A comparison of polarized and non-polarized human endometrial monolayer culture systems on murine embryo development

BACKGROUND: Co-culture of embryos with various somatic cells has been suggested as a promising approach to improve embryo development. Despite numerous reports regarding the beneficial effects of epithelial cells from the female genital tract on embryo development in a co-culture system, little is known about the effect of these cells when being cultured under a polarized condition on embryo growth. Our study evaluated the effects of in vitro polarized cells on pre-embryo development. METHODS: Human endometrial tissue was obtained from uterine specimens excised at total hysterectomy performed for benign indications. Epithelial cells were promptly isolated and cultured either on extra-cellular matrix gel (ECM-Gel) coated millipore filter inserts (polarized) or plastic surfaces (non-polarized). The epithelial nature of the cells cultured on plastic was confirmed through immunohistochemistry, and polarization of cells cultured on ECM-Gel was evaluated by transmission electron microscopy (TEM). One or two-cell stage embryos of a superovulated NMRI mouse were then flushed and placed in culture with either polarized or non-polarized cells and medium alone. Development rates were determined for all embryos daily and statistically compared. At the end of the cultivation period, trophectoderm (TE) and inner cell mass (ICM) of expanded blastocysts from each group were examined microscopically. RESULTS: Endometrial epithelial cells cultured on ECM-Gel had a highly polarized columnar shape as opposed to the flattened shape of the cells cultured on a plastic surface. The two-cell embryos cultured on a polarized monolayer had a higher developmental rate than those from the non-polarized cells. There was no statistically significant difference; still, the blastocysts from the polarized monolayer, in comparison with the non-polarized group, had a significantly higher mean cell number. The development of one-cell embryos in the polarized and non-polarized groups showed no statistically significant difference. CONCLUSION: Polarized cells could improve in vitro embryo development from the two-cell stage more in terms of quality (increasing blastocyst cellularity) than in terms of developmental rate

Investigating the Effect of Laser Assisted Hatching on the Development and Quality of Vitrified-Warmed 4-Cell Stage Mouse Embryos

Objective: The aim of this study was to investigate the effect of laser assistedhatching on the development and quality of vitrified-warmed 4-cell stage mouseembryos.Materials and Methods: The vitrified-warmed 4-cell mouse embryos were dividedinto two groups: control group (without laser assisted hatching) and experiment group(with laser assisted hatching). All embryos in both groups were cultured in sequentialmedia containing G1TMver3 and G2TMver3. Afterward, all expanded blastocystswere randomly selected and stained with differential (for cellularity) and TUNEL (forcell death) methods.Results: On day 1(24hrs) of culture, the difference between the control and theexperimental groups was insignificant in the rate of blastocyst formation. But onday 2(48hrs) of culture, 87.61% of embryos in the experimental group reached theblastocyst stage. This rate did not increase significantly as compared to the controlgroup (78.14%). Finally on day 3 (72 hrs), the rate of blastocyst formation reached94.40% and 81.75%, respectively, in both the experimental and control groups.The difference between these two groups were significant (p<0.05). The numberof blastomeres and apoptotic cells were similar in the experimental and controlgroups.Conclusion: The laser assisted hatching has no decreasing effect on cellularity, butit has increasing effect on incidence of cell death. In addition, the assisted hatchingsignificantly increases the blastocyst formation rate of intact vitrified-warmed 4-cellstage mouse embryos

Comparison of in vitro culture of preantral follicle isolated from vitrified-warmed mouse ovaries with fresh follicles in culture media

Background: Vitrification is a simple and ultra rapid technique for the conservation of fertility. In this study, we compare the in vitro development of preantral follicles obtained from fresh specimens with vitrified- warmed mouse ovaries.Materials and Methods: This experimental study was carried out on 20, 14-day-old female mice (NMRI). Ovaries were vitrified with a solution containing ethylene glycol, ficoll 70 and sucrose in PB1 (EGFS40) for 5 min, and transferred directly into liquid nitrogen and stored for one week. Fast warming was done by descending sucrose at room temperature. Preantral follicles with 100-130 µm in diameter were mechanically isolated from fresh and vitrified-warmed ovaries and cultured in α-minimum essential medium (α-MEM) supplemented with 5 fetal bovine serum (FBS), 100 mIU/ml rFSH, 1 ITS, and 20ng/ml mrEGF in vitro for 10 days. Diameter of follicle, and, survival rate and number of antral follicles in both groups were compared using t-test and chi-square test, respectively.Results: Isolated follicles from the vitrified and nonvitrified groups survived and grew in vitro culture. On the day 6 survival rates in the vitrified and fresh control groups were 72.1 and 78.6, and, on the day 10, they were 66.9 and 72.6, respectively. Follicle antrum formation was 37.5 in the vitrified group while it was 43.5 in the fresh group in the 10th day. On the day 2, the mean diameters of fresh and vitrified follicles were 158.11 ± 11.23 and 155.48 ± 8.35 and on the day 4, they were 201.56 ± 9.87 and 193.42 ± 8.46, respectively. There was no significant difference between the control and vitrified groups in these variables (p>0.05).Conclusion: Taken together, cryopreservation of the ovary by vitrification seems to be a promising method to preserve ovarian follicles

The Arrangement of Microtubules in Embryos Derived from Mice Young, Old and Reconstructed Oocytes

Objective: To study the structure and distribution of microtubules in embryos derivedfrom young, old and reconstructed oocytes.Materials and Methods: Embryos obtained from old (50 embryos), young(50 embryos) and reconstructed oocytes (10 embryos) were studied byimmunocytochemistry. The microtubule structures of the embryos were studied byusing fluroscent microscopy with FITC-PI filter and polyclonal antibody against alfatubulin.Results: The spindle structure of MII young oocyte and the obtained embryos werenormal with the suitable condensation. There was no contact between chromosomeand spindle in old Oocytes as well as the obtained embryos, in addition, the spindlewas extended in old group. In reconstructed embryos, thin and scattered filamentswere observed.Conclusion: This study reveals that the arrangement of microtubules inreconstructed embryos was caused by repeating of injection and oocytemanipulation. Also, interactions between karyoblast, cytoplasm and microtubulsmay not be suitable. This may be caused by low fertilization in these oocytes

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

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. © 2020 Elsevier Inc

Long term effect of vasectomy on rat prostate tissue

History and Objectives: vasectomy is one of the prevalent method approved by the Ministry of Health and Medical Education for prevention of male reproductive competency. Due to discrepancies in the reported cases of the effect of vasectomy on the testicular tissue of human and animal models, in order to determine the long term affected of vasectomy on the general appearance and histology of testicular tissue, the present study was carried out in Tarbiat Modares university in 1995. Materials and Methods: An experimental study on 14 Wistar rats was performed. They were randomly divided equally into 2 vasectomy and shame operated groups: A bilateral surgery was performed on all rats. Changes in the testicular tissue were assessed by taking histologic samples (With hematoxillin-eosin, PAS and trichrome mission staining) 6 months after the surgery. Weight and volume of testis, seminiferous tubule diameter, epithelium thickness of seminiferous tubule, testicular volume percentage and number of epithelial germinal sertoli cell of seminiferous tubule were recorded. Data gathered from 2 groups was compared and statistical analysis was done. Results: vasectomy induces changes in increased folding of membrane of seminiferous tubule, vacuolization of epithelium seminiferous tubule and desquamation of immature germ cells, in vasectomized group. Comparison of the changes of variables under investigation in 2 groups indicates that there is a significant difference between the 2 groups. Testicular weight (1.2±0.2 in vasectomized group compared to 1.5±0.2 g in control group), testicular volume (1.1±0.2 cm³ compared to 1.4±0.2 cm³) (P<0.0005), seminiferous tubular thickness 225±53 micron compared to 78±15.1 micron) (P<0.0001) showed significant reduction. In addition, testicular volume percentage and number of germ cells in 2 groups showed significant difference (P<0.0001). However, number of sertoli cells did not changed significantly. Conclusion: Vasectomy induces significant changes in the rat testicular tissue which further study is required to elucidate the underlying mechanism and extrapolation of the results to humans

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

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. © 2020 Elsevier B.V

A Review on the Activity of Angiogenic and Apoptotic Factors in Transplanted Ovarian Tissue

Many changes of female reproductive system are based on the vascular activity and blood flow. In ovarian follicular development, re-anastomosis is critical and permits cells and oocytes to utilize nutrients as well as the hormones. Moreover, it plays a main role in corpus luteum formation. Female reproductive system undergoes several angiogenesis programs which are considered essential in order to reach a follicle to preovulatory stage and to set up a network between granulosa cells and blood vessels through the theca layer. Up to the first 3 days after transplantation and reanastomosis start, a large number of follicles especially advanced types, begin an early death due to their severe dependence on nutrients of blood supply. Loss of the nutritional sources of granulosa cells leads to a decrease in survival and supporting factors and thus, cell death increases. Considering the importance of this issue, the present study intends to investigate the activity of angiogenic and apoptotic factors in transplanted ovarian tissue