Improvement of Mouse Preantral Follicle Survival and Development following Co-Culture with Ovarian Parenchyma Cell Suspension.

BACKGROUND: The parallel and continued improvements in both infertility treatment and the management of malignancy cases have brought to the forefront the potential for fertility preservation. Using ovarian follicular resources can effectively improve reproductive capacity and prevent infertility. The primary aim of this research was to try to generate an appropriate in vivo environment for the growth of the mouse follicles. Hence, the possible effects of the ovarian parenchyma cell suspension were explored on the growth and maturation of preantral follicles in vitro. MATERIALS AND METHODS: In this experimental study, ovarian parenchymal cells were mechanically dissociated from preantral follicles of 12-14 days-old NMRI mice and then divided into 5 experimental groups (G1: Control, G2: Fresh follicle with fresh parenchyma cell suspension, G3: Vitrified-warmed follicle with fresh parenchyma cell suspension, G4: Fresh follicle with frozen-thawed parenchyma cell suspension, and G5: Vitrified-warmed follicle with frozenthawed parenchyma cell suspension). The diameter of the follicles and immature oocytes, viability, antrum formation, resumption of meiosis, in vitro fertilization (IVF), and Gdf9, Bmp6, and Bmp15 gene expression were examined on different periods. RESULTS: The diameter of the follicles and the oocytes on days 4 and 8, as well as the survival rate of the follicles up to day 12, were significantly higher in G2 and G4 compared to the Ctrl group (G1: 73.66%, G2:87.99%, G3: 82.70%, G4: 94.37%, and G5: 78.59%). Expression of growth marker genes for G3, and G5 groups was significantly higher than other groups, which indicated the protective effects of parenchyma cell suspension on follicles damaged by vitrification solutions. CONCLUSION: The growth, survival, and maturation of preantral follicles could be enhanced by co-culturing them with ovarian parenchyma cells. Further studies are needed to optimize the conditions for a successful parenchyma cell suspension-induced in vitro maturation (IVM) to occur in infertility clinics.

Fibrin Scaffold Incorporating Platelet Lysate Enhance Follicle Survival and Angiogenesis in Cryopreserved Preantral Follicle Transplantation.

BACKGROUND: Transplantation of cryopreserved follicles can be regarded as a promising strategy for preserving fertility in cancer patients under chemotherapy and radiotherapy by reducing the risk of cancer recurrence. The present study aimed to evaluate whether fibrin hydrogel supplemented with platelet lysate (PL) could be applied to enhance follicular survival, growth, and angiogenesis in cryopreserved preantral follicle grafts. MATERIALS AND METHODS: Preantral follicles were extracted from 15 four-week-old NMRI mice, cryopreserved by cryotop method, and encapsulated in fibrin-platelet lysate for subsequent heterotopic (subcutaneous) auto-transplantation into the neck. Transplants were assessed in three groups including fresh follicles in fibrin-15%PL, cryopreserved follicles in fibrin-15%PL, and cryopreserved follicles in fibrin-0% PL. Two weeks after transplantation, histological, and immunohistochemistry (CD31) analysis were applied to evaluate follicle morphology, survival rate, and vascular formation, respectively. RESULTS: Based on the results, fibrin-15% PL significantly increased neovascularization and survival rate (SR) both in cryopreserved (SR=66.96%) and fresh follicle (SR=90.8%) grafts, compared to PL-less fibrin cryopreserved transplants (SR=28.46%). The grafts supplemented with PL included a significantly higher percentage of preantral and antral follicles. Also, no significant difference was observed in the percentage of preantral follicles between cryopreserved and fresh grafts of fibrin-15% PL. However, a significantly lower (P=0.03) percentage of follicles (23.37%) increased to the antral stage in cryopreserved grafts of fibrin-15%PL, compared to fresh grafts (35.01%). CONCLUSION: The findings demonstrated that fibrin-PL matrix could be a promising strategy to improve cryopreserved follicle transplantation and preserve fertility in cancer patients at the risk of ovarian failure.

The in vitro effect of chick embryo extract on mice pre-antral follicles.

Chick embryo extract (CEE) contains a variety of growth factors which may improve in vitro follicle growth. Therefore, the effect of CEE on mouse pre-antral follicle culture was evaluated. Different percentages of CEE (0, 0.50%, 1.00%, 5.00% and 10.00%) were added to culture medium. Hence, the osmolarity of media was measured. Pre-antral follicles with diameter of 120-150 µm were isolated from 12-14 days old mouse ovary and cultured for 12 days. After culture, the maturation rate was assessed. Granulosa cells viability was evaluated using MTT test and estradiol levels were evaluated using related radio-immunoassay (RIA). Genes expression (BMP15 and ALK6) was also evaluated. The osmolarity of media and granulosa cells viability were the same in all groups. Estradiol level in group with 10.00% CEE was significantly decreased compared to the control group. After 12 days culture, the percentage of antral follicles development was significantly higher in the group with 5.00% CEE compared to control group. The percentage of metaphase II and germinal vesicle breakdown oocytes was significantly higher in group 5.00% CEE compared to control group. The expression of BMP15 gene in antral follicles in 5.00% CEE and control groups was significantly lower compared to pre-antral follicles. However, the expression of ALK6 gene in antral follicles in 5.00% CEE and control groups was not significantly different compared to pre-antral follicles. The increasing effect of CEE on follicle viability with keeping normal gene expression indicates that addition of proper percentage of CEE to culture media improves culture conditions, making it a possible choice to be used as a follicular growth enhancer in infertility clinics.

The effect of Verapamil on ischaemia/reperfusion injury in mouse ovarian tissue transplantation.

One of the challenges that must be overcome during ovarian tissue transplantation is Ischaemia/Reperfusion Injury (IRI). The most important hypothesis explaining the cellular events in I/R processes are calcium overload and oxygen free radicals constitute. Here, we study the effect of verapamil on IRI, and consequently on follicle survival during ovarian transplants in an autograft model. Female mice were randomly assigned into three groups in order to ovarian autotransplantation as follow: Group 1 (Control group), Group 2 (Transplanted group) and Groups 3 (Transplanted + Verapamil group). The grafted ovaries were collected at 3, 7 and 14 days after transplantation for evaluation of follicle content and morphology, apoptosis and Malondialdehyde (MDA) concentration. The results showed that verapamil treatment significantly preserved primordial follicular reserve and reduced the number of degenerated follicles compared to the transplanted group (P < 0.05). MDA levels were significantly higher on the 14th day after transplantation, in group 2 than in group 3. In conclusion, verapamil treatment is effective for the preservation of the follicular pool and reducing tissue damage induced by transplantation of ovarian tissue.

An in vitro study on oocyte and follicles of transplanted ovaries treated with vascular endothelial growth factor.

OBJECTIVE: Retrieval of high quality follicles and oocytes from transplanted ovaries is essential for higher fertility preservation efficiency. The effect of vascular endothelial growth factor (VEGF) was evaluated on the survival rate of preantral follicles following ovarian transplantation. MATERIAL AND METHODS: Prepubertal female mice were divided to 6 groups including: control (C), transplanted with no VEGF treatment (T) and transplanted with different dosages of VEGF [0.5 µg/mL (TV1), 1 µg/mL (TV2), 2 µg/mL (TV3), and 4 µg/mL (TV4)]. Twenty-one days later, the left ovaries were removed and transplanted on gluteal muscle. Each dose was injected directly into transplanted ovary. Twenty-one days after transplantation, the ovaries were taken, and follicles and cumulus-oocyte-complexes (COCs) were released using 26-gauge needles with a stereo microscope. The number of healthy COCs, matured oocytes, and in vitro developed embryos after fertilization in vitro were evaluated to determine the best dose of VEGF. Follicle number and follicular growth was evaluated relative to the dose of VEGF provided. Transplantation and VEGF treatment with the best dose was performed as mentioned above and in vitro follicle growth in transplanted ovaries was compared with opposite ovaries (OPP). RESULTS: COC retrieval was significantly lower in the transplanted groups compared with the control group (p<0.05). The percentage of metaphase II oocytes was significantly lower in the group treated with 4 µg/mL VEGF compared with the controls (p<0.01). In the TV2 (1 µg/mL) and TV3 (2 µg/mL) groups, the percentages of morula and blastocysts were significantly improved compared with the T group (p<0.01). In the OPP group, the number of follicles was significantly higher compared with the transplanted groups (p<0.01). CONCLUSION: The improving effect of VEGF on in vitro maturation and in vitro development outcome indicates that VEGF administration may increase transplantation efficiency for fertility preservation.

Function of vitrified mouse ovaries tissue under static magnetic field after autotransplantation.

This study was designed to investigate the effects of applying 1 mT static magnetic field (SMF) during the vitrification process, on the viability of ovarian follicles after vitrification-warming and autotransplantation. The study was conducted in two phases. In the first phase, ovaries of female NMRI mice (6 to 8 weeks old) were randomly divided into three groups: 1- Freshly isolated ovaries fixed in Bouin solution (control group), 2- Ovaries vitrified-warmed without exposure to magnetic field (V1 group) and 3- Ovaries exposed to magnetic field during equilibration step of the vitrification process (V2 group). In the second phase, the vitrified (V1 and V2 groups) and fresh ovarian tissues were autografted into the back muscles of the mice from which the ovaries were extracted. In both phases, morphological aspects and molecular characteristics of active-apoptotic caspase-3 antibody were evaluated. Results indicated the lower percentages of morphologically intact primordial, primary and antral follicles in the V1 group (67.6, 49.5 and 17.6%, respectively) than those of control (97.3, 85.4 and 42.1%, respectively) and V2 (94.1, 78.8 and 40.9%, respectively) groups. In addition, the mean percentages of morphologically intact follicles in the V1 group were statistically lower than those in other groups, after transplantation. The rate of apoptosis in preantral follicles of the V1 group was significantly higher than that in the other groups. It was concluded that exposure of mice ovaries to SMF during vitrification resulted in greater resistance to injuries.

Comparison of Allotransplantation of Fresh and Vitrified Mouse Ovaries to The Testicular Tissue under Influence of The Static Magnetic Field.

OBJECTIVES: The aim of this study was to investigate the effects of static magnetic field (SMF) during transplantation of the ovarian tissue into the testis. MATERIALS AND METHODS: In this experimental study, ovaries of 6- to 8-week-old female Naval Medical Research Institute (NMRI) mice were randomly divided into four groups: i. Fresh ovaries were immediately transplanted into the testicular tissue (FOT group), ii. Fresh ovaries were exposed to the SMF for 10 minutes and then transplanted into the testicular tissue (FOT+ group), iii. Vitrified-warmed ovaries were transplanted into the testicular tissue (VOT group), and iv. Vitrified-warmed ovaries were transplanted into the testicular tissue and the transplantation site was then exposed to the SMF for 10 minutes (VOT+ group). RESULTS: The lowest percentages of morphologically dead primordial follicles and the highest percentages of morphologically intact primordial follicles were seen in the FOT+ group (4.11% ± 2.88 and 41.26% ± 0.54, respectively). Although the lowest significant percentage of maturation, embryonic development and fertility was observed in the VOT group as compared to the other groups, the difference in the fertility rate was not significant between the VOT and VOT+ groups. Estrogen and progesterone concentrations were significantly higher in the FOT+ group than those of the control mice. CONCLUSIONS: It is concluded that, exposure of the vitrified-warmed ovaries to SMF retains the structure of the graft similar to that of fresh ovaries.

Auto-transplantation of whole rat ovary in different transplantation sites.

This study was carried out to assess the different ovarian transplantation sites after short-time autografting. Female rats were randomized into five groups, with six rats in each group, including control (intact), cervical subcutaneous transplanted (CST), back subcutaneous transplanted (BST), subfascial transplanted (SFT) and intramuscular transplanted (IMT) groups. In all experimental groups, the right ovary was removed and transplanted into different sites. After three weeks, ovaries were removed for morphology assessment, follicular counting and the rates of corpus luteum (CL) and cyst formation. Transplanted ovaries in BST and SFT groups were full of cysts and did not have sufficient numbers of intact follicles and were excluded from experiments. In IMT and CST groups, re-anastomosis, follicular development and good homogeneity of the stromal tissue were seen. However, the difference in intact antral follicles between CST (7.92 ± 0.02%) and CST-Op (opposite ovary of CST group) (30.99 ± 0.03%) was significant as well as the difference between CST (7.92 ± 0.02%) and control (10.08 ± 0.01%) groups. In addition, the number of intact primordial follicles in the CST-Op (16.58 ± 0.02%) group was significantly less than that of the control (40.40 ± 0.03%) group. Interestingly, the number of CL was significantly increased in the CST-Op (11.71 ± 0.01%) and IMT-Op (9.16 ± 0.02%) groups compared to the control and experimental groups. Although both intramuscular and subcutaneous sites effectively preserved ovarian follicles after three weeks, cervical subcutaneous site was better suited for auto-transplantation in rat.

Effects of Crocin Supplementation during In Vitro Maturation of Mouse Oocytes on Glutathione Synthesis and Cytoplasmic Maturation.

BACKGROUND: Crocin is an active ingredient of saffron (Crocus sativus L.) and its antioxidant properties have been previously investigated. This carotenoid scavenges free radicals and stimulates glutathione (GSH) synthesis; consequently, it may protect cells against oxidative stress. The aim of this research is to protect oocytes from oxidative stress by the addition of a natural source antioxidant. MATERIALS AND METHODS: In the present in vitro experimental study, we collected cumulus oocyte complexes (COCs) from mouse ovaries of euthanized, 6-8 week-old female Naval Medical Research Institute (NMRI) mice. Oocytes were subjected to in vitro maturation (IVM) in the presence of either crocin (5 or 10 µg/ml), 5 mM buthionine-[S-R]- sulfoximine (BSO), or the combination of crocin plus BSO. Oocytes that matured in vitro in a medium without crocin or BSO supplements were considered as controls. Following 16-18 hours of IVM, matured oocytes (n=631) were fertilized by capacitated sperm from NMRI male mice, and cultured in vitro for up to 96 hours to assess preimplantation embryonic development. The levels of GSH in metaphase II (MII) oocytes after IVM (n=240) were also assessed by the 5, 5-dithio-bis (2-nitrobenzoic acid) (DTNB)-GSH reductase recycling assay. RESULTS: Supplementation of IVM media with 10 µg/ml crocin significantly (P<0.05) increased nuclear maturation, preimplantation development and GSH concentrations compared with the control group. Maturation of oocytes in IVM medium supplemented with BSO alone or the combination of 5 µg/ml crocin and BSO drastically decreased GSH concentrations and subsequently resulted in low rates of maturation, fertilization and blastocyst development. However, the combination of 10 µg/ml crocin with 5 mM BSO increased the level of nuclear maturation which was comparable to the control group. CONCLUSION: Supplementation of IVM media with crocin can improve nuclear maturation rates and subsequent developmental potential of mouse oocytes. This may occur by its beneficial effect in increasing GSH concentrations in MII oocytes.

The effects of platelet lysate on maturation, fertilization and embryo development of NMRI mouse oocytes at germinal vesicle stage.

Purpose: Improving in vitro maturation could increase the rate of pregnancy from oocytes matured in vitro. Consequently, patients will be prevented from using gonadotropin with its related side effects. In this study, the maturation medium was enriched by platelet lysate (PL), then maturation and subsequent developments were monitored. Methods: Oocytes at germinal vesicle stage with cumulus cells (cumulus-oocyte complex) and without cumulus cells (denuded oocytes) were obtained from mature female mice. The maturation medium was enriched by 5 and 10 % PL and 5 % PL + 5 % fetal bovine serum (FBS) as experimental groups; the control groups' media consisted of 5 and 10 % FBS. After 18 h, the matured oocytes were collected and, after fertilization, subsequent development was monitored. Results: The rates of maturation, fertilization and 2-cell embryo development for the denuded oocyte groups in experimental media 5 % PL and 5 % PL + 5 % FBS were significantly higher than those of the control groups (P < 0.05), while the results for the cumulus-oocyte complex groups were similar between the experimental groups and control groups. Conclusions: The results of this study indicated that platelet lysate could improve the maturation rate in the absence of granulosa cells compared to media with FBS. This extract also had positive effects on fertilization and embryo development.

Follicle Development of Xenotransplanted Sheep Ovarian Tissue into Male and Female Immunodeficient Rats.

BACKGROUND: This study aimed to assess follicle survival after xenotransplantation of sheep ovarian tissue into male and female immunodeficient rats. We evaluated the effects of gonadotropin treatment on follicular development in the transplanted tissue. MATERIALS AND METHODS: In this experimental study, sheep ovarian cortical strips were transplanted into the neck back muscles of 8 male and 8 female immunodeficient, castrated rats. Fourteen days after surgery, each rat was treated with human menopausal gonadotropin (hMG) for 9 weeks. One day after the last injection, ovarian tissues were removed and fixed for histology assessment. Histology analyses were performed before and after grafting. Estradiol (E2) levels were measured before and after gonadectomy, and at the end of the experiment. The control group consisted of 7 male and 7 female noncastrated/non-grafted rats and the sham group comprised 7 male and 7 female castrated/ non-grafted rats for comparison of serum E2 concentrations. RESULTS: The percentage of primordial follicles decreased after transplantation in male (25.97%) and female (24.14%) rats compared to the control group (ovarian tissue nongrafted; 37.51%). Preantral follicles increased in the male (19.5%) and female (19.49%) transplanted rats compared to the control group (11.4%). Differences in antral follicles between male (0.06 ± 0.0%) and female (0.06 ± 0.0%) rats were not noticeable compared to control (1.25 ± 0.0%) rats. We observed a significantly higher percent of mean E2 secretion in grafted males compared to grafted females (P˂0.05). CONCLUSION: Despite significant differences in E2 secretion between xenografted male and female rats, we observed no statistical differences in terms of follicular development.

N-acetylcysteine improves function and follicular survival in mice ovarian grafts through inhibition of oxidative stress.

The effect of N-acetylcysteine (NAC) on mouse ovary heterotopic autotransplantation was investigated. Mice (age 4-5 weeks) were divided into the following groups: control; autograft plus NAC (150 mg/kg daily intraperitoneal injection) and autograft plus saline (n = 6 per group). Groups were treated from 1 day before until 7 days after transplantation. After 28 days, ovary compartments were estimated stereologically. Plasma malondialdehyde, progesterone, oestradiol concentrations and the percentage of apoptotic follicles were measured to evaluate the rate of oxidative stress and ovarian graft function. The mean total volume of ovary, cortex and the number of follicles was significantly higher (all P < 0.001) in the autografts plus NAC group compared with the saline group. In the autografts plus NAC group, the mean percentage of apoptotic follicles (P < 0.001) and malondialdehyde concentration (P < 0.001 day 7; P < 0.05 day 28) were significantly lower, whereas oestradiol concentration was significantly higher (P < 0.05) compared with the saline group. Although NAC cannot compensate the above parameters to the control level, it considerably improves follicular survival and development and also the structure and function of transplanted ovaries, through reducing oxidative stress and apoptosis.

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.

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.

Effects of erythropoietin on ischemia, follicular survival, and ovarian function in ovarian grafts.

Ovarian tissue transplantation is performed to preserve fertility in patients undergoing chemotherapy and radiotherapy. However, ischemia/reperfusion (IR) injury and free radical production occurring during the revascularization of the transplanted tissue are the major limitations of this procedure. The aim of this study was to investigate the effect of erythropoietin (EPO) as an antioxidant on oxidative stress and ovary survival following transplantation. The Naval Medical Research Institute (NMRI) mice (4-5 weeks old) were divided into three groups (six mice per group): control; autograft+saline, and autograft+EPO (500  IU/kg i.p.). After 28 days, ovary compartments were estimated stereologically. DNA fragmentation and plasma malondialdehyde (MDA), progesterone, and estradiol (E2) concentrations were also evaluated. The results were analyzed using one-way ANOVA and Tukey's test, and the means were significantly different at P<0.05. The mean total volume of ovary, cortex, and medulla and the number of follicles increased significantly in the autograft+EPO group (P<0.01). Apoptosis rate in the autograft+EPO group was lower than that in the autograft+saline group. The concentration of MDA decreased significantly in the autografted EPO-treated group than in the autografted saline-treated group (P<0.01). The concentration of E2 increased significantly in the autograft+EPO group than in the autograft+saline group (P<0.01). EPO reduced IR injury, increasing follicle survival and function in grafted ovaries. Free Persian abstract A Persian (Farsi) translation of the abstract is freely available online at http://www.reproduction-online.org/content/147/5/733/suppl/DC1.

Evaluation effects of allopurinol and FSH on reduction of ischemia-reperfusion injury and on preservation of follicle after heterotopic auto-transplantation of ovarian tissue in mouse.

Purpose: Allopurinol and FSH injection are applied to reduce ischemia-reperfusion injury and to increase survival rate for ovarian follicles after ovarian heterotopic autotransplantation in mice. Methods: Ovarian tissues from 6-week-old mice were grafted into back muscle then collected after 3 weeks. A total of five groups were included in this experiment as follows: control group (n = 5), sham-operated group (n = 5), allopurinol treatment group (AP) (n = 5), follicle stimulating hormone (FSH) treatment group (n = 5), as well as, allopurinol and FSH treatment group (APF) (n = 5). We investigated survival, number and development of follicles, vaginal cytology along with plasma malondialdehyde (MDA) concentration in grafted ovary. Results: Total follicles count significantly increased in APF group compared with other treatment groups (p < 0.05). MDA concentration significantly decreased in AP group and APF treatment group compared with sham-operated group. In AP group, vaginal smears showed presence of cornified epithelial cells three-five day after surgery. Conclusions: We demonstrated that allopurinol, as a XO inhibitor, plays an important role in order to decrease ischemia injury and to increase survival rate for follicles. Also, FSH, as a folliculogenesis and angiogenesis factor, increases development of follicles. It seems that allopurinol can cause re-establishing hypothalamus-pituitary axis and finally can restore estrous cycle earlier than for the sham operated group, so it explains the increasing survival rate for follicles.

The influence of amifostine administration prior to cyclophosphamide on in vitro maturation of mouse oocytes.

PURPOSE: The protective effect of amifostine against cyclophosphamide (CP) was evaluated on mouse oocytes. MATERIALS AND METHODS: Female mice were divided into four groups as follows: group1: cyclophosphamide (CP) (75 mg/kg, i.p) injection, group2: amifostine (250 mg/kg, i.p) injection, group3: amifostine (250 mg/kg, i.p) administered prior to CP (75 mg/kg, i.p) injection, Control group with injection of saline. About 21 days after injection, in vitro maturation (IVM) of oocytes was recorded. Furthermore the percentage of aneuploid oocytes was determined. RESULTS: In the CP group IVM rate was significantly decreased and aneploidy rate was significantly increased when compared to other groups (p < 0.05). With the administration of Amifostine prior to CP injection IVM rate was increased and aneploidy rate was decreased. DISCUSSION: Depletion in IVM rate with administration of CP is due its adverse effects on oocyte quality. Amifostine administration prior to CP injection appears to modulate deleterious effects of CP on oocytes.

The effect of intra-peritoneal administration of Papaver bracteatum Lindl. extract on development of NMRI mice oocytes treated with Doxorubicin.

PURPOSE: The effect of water-alcohol Papaver bracteatum Lindl. extract on development of mice oocytes treated with Doxorubicin (dox) was examined in this study. METHODS: The mice were classified into four groups. Control group, mice injected intraperitoneally (IP) with saline. Extract group alone, mice treated with 200 mg/kg of body weight (bw), IP, twelve consecutive days. Dox group alone, mice were given dox, IP, 10 mg/kg bw. Experimental group treated with extract and dox together. Effect of the extract on the development of mice oocytes treated with dox were evaluated through assisted reproductive technology techniques (ARTs). RESULTS: Developmental rate and blastocyst formation was improved by using the extract. A significant increase in in vitro developmental competence in comparison with dox group (P < 0.05) was observed. CONCLUSIONS: The results of this study indicated that P. bracteatum Lindl. extract could prevent dox toxicity of dox affecting both follicle or oocytes, and therefore it can result in improved embryo development which was observed in mice treated with dox plus P. bracteatum Lindl. compared to mice treated with dox alone.

Superovulation, in vitro fertilization (IVF) and in vitro development (IVD) protocols for inbred BALB/cJ mice in comparison with outbred NMRI mice.

PURPOSE: To study assisted reproductive technology (ART) protocols including superovulation, in vitro fertilization (IVF) and in vitro development (IVD) for BALB/cJ mice in comparison with a common ART protocol for NMRI mice. METHODS: Adult NMRI and BALB/cJ mice were superovulated using a 48 h G-interval. In order to find a more suitable G-interval for the BALB/cJ strain, G-intervals including 44, 46 and 50 h were also examined. Superovulation rates were recorded in all groups. IVF rate of BALB/c oocytes in T6 and mHTF media were compared. IVD rates of BALB/cJ zygotes in mHTF, T6 and G1V5/G2V5 media were compared. In addition, IVF and IVD rates of BALB/cJ and NMRI oocytes were compared in T6 medium during IVF-IVD procedures. RESULTS: In BALB/cJ mice the highest superovulation rates were observed with 44-46 h G-intervals. However, with a 48 h G-interval, superovulation rates were significantly lower in BALB/cJ compared to NMRI mice (p < 0.05). mHTF medium significantly increased in vitro fertilization of BALB/cJ oocytes compared to T6 medium (p < 0.05). Fertilization rate of NMRI oocytes was significantly higher than BALB/cJ oocytes in T6 medium (p < 0.05). The BALB/cJ embryo IVD was significantly higher in G1/G2 medium compared to mHTF and T6 media (p < 0.01). CONCLUSIONS: Superovulation with 48 h G-interval and using T6 during all in vitro procedures produces embryos more efficiently for NMRI mice than for BALB/cJ mice. For BALB/cJ mice, a protocol including superovulation with a 44-46 h G-interval, using mHTF during IVF and G1V5/G2V5 medium during IVD, may improve in vitro embryo production.

Effects of Saffron (Crocus sativus L.) Aqueous Extract on In vitro Maturation, Fertilization and Embryo Development of Mouse Oocytes.

OBJECTIVE: Lower pregnancy rates of in vitro matured oocytes compared to those of in vivo stimulated cycles indicate that optimization of in vitro maturation (IVM) remains a challenge. Reduced developmental competence of in vitro matured oocytes shows that current culture systems for oocyte maturation do not adequately support nuclear and/or cytoplasmic maturation. Therefore this study evaluates the effects of different concentrations of saffron (Crocus sativus L.) aqueous extract (SAE), as an antioxidant agent on IVM of immature mouse oocytes. MATERIALS AND METHODS: In this experimental study ,cumulus-oocyte complexes (COCs) were collected from 6-8 weeks old novel medical research institute (NMRI) female mice ovaries. COCs were cultured in IVM medium supplemented with 0 (control), 5, 10, 20 and 40 µg/ml of SAE in 5% CO2 at 37℃. The rates of maturation, fertilization and development were recorded. ANOVA and Duncan's protected least significant test, using the SAS program was applied for all statistical analysis. RESULTS: The maturation rate was significantly higher in all groups treated with different concentrations of SAE compared with the control group (p<0.05). However, the lower concentrations of SAE (10 and 5 µg/ml) in maturation medium respectively increased the fertilization rate of oocytes and in vitro developmental competence when compared with the control group (p<0.05). CONCLUSION: The results of this study indicate that lower concentrations of SAE are more appropriate to be added to maturation medium when compared with other experimental and control groups. Generally, we conclude that addition of appropriate amounts of natural extracts such as SAE to maturation medium improves oocyte maturation and embryo development.

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

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.