes and proteins expression upon endometrium in the PCOS experimental mouse model was the main goal of this study. 30 NMRI female mice were equally divided into control, experimental [PCOS; received estradiol valerate [40 mg/kg]] and sham group [received; olive oil]. After 8 weeks, each group was hyper stimulated by 7 IU PMSG and then, after 48hrs, 7 IU HCG was injected. Vaginal plaque was checked. After 5 days, Progesterone and estradiol levels and endometrial tissues were investigated to evaluate of alpha4, alphav, beta1 and beta3 integrins gene and protein by qPCR method and immunohistochemistry, respectively. Tissue samples were assessed and showed that level of progesterone was significantly decreased in PCOS group. Results of molecular part in the amount of alphav, beta3, beta1 and alpha4 gene expressions showed a great difference in beta3 and alphav genes expressions between experimental groups, alphav, beta3, alpha4 and beta1 proteins in the endometrial stroma in the control group were expressed, but they were not detected in PCOS group. According to the results, integrins had different expression patterns in different areas of the endometrium; such as epithelial and stromal. It seems that in PCOS, this pattern has changed and the results might have a great influence on implantation failure. Therefore, this study suggests that a great attention to this problem may be essential in patients who are involved

Animals, Laboratory , Integrin alpha4 , Integrin alpha5 , Integrin beta3 , Integrin beta1 , Integrins , Endometrium , Mice , Gene Expression , Embryo Implantation
IJRM-Iranian Journal of Reproductive Medicine. 2014; 12 (7): 459-466
in English | IMEMR | ID: emr-159480


Catsper proteins are responsible for entering Ca[2+] to the cell and play an important role in sperm motility and male fertility. Antioxidants are vital for sperm motility too. Escanbil [Calligonum] extract possess some of the important antioxidant like Catechin and Quercetin. Here we investigated the effects of Escanbil [Calligonum] extract on the sperm parameters and the expressing of Catsper gene in aging male mice. In this animal study, firstly, dose response was performed by using these three doses of Escanbil [Calligonum] [10, 30 and 50 mg/kg]. 5 mice in each group were considered and Intra Peritoneal injection was done for 5 weeks. the sperm parameters analyzed and Terminal deoxynucleotidyl transferase dUTP nick end labeling [TUNEL]staining was done. 30 mg/kg dose was considered as optimum dose. Secondly: fifteen aging male mice [11-13 months] were divided into three groups: control, sham and experiment. The experiments were injected Intra peritonealy with Escanbil [Calligonum] extract [30mg/kg] weekly for up to 5 weeks. The sham group was injected Intra Peritoneal [DMSO]. Sperm parameters were analyzed. Expression of Catsper genes was analyzed by Real time PCR. Our results showed that after Escanbil [Calligonum] treatment [30 mg/kg], the sperm parameters were improved in experimental group [p<0.05]. Our data showed that there was a statistical significance difference between the expressions of Catsper 2, 4 in aging experiment group comparison with aging control group [p<0.05]. We investigated that the Escanbil [Calligonum] extract [30 mg/kg] can improve sperm parameters and change the expression of Catsper genes in aging male mice. This herbal extract can be used as an antioxidant component for clinical usages

Modares Journal of Medical Sciences, Pathobiology. 2013; 16 (2): 25-38
in Fa | IMEMR | ID: emr-133252


Diabetic neuropathy leads to axonal transport abnormalities. However its mechanism and the beneficial effects of exercise on these abnormalities are not well documented. The present study aims to investigate KIF1B mRNA in spinal cord sensory neuron tissue of Wistar male rats with diabetic neuropathy following endurance training. We randomly assigned 12 male Wistar rats into three groups: diabetic trained, diabetic untrained and healthy control. Intraperitoneal injection of a STZ [streptozotocin] solution [45 mg/kg] was used to induce diabetes. At two weeks after STZ injections, the mechanical allodynia and thermal hyperalgesia tests demonstrated the presence of diabetic neuropathy. A moderate endurance training protocol was performed for a sixweek period. At 24 hours after the final training session, the rats were sacrified and the L4-L6 sensory neurons of the spinal cord tissue were removed. KIF1B mRNA expression was performed using real time-PCR. Diabetic neuropathy led to increased KIF1B gene expression in the diabetic untrained group compared with the intact control group [p=0.03]. Compared with the diabetic untrained group, training significantly decreased KIF1B gene expression [P<0.05] and blood glucose levels [P=0.0001] in the diabetic trained group. KIF1B mRNA up-regulation in sensory neurons of STZ-diabetic rats is a factor which can be involved in abnormal axonal transport. Endurance training as a nonpharmacotherapy strategy can modulate and return KIF1B to approximate normal levels.

Modares Journal of Medical Sciences, Pathobiology. 2013; 16 (2): 85-94
in Fa | IMEMR | ID: emr-133257


This study presents an efficient, cost-effective method to improve proliferation and colonization of spermatogonial stem cells [SSCs] in vitro. Isolated SSCs from neonate mice were cultured in DMEM culture medium with 10% fetal bovine serum [FBS]. In the first phase of the study, the temperature was controlled by low intensity pulsed ultrasound stimulation [LIPUS] of the plate that contained the culture medium. In the next phase, SSCs were stimulated by LIPUS with 200 mW/cm[2] with 20% and 40% duty cycle for five days. Proliferation and colonization of SSCs were on the seventh day. LIPUS treatment of mouse SSCs increased the proliferation rate and colonization of SSCs in the experimental groups compared to the control group. Average proliferation rate in the 20% duty cycle group was 1.46 +/- 0.06, in the 40% duty cycle group it was 2.00 +/- 0.1 and for the control group, it was 1.26 +/- 0.06. The average number of colonies in the 20% duty cycle group was 24 +/- 7.7, whereas the 40% duty cycle group had 62 +/- 1.4 colonies and the control group had an average of 19 +/- 5.5 colonies. Average colony diameters were as follows: 186.6 +/- 2.07 micro m [20% duty cycle group], 185.3 +/- 4.4 micro m [40% duty cycle group] and 190.0 +/- 2.0 micro m [control group]. Our results showed a significant increase in proliferation rate and number of colonies in the experimental groups compared to the control group [P<0.05], whereas no significant differences were observed between groups in colony diameters. These results suggested that LIPUS treatment can be an efficient, cost effective method to improve proliferation and colonization of SSCs during in vitro culture.

Modares Journal of Medical Sciences, Pathobiology. 2013; 16 (1): 41-54
in Fa | IMEMR | ID: emr-132911


Antioxidants are essential for sperm motility. Calligonum extract possesses the important antioxidants catechin and quercetin. This study investigates the effects of calligonum extract on sperm parameters and the rate of apoptosis in testes of aging male mice. We initially performed a dose response test with using three doses of calligonum [10 mg/kg, 30 mg/kg and 50 mg/kg]. A total of 25 aging male mice [11-13 months] were divided into the following groups of five mice each: control, sham and three experimental groups. The experimental groups received IP injections of calligonum extract [10 mg/kg, 30 mg/kg, 50 mg/kg] weekly for up to five weeks. The sham group received IP injections of DMSO. At the end of the injection period, mice were sacrificed and sperm parameters analyzed. To determine apoptosis in testes, we performed TUNEL staining. Our results showed that after calligonum treatment, there were improved sperm parameters in the 30 mg/kg-treated group compared to the other groups [P

Animals, Laboratory , Spermatozoa , Plant Extracts , Apoptosis , Testis , Mice
Journal of Family and Reproductive Health. 2010; 4 (3): 105-107
in English | IMEMR | ID: emr-113420


To evaluate the potentiality of OVCAR-3 cell line of ovarian adenocarcinoma as a xenograft model for ovarian adenocarcinoma in nude mice. The cell line isolated from advanced human ovarian adenocarcinoma, were inoculated to eight nude mice and two months later. Established tumors were transferred to pathology laboratory to be prepared by H and E staining and immunohistochemical staining with CA125 antibody. Study of H and E slides showed advanced adenocarcinoma. The CA125 Tumor marker was also positive in tumoral tissue. Established tumors showed excellently the characteristics of ovarian adenocarcinoma. This model can be used to evaluate new treatment strategies