The Effects of Endometrial Mesenchymal Stem Cells on The In Vitro Maturation of Germinal Vesicle Oocytes in Hanging Drop and Sodium Alginate Hydrogel Co-Culture Systems.

BACKGROUND: The aim of this study is to investigate the co-culture effects of human endometrial mesenchymal stem cells (EnMSCs) with mouse oocytes to enhance their maturation and development by using the hanging drop and sodium alginate hydrogel methods. MATERIALS AND METHODS: In this experimental study, we prepared human EnMSCs (2.5×105 cells/mL) and co-cultured them with partially denuded mouse oocytes by the hanging drop (n=120) and sodium alginate hydrogel (n=120) methods. Control oocytes (n=230, total) were cultured in both systems in the absence of human EnMSCs for 18 hours. Both survival and maturation rates of the oocytes were analysed morphologically. After insemination with capacitated sperm, the fertilization and development of the embryos up to the blastocyst stage were assessed and compared statistically for all of the study groups via one-way ANOVA and the t tests. RESULTS: Oocytes cultured in the hanging drop method had a significantly higher survival rate than their control group (92.60 ± 4.36% vs. 84.20 ± 3.12%, P=0.018). There were no significant differences between the two experimental groups in terms of survival. The mean percent of oocytes that reached the metaphase II (MII) stage was 64.35 ± 3.19% and fertilised was 62.25 ± 4.43% in the hanging drop method; these rates were 63.43 ± 1.92% and 58.14 ± 4.14 in sodium alginate hydrogel method, respectively. These rates were higher than their controls (P<0.050), but there were no statistical differences between the two experimental groups (P>0.050). Among the studied groups, the highest significant blastocyst rate (32.55 ± 2.18%) was observed in the hanging drop experimental group (P=0.0017). CONCLUSION: The results of this study show that human EnMSCs improve the survival, maturation, and development rates of oocytes and they could have future clinical applications.

Exploring the Role of Hyaluronic Acid in Reproductive Biology and Beyond: Applications in Assisted Reproduction and Tissue Engineering.

Hyaluronic acid (HA) plays a prominent role in various aspects of reproductive biology and assisted reproductive technologies (ART). This review describes the multifaceted influence of HA, ranging from primordial germ cell migration, ovarian follicle development, and ovulation in females to sperm structure, physiology, motility, and capacitation in males. In addition, HA also plays an important role in fertilization and promotes embryo implantation by mediating cellular adhesion and communication within the uterus. Against this physiological background, the review examines the current applications of HA in the context of ART. In addition, the article addresses the emerging field of reproductive tissue engineering, where HA-based hydrogels offer promising perspectives as they can support the development of mature oocytes and spermatogenesis in vitro. Overall, this review highlights the integral role of HA in the intricate mechanisms of reproductive biology and its growing importance for improving ART outcomes and the field of tissue engineering of the reproductive system.

Maternal nutrition and fetal imprinting of the male progeny.

The global population as well as the demand for human food is rapidly growing worldwide, which necessitates improvement of efficiency in livestock operations. In this context, environmental factors during fetal and/or neonatal life have been observed to influence normal physical and physiological function of an individual during adulthood, and this phenomenon is called fetal or developmental programming. While numerous studies have reported the impact of maternal factors on development of the female progeny, limited information is available on the potential effects of fetal programming on reproductive function of the male offspring. Therefore, the objective for this review article was to focus on available literature regarding the impact of maternal factors, particularly maternal nutrition, on reproductive system of the male offspring. To this end, we highlighted developmental programming of the male offspring in domestic species (i.e., pig, cow and sheep) as well as laboratory species (i.e., mice and rat) during pregnancy and lactation. In this sense, we pointed out the effects of maternal nutrition on various functions of the male offspring including hypothalamic-pituitary axis, hormonal levels, testicular tissue and semen parameters.

Cryopreservation of Limited Sperm Using A Combination of Sucrose and Taurine, Loaded on Two Different Devices, and Thawed at Two Different Temperatures.

BACKGROUND: Cryopreservation of sperm is essential for patients with low sperm counts and couples undergoing infertility treatment. The aim of this study was to compare the effects of Taurine (T) and Sucrose (S) in individual sperm cryopreservation utilizing cryotop and petri dish and thawing at 37 and 42°C. MATERIALS AND METHODS: In this experimental study, 17 normospermic semen samples were processed using the "Swim-up" procedure and progressively motile sperm were then isolated from these samples using an inverted microscope. Sperm were added to droplets of "sucrose medium" with 25 mM Taurine antioxidant (S+T) and the commercial cryoprotectant "Sperm Freeze" (CPA), loaded on a petri dish and cryotop. After rapid freezing of the samples, they were thawed at two different temperatures (37°C and 42°C), and the sperm classical parameters, viability, and DNA fragmentation were assessed. RESULTS: Statistical analysis displayed a significant increase in total and progressive motility in individual sperm freezing on cryotop with CPA and thawing at 42°C (P<0.05). Other parameters did not show any differences between the CPA and S+T groups and two thawing temperatures in either of the cryopreservation methods. CONCLUSION: Although, both cryoprotectants (CPA and S+T) may preserve individual sperm effectively using cryotop, the CPA and thawing at 42°C showed a better effect on the motility percentage of the small number of sperm.

Successful 3D culture and transplantation of mouse isolated preantral follicles in hydrogel of bioengineered Wharton's jelly.

MAIN OBJECTIVE: Due to Human Wharton's Jelly (HWJ) could be applied in tissue engineering as a bio scaffold, the present study was conducted to investigate the effects of HWJ hydrogel on in vitro culture and auto-transplantation of mouse ovarian follicles. MATERIALS AND METHODS: HWJ was isolated from umbilical cord and decellularized with SDS/Tris/EDTA. DNA, Collagen and Glycosaminoglycans (GAGs) were measured. Decellularized Wharton's Jelly (DWJ) was dissolved to make Wharton's Jelly Hydrogel (WJH), and composited with Alginate (ALG) (1.5%) in equal ratio (WJH+ALG). Then, mouse preantral follicles were isolated and encapsulated in 10µL droplets of WJH and randomly considered for both 14 days culture and auto-transplantation. RESULTS: Collagen, GAGs and DNA evaluations showed majority of WJ cells have been removed and MTT approved no toxicity. Degradation rate and rheological analysis represented optimal hydrogel compatibility. The data from in vitro culture revealed significant antral formation in WJH+ALG (P≤0.05). In transplantation, follicles failed to survive in ALG; however, survived in WJH+ALG to antral stage (P<0.05). VEGF and CD34 had greater expression in WJH+ALG than ALG (P< 0.05). CONCLUSION: Wharton's jelly hydrogel and Alginate compound is interesting composite for successful development of mouse preantral follicles in both 3D in vitro culture and transplantation.

Co-Culture of Mouse Blastocysts on A Human Recellularized Endometrial Scaffold: An In Vitro Model for Future Implantation Studies.

OBJECTIVE: This study evaluates the interaction of mouse blastocysts as a surrogate embryo on a recellularized endometrial scaffold by seeding human endometrial mesenchymal cells (hEMCs). MATERIALS AND METHODS: In this experimental study, prepared decellularized human endometrial tissues were characterized by morphological staining, DNA content analysis, and scanning electron microscopic (SEM) analysis. The scaffolds were subsequently recellularized by hEMCs. After seven days of cultivation, the mouse blastocysts were co-cultured on the recellularized scaffolds for 48 hours. Embryo attachment and implantation within these scaffolds were evaluated at the morphological, ultrastructural, molecular, and hormonal levels. RESULTS: There was no morphological evidence of cells and nuclei in the decellularized scaffold. DNA content significantly decreased by 89.92% compared to the control group (P<0.05). Both decellularized and native tissues had similar patterns of collagen bundles and elastin fibers, and glycosaminoglycan (GAGs) distribution in the stroma. After recellularization, the hEMCs attached to the scaffold surface and penetrated different parts of these scaffolds. In the co-cultured group, the embryo attached to the surface of the scaffold after 24 hours and penetrated the recellularized endometrial tissue after 48 hours. We observed multi-layered organoid-like structures formed by hEMC proliferation. The relative expressions of epithelial-related genes, ZO-1 and COL4A1, and SSP1, MMP2, and PRL, as decidualizationrelated genes, were significantly higher in the recellularized group on day 9 in the presence of the embryo compared to the other groups (P<0.05). Beta human chorionic gonadotropin (ß-hCG) and prolactin were statistically increased in the recellularized group on day 9 group (P<0.05). CONCLUSION: hEMCs and mouse embryo co-cultured on a decellularized endometrial scaffold provides an alternative model to study embryo implantation and the earlier stage of embryo development.

Characterization of altered microRNAs related to different phenotypes of polycystic ovarian syndrome (PCOS) in serum, follicular fluid, and cumulus cells.

OBJECTIVE: Polycystic ovarian syndrome (PCOS) is a metabolic syndrome in which steroidogenesis, folliculogenesis, and cellular adhesion play crucial roles in its prognosis. These pathways are controlled and regulated by some small non-coding RNAs called microRNAs (miRs). Several miRs have differential expression in PCOS compared to healthy women, and their dysregulation suggests important roles of miRs in PCOS pathophysiology. However, the role of miRs is still unclear, especially in various phenotypes of PCOS. MATERIALS AND METHODS: This study was conducted to evaluate the diagnostic potential of miR-212-3p, miR-490-5p, miR-647, and miR-4643 in different subtypes of PCOS. Accordingly, nineteen PCOS patients with different subtypes based on Rotterdam criteria (A: 8, B: not detected in this study, C: 5, and D: 6 patients) and six control age and BMI matched women under ICSI treatment were selected. The relative expression of miRs was then measured in blood serum before hormonal treatment (S1) and before ovum pickup (S2), follicular fluid (FF), and cumulus cells (CC) in all subjects. Also, the expression of miRs predicted target genes (AMH, AR, CYP11A1, CYP17A1, CYP19A1, GDF9, and HSD17B12) were done in the CC of understudy groups. RESULTS: In general, the results indicated that PCOS significantly increased the expression of miR-212-3p, miR-490-5p, and miR-4643 in FF and CCs compared to control. Although these miRs tend to increase in serum 1 of the PCOS patients, the differences were insignificant. However, there was a significant reduction in the expression of miR-647 in FF and CCs between PCOS vs. control. In addition, the miRs had significantly different expressions in various phenotypes of PCOS. For example, high levels of miR-647 in S2 and low levels of miR-490 in FF and miR-212 in CC can differentiate phenotype A from the other. Also, upregulation of miR-212 in FF and miR-4643 in S1 and low levels of this miR in FF can specifically differentiate subtype A from D. On the other hand, high levels of miR-4643 in FF and miR-490 in CC and lower titter of miR-647 can distinguish subtype C from the other. On the other hand, high levels of AMH, AR, CYP11, CYP17, and HSD17 in the hyperandrogenic PCOS and upregulation of CYP19A1 in the hypoandrogenic group can validate the role of selected miRs in the prognosis of PCOS. CONCLUSION: Characterization of altered microRNAs in serum, FF, and CCs and their targets in CC showed that the miRs might play critical roles in steroidogenesis and folliculogenesis. These miRs may be used for molecular classification of PCOS subtypes and as biomarkers for PCOS diagnosis.

The effect of mTOR activation and PTEN inhibition on human primordial follicle activation in ovarian tissue culture.

PURPOSE: The effect of PTEN inhibitor (Bpv(HOpic); Bpv) and mTOR activators (phosphatidic acid (PA) and propranolol (PP)), were evaluated on the activation and subsequent development of human primordial follicles in ovarian tissue culture. METHODS: Slow frozen-thawed human ovarian cortical strips were incubated for 24 h in different groups: (1) Control (base medium), (2) Bpv (100 µM), (3) PA (200 µM), (4) PA + PP (50 µm), and (5) Bpv + PA + PP. Afterward, the medium was exchanged, and all groups were cultured without stimulators for 6 additional days. The proportion of normal and degenerated follicles, estradiol secretion, and expression of RPS6, FOXO3a, and AKT proteins was evaluated and compared between groups. RESULTS: After 24 h of culture, there was no significant difference between the proportion of primordial and growing follicles in either of the experimental groups. This non-significant change was also observed for the phosphorylated protein to total protein ratios of RPS6, FOXO3a, and AKT proteins. After 7 days of culture, the proportion of the transitional follicles was significantly higher in comparison to the primordial follicles for the PA, PA + PP, and Bpv + PA + PP groups. The estradiol level was significantly higher on the last day compared to the first day, in PA, PA + PP, and Bpv + PA + PP groups. Hormonal secretion was significantly higher in the PA and PA + PP groups and lower in the Bpv and Bpv + PA + PP groups compared to the control on day 7 of culture. CONCLUSION: Temporary in vitro treatment of human ovarian tissue with mTOR activators enhances the initiation of primordial follicle development and positively influences steroidogenesis after short-term culture.

Correction: A testis-derived macroporous 3D scaffold as a platform for the generation of mouse testicular organoids.

Correction for 'A testis-derived macroporous 3D scaffold as a platform for the generation of mouse testicular organoids' by Tohid Rezaei Topraggaleh et al., Biomater. Sci., 2019, 7, 1422-1436, DOI: 10.1039/C8BM01001C.

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.

Preimplantation Genetic Screening and The Success Rate of In Vitro Fertilization: A Three-Years Study on Iranian Population.

OBJECTIVE: In vitro fertilization (IVF) is one of the most efficient approaches within the context of assisted reproductive technology (ART) to treat infertility. High pregnancy rates have become the major index of successful IVF in clinical studies. It is not clear yet which factors are certainly responsible for IVF success, as various outcomes were obtained in different IVF centers with different settings. In this study, we aimed to address controversies in the interpretation of promising results of IVF with respect to preimplantation genetic screening (PGS). MATERIALS AND METHODS: In this retrospective case series study, we built a dataset containing data from 213 IVF patient candidates for PGS (654 embryos) with blastomere biopsy at day 3 and trophectoderm biopsy in day 5, referred to Royan Institute, Tehran, Iran from 2015 to 2018. Next, the data were analyzed to find influential factors affecting success rate of ART cycles. RESULTS: Data analyses showed that regardless of PGS indications (ART failures, recurrent miscarriage, chromosomal abnormalities, etc.), the pregnancy rate is influenced by maternal and embryonic factors such as the age of mother as well as quantity and quality of transferred embryos. Furthermore, genotyping of embryos using array comparative genomic hybridization (aCGH) depicted the highest rate of chromosomal aberrations for chromosomes 1, 16 and 19 while the lowest frequency for chromosomes 11 and 17. Similarly, we detected 463 genetically abnormal embryos by aCGH, among which only 41.9% could be detected by classical fluorescent in situ hybridization (FISH) method. CONCLUSION: This study not only highlighted the advantages of aCGH over the FISH method in detection of chromosomal abnormalities, but also emphasized the importance of genetic abnormality as an indication for determination of IVF success rate.

Human embryonic stem cell-derived mesenchymal stem cells improved premature ovarian failure.

BACKGROUND: Premature ovarian failure (POF) affects many adult women less than 40 years of age and leads to infertility. According to previous reports, various tissue-specific stem cells can restore ovarian function and folliculogenesis in mice with chemotherapy-induced POF. Human embryonic stem cells (ES) provide an alternative source for mesenchymal stem cells (MSCs) because of their similarities in phenotype and immunomodulatory and anti-inflammatory characteristics. Embryonic stem cell-derived mesenchymal stem cells (ES-MSCs) are attractive candidates for regenerative medicine because of their high proliferation and lack of barriers for harvesting tissue-specific MSCs. However, possible therapeutic effects and underlying mechanisms of transplanted ES-MSCs on cyclophosphamide and busulfan-induced mouse ovarian damage have not been evaluated. AIM: To evaluate ES-MSCs vs bone marrow-derived mesenchymal stem cells (BM-MSCs) in restoring ovarian function in a mouse model of chemotherapy-induced premature ovarian failure. METHODS: Female mice received intraperitoneal injections of different doses of cyclophosphamide and busulfan to induce POF. Either human ES-MSCs or BM-MSCs were transplanted into these mice. Ten days after the mice were injected with cyclophosphamide and busulfan and 4 wk after transplantation of the ES-MSCs and/or BM-MSCs, we evaluated body weight, estrous cyclicity, follicle-stimulating hormone and estradiol hormone concentrations and follicle count were used to evaluate the POF model and cell transplantation. Moreover, terminal deoxynucleotidyl transferase mediated 2-deoxyuridine 5-triphosphate nick end labeling, real-time PCR, Western blot analysis and immunohistochemistry and mating was used to evaluate cell transplantation. Enzyme-linked immunosorbent assay was used to analyze vascular endothelial growth factor, insulin-like growth factor 2 and hepatocyte growth factor levels in ES-MSC condition medium in order to investigate the mechanisms that underlie their function. RESULTS: The human ES-MSCs significantly restored hormone secretion, survival rate and reproductive function in POF mice, which was similar to the results obtained with BM-MSCs. Gene expression analysis and the terminal deoxynucleotidyl transferase mediated 2-deoxyuridine 5-triphosphate nick end labeling assay results indicated that the ES-MSCs and/or BM-MSCs reduced apoptosis in the follicles. Notably, the transplanted mice generated new offspring. The results of different analyses showed increases in antiapoptotic and trophic proteins and genes. CONCLUSION: These results suggested that transplantation of human ES-MSCs were similar to BM-MSCs in that they could restore the structure of the injured ovarian tissue and its function in chemotherapy-induced damaged POF mice and rescue fertility. The possible mechanisms of human ES-MSC were related to promotion of follicular development, ovarian secretion, fertility via a paracrine effect and ovarian cell survival.

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.

Applicability of Hyaluronic Acid-Alginate Hydrogel and Ovarian Cells for In Vitro Development of Mouse Preantral Follicles.

OBJECTIVE: In the present study, the applicability of hyaluronic acid-alginate (HAA) hydrogel and ovarian cells (OCs) for the culture of mouse ovarian follicles were investigated and compared with those of alginate (ALG) and fibrin-alginate (FA) hydrogels. MATERIALS AND METHODS: In the first step of this experimental study, mechanically isolated preantral follicles from the ovaries of two-week-old mice were encapsulated in the absence or presence of OCs in ALG, HAA, and FA hydrogels and cultured for 14 days. The morphology, diameter, survival and antrum formation rates of the follicles and the maturation and quality of the oocytes were evaluated during culture. In the second step, preantral follicles were cultured similar to the first step, but for 13 days, and their gene expressions and hormonal secretion were assessed on the last day of culture. RESULTS: In the absence of OCs, higher numbers of ALG- and HAA-encapsulated follicles reached the antral stage compared to FA-encapsulated follicles (P<0.05). However, a higher percentage of HAA-developed oocytes resumed meiosis up to the germinal vesicle breakdown (GVBD)/metaphase II (MII) stages in comparison with ALG-developed oocytes (P<0.05). HAA-encapsulated follicles had significant overexpression of most of the growth and differentiation genes, and secreted higher levels of estradiol (E2) compared to ALG- and FA-encapsulated follicles (P<0.05). The co-culture condition increased the diameter of ALG-encapsulated follicles on day 13 of culture (P<0.05). It also increased the survival and maturation rates of ALG- and FA-encapsulated follicles, respectively (P<0.05). The co-culture condition improved cortical granule distribution in all groups, increased E2 and progesterone (P4) secretions in the ALG and FA groups, and androstenedione (A4) secretion in the FA group (P<0.05). CONCLUSION: The present study results show that HAA hydrogel is a promising hydrogel for follicle culture. OCs utilization could ameliorate the culture conditions regardless of the type of hydrogel.

The anti-oxidant roles of Taurine and Hypotaurine on acrosome integrity, HBA and HSPA2 of the human sperm during vitrification and post warming in two different temperature.

Despite advances in vitrification techniques for sperm cryopreservation, cryo-damages of sperm caused by generation of reactive oxygen species (ROS) continue to impede implementation of this technique. This study analyses the effects of taurine and hypotaurine as anti-oxidants during vitrification of human sperms. The study was performed in two steps. In the first step, 20 normospermic semen samples were vitrified in the presence of varying concentrations of taurine and hypotaurine, and their effects as anti-oxidant agents on classical sperm parameters, hyaluronan-binding assay (HBA), lipid peroxidation (LPO) and acrosome reaction (AR) were studied. Statistical analyses showed that the sperm parameters in all vitrified groups decreased significantly (P < 0.05) compared to the fresh group. However, HBA and acrosome integrity in vitrified groups containing taurine and 50 mM of hypotaurine were better than in the control group (P < 0.05). The morphology of the vitrified group was good only in the group that contained 50 mM of hypotaurine (P < 0.05). Based on the results from the first step, 50 mM of hypotaurine was considered the ideal anti-oxidant formulation and further tests were carried out on 10 normospermic semen samples with this protecting agent. In addition to the mentioned parameters, the expression of heat shock proteinA2 (HSPA2) was studied in the vitrified group with 50 mM hypotaurine, warmed under two different warming temperatures 37 and 42 °C. 50 mM Hypotaurine was found to equally improve motility, morphology, HBA, and AR after warming at 37 °C and 42 °C (P < 0.05). However, at both warming temperatures, the expression of HSPA2 was reduced in all vitrified groups comparing to the fresh group (P < 0.05). In conclusion, taurine and hypotaurine antioxidants, especially 50 mM hypotaurine, are able to reduce deleterious cryo-injuries on morphology, acrosome and HBA and improve sperm recovery at both warming temperatures (37 and 42 °C). However, they do not have any protective action on expression of HSPA2.

Induction of Mouse Peritoneum Mesenchymal Stem Cells into Germ Cell-Like Cells Using Follicular Fluid and Cumulus Cells-Conditioned Media.

The peritoneum mesothelium lines body cavities and has the same origin as ovarian surface epithelium with probable existence of peritoneum mesenchymal stem cells (PMSCs). In the present research, PMSCs were isolated from peritoneum and differentiated into ovarian cell-like cells using human follicular fluid (HFF) and human cumulus-conditioned medium (HCCM). Anterior abdominal wall and intestinal peritoneum explants were used for cells isolation and cultured in Dulbecco's modified Eagle's medium. After passage 3, purified PMSCs were assessed for morphology, proliferation rate, and cell viability. Then, isolated PMSCs underwent two characterization procedures: (1) differentiation to mesodermal lineage and (2) expression of mesenchymal (CD90 and CD44) and epithelial cell (CK19) markers. The characterized PMSCs were differentiated into ovarian cell-like cells using 10% HFF and 50% HCCM for 21 days, and the expressions of oocyte (Zp3, Gdf9), germ cell (Ddx4, Dazl), granulosa cell (Amh), and theca cell (Lhr) markers were assessed using real-time polymerase chain reaction and immunocytofluorescence assay. Both anterior abdominal wall and intestinal peritoneum mesenchymal stem cells (AP-MSCs and IP-MSCs) showed mesenchymal characters and differentiated to adipocyte and osteocyte. AP-MSCs expressed mesenchymal- and epithelial cell-specific markers more than IP-MSCs and showed an analytically better proliferation rate. The induced AP-MSCs and IP-MSCs were expressed as germ and oocyte cell-specific markers, and this expression increased in the third week of culture. In both groups of AP-MSCs and IP-MSCs, the expressions of Gdf9, Zp3, Ddx4, Dazl, and Amh genes under just HCCM induction showed upregulation significantly on the 21st day of culture compared with day 0. But in protein synthesis of all mentioned genes, both HFF and HCCM had equal induction effect on the 21st day of culture against the 0th day. In addition, LHR was not expressed in any groups. Finally, in both characterization and differentiation procedures, the AP-MSCs respond to inducers better than IP-MSCs.

A testis-derived macroporous 3D scaffold as a platform for the generation of mouse testicular organoids.

Extracellular matrix-derived scaffolds provide an efficient platform for the generation of organ-like structures. Successful development of testicular organoids (TOs) with the capability of supporting complete spermatogenesis has not been reported yet. Here, we have developed an optimized method for the decellularization of ram testicular tissue fragments. Our findings showed that testicular fragments treated with a serial combination of Triton X-100 and SDS in PBS for 48 h resulted in the efficient removal of cellular materials and retention of the extracellular matrix (ECM) components. In order to fabricate testis-derived scaffolds (TDSs), the testicular ECM (T-ECM) was digested in acid/pepsin, followed by neutralization of pre-gel solution to form a hydrogel. Then, the hydrogels were freeze-dried and cross-linked using a chemical method. To reach the optimal concentration for the T-ECM in the fabrication of TDSs, the scaffold properties including porosity, pore size, swelling behavior, and degradation were evaluated. Our study suggested that 25 mg ml-1 of the T-ECM is the best concentration for the fabrication of macroporous TDSs for demonstrating lower pore size, homogeneously distributed pores, and a higher swelling ratio. Furthermore, inoculation of neonatal mouse testicular cells onto TDSs resulted in the generation of multicellular TOs in which the differentiation of spermatogonial cells into post-meiotic cells was confirmed. Hormonal analysis of TDSs revealed the functionality of TOs in the secretion of testosterone and inhibin B. The current study also demonstrated that macroporous TDSs could provide a novel platform for testicular tissue engineering and in vitro spermatogenesis.

Human Ovarian Theca-Derived Multipotent Stem Cells Have The Potential to Differentiate into Oocyte-Like Cells In Vitro.

OBJECTIVE: In this study, we have examined human theca stem cells (hTSCs) in vitro differentiation capacity into human oocyte like cells (hOLCs). MATERIALS AND METHODS: In this interventional experiment study, hTSCs were isolated from the theca layer of small antral follicles (3-5 mm in size). Isolated hTSCs were expanded and cultured in differentiation medium, containing 5% human follicular fluid, for 50 days. Gene expressions of PRDM1, PRDM14, VASA, DAZL, OCT4, ZP1, 2, 3 GDF9, SCP3 and DMC1 were evaluated by quantitative reverse transcription polymerase chain reaction (qRT-PCR) on days 0, 18, and 25 after monoculture as well as one week after co-culture with human granulosa cells (hGCs). In addition, GDF9, OCT4, DAZL, VASA, and ZP3 proteins were immune-localized in oocyte-like structures. RESULTS: After 16-18 days, the color of the medium became acidic. After 25 days, the cells started to differentiate into round-shaped cells (20-25 µm diameter). One week after co-culturing with hGCs, the size of the round cells increased 60 to70 µm and convert to hOLCs. However, these growing cells expressed some primordial germ cell (PGC)- and germ cell genes (PRDM1, PRDM14, VASA, DAZL, and OCT4) as well as oocyte specific genes (ZP1, 2, 3 and GDF9), and meiotic-specific markers (SCP3 and DMC1). In addition, GDF9, OCT4, DAZL, VASA, and ZP3 proteins were present in hOLCs. CONCLUSION: To sum up, hTSCs have the ability to differentiate into hOLCs. This introduced model paved the way for further in vitro studies of the exact mechanisms behind germ cell formation and differentiation. However, the functionality of hOLCs needs further investigation.

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

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.

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

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 ß-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, ß-CATENIN, FZD-2 and GSK-3ß 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 ß-CATENIN staining, while control and vitrification groups only showed ß-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.