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.

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.

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.

Fertility Preservation in Cancer Patients: In Vivo and In Vitro Options.

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

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.

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.

Comparative study between intact and non-intact intramuscular auto-grafted mouse ovaries.

Anticancer treatments often lead to ovarian failure and infertility. Cryopreservation and subsequent transplantation of the ovaries is one of the solutions that has been adopted as a means of preserving fertility, but primitive ischaemia in the grafted ovary that damages the oocyte pool is considered to be a possible problem. In order to improve blood supply and follicle preservation, two incisions were made in the ovaries before an intramuscular auto-grafting procedure and these non-intact ovaries were compared with the intact ovaries that were also auto-grafted intramuscularly. Follicle numbers and apoptosis were examined in intact and non-intact groups after 1, 2 and 3 weeks post-grafting. The results were compared with the control ovaries, which were not incised and grafted. Although follicle survival in both grafted groups was lower than in the controls (P

The effect of leukemia inhibitory factor and coculture on the in vitro maturation and ultrastructure of vitrified and nonvitrified isolated mouse preantral follicles.

OBJECTIVE: To develop a system for in vitro maturation of preantral follicles isolated from vitrified and nonvitrified mouse ovaries by leukemia inhibitory factor (LIF) and coculture with cumulus cells. DESIGN: Experimental study. SETTING: University-based research laboratory. ANIMAL(S): Twelve- to 14-day-old National Medical Research Institute female mice. INTERVENTION(S): Vitrification of mouse ovaries and in vitro maturation of follicles. MAIN OUTCOME MEASURE(S): The growth, maturation, steroidogenesis, alkaline phosphatase activity, and ultrastructure of preantral follicles derived from vitrified and nonvitrified mouse ovaries and the developmental capacity of embryos obtained from metaphase II to blastocyst stage. RESULT(S): The follicular diameters were increased in the presence of LIF in the simple culture, coculture, and vitrified coculture groups. The survival and developmental rates of follicles were not significantly different between groups. The E(2) production was increased in all groups during the culture period. Alkaline phosphatase activity was observed in the follicles of cryosectioned slices and cultured preantral follicles. There was no remarkable change in ultrastructural maturation features of cultured follicles. CONCLUSION(S): LIF alone or in combination with the coculture system increased the growth of cultured preantral follicles but had no effect on their maturation; the ultrastructure, growth, and maturation of vitrified follicles were the same in the cultured groups and in the controls.