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 ìm diameter]. One week after co-culturing with hGCs, the size of the round cells increased 60 to70 ìm 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

impact of genetic variation and gene expression level of the follicle-stimulating hormone receptor on ovarian reserve

Objective: ovarian reserve is defined as the capacity of the ovary to provide fertile oocytes. Diminished ovarian reserve [DOR] is a disorder in which ovaries are prone to go through early menopause. Where this loss of function occurs before the age of 40, it results in the premature ovarian failure [POF] disease. Throughout folliculogenesis, the follicle-stimulating hormone receptor [FSHR] starts a signaling cascade in the granulosa cells where its inactivation leads to the arrest of follicle maturation and therefore adversely affects ovarian reserve. The aim of this study was to investigate the association of genetic variation [polymorphisms and inactivating mutations] of FSHR with POF and DOR

Materials and Methods: this case-control study comprised 84 POF, 52 DOR and 80 fertile Iranian women. To determine the presence of the 566C>T mutation and the -29G>A polymorphism in FSHR, PCR-RFLP method was used. SSCP-sequencing was used to identify any allelic variants in exon 10. The expression of human FSHR at the transcript level was also compared between DOR and fertile controls by real time-polymerase chain reaction [PCR]

Results: the 566C>T polymorphism was normal in all the cases. All genotypes of -29G>A and 919G>A [exon 10] polymorphisms were observed. Statistically significant differences were seen in the genotypic distribution of both polymorphisms when comparing the control group with the DOR patient group. A decrease was observed in FSHR expression of DOR patients compared with the control group but was not significant

Conclusion: we conclude that the -29G>A and 919G>A polymorphisms in FSHR may be associated with DOR. Although these polymorphisms had significant differences at the genic level, no significant variation was found at the transcript level

Temporal gene expression and DNA methylation during embryonic stem cell derivation

Objective: Dual inhibition of mitogen-activated protein kinase [MAPK] kinase [also known as MEK] and transforming growth factor beta [TGFbeta] type I receptors by PD0325901 and SB431542, known as R2i has been introduced as a highly efficient approach to the generation of mouse embryonic stem cells [ESC]. In the present study, we investigated the molecular mechanisms underlying ESC derivation in the R2i condition

Materials and Methods: In this experimental study, zona-free whole E3.5 blastocysts were seeded on mouse embryonic fibroblast [MEF] feeder cells in both R2i and serum conventional media. The isolated inner cell mass [ICM], ESCs and the ICM-outgrowths were collected on days 3, 5 and 7 post-blastocyst culture for quantitative real timepolymerase chain reaction [qRT-PCR] analysis as well as to assess the DNA methylation status at the time points during the transition from ICM to ESC

Results: qRT-PCR revealed a significantly higher expression of the pluripotency-related genes [Oct4, Nanog, Sox2, Rex1, Dppa3, Tcf3, Utf1, Nodal, Dax1, Sall4 and beta-Catenin] and lower expression of early differentiation genes [Gata6, Lefty2 and Cdx2] in R2i condition compared to the serum condition. Moreover, the upstream region of Oct4 and Nanog showed a progressive increase in methylation levels in the upstream regions of the genes following in R2i or serum conditions, followed by a decrease of DNA methylation in ESCs obtained under R2i. However, the methylation level of ICM outgrowths in the serum condition was much higher than R2i, at levels that could have a repressive effect and therefore explain the absence of expression of these two genes in the serum condition

Conclusion: Our investigation revealed that generation of ESCs in the ground-state of pluripotency could be achieved by inhibiting the MEK and TGF-beta signaling pathways in the first 5 days of ESC derivation

Attitude of a sample of Iranian researchers toward the future of stem cell research

Objective: Stem cells that have unlimited proliferation potential as well as differentiation potency are considered to be a promising future treatment method for incurable diseases. The aim of the present study is to evaluate the future trend of stem cell researches from researchers' viewpoints

Materials and Methods: This was a cross-sectional descriptive study on researchers involved in stem cell research at Royan Institute. We designed a questionnaire using a qualitative study based on expert opinion and a literature review. Content validity was performed using three rounds of the Delphi method with experts. Face validity was undertaken by a Persian literature expert and a graphics designer. The questionnaire was distributed among 150 researchers involved in stem cell studies in Royan Institute biology laboratories

Results: We collected 138 completed questionnaires. The mean age of participants was 31.13 +/- 5.8 years; most [60.9%] were females. Participants [76.1%] considered the budget to be the most important issue in stem cell research, 79.7% needed financial support from the government, and 77.5% felt that charities could contribute substantially to stem cell research. A total of 90.6% of participants stated that stem cells should lead to commercial usage which could support future researches [86.2%]. The aim of stem cell research was stipulated as increasing health status of the society according to 92.8% of the participants. At present, among cell types, importance was attached to cord blood and adult stem cells. Researchers emphasized the importance of mesenchymal stem cells [MSCs] rather than hematopoietic stem cells [HSCs, 57.73%]. The prime priorities were given to cancer so that stem cell research could be directed to sphere stem cell research whereas the least preference was given to skin research

Conclusion: Regenerative medicine is considered the future of stem cell research with emphasis on application of these cells, especially in cancer treatment

[ review on the activity of hormones and growth factors after ovarian tissue transplantation]

The majority of cancer treatments are invasive. Gonadal injuries cause reductions in fertility which results in lack of hope for conception in cancer patients and frustration for their partners. Fortunately, current advancements in cryopreservation and transplantation sciences regarding fertility preservation lead to cryostorage of gonads and preservation prior to the onset of chemo- and radiotherapy treatments. Accordingly in women, the main goal of ovarian cryopreservation is establishment of fertility and hormonal cycle restoration after auto-transplantation. Although the history of ovarian transplantation dates back to the 19[th] century, there are reports of live human births following ovarian tissue cryopreservation and transplantation since the past 100 years. Despite this success and additional research in the field of ovarian cryopreservation and transplantation, numerous questions remain unanswered. Among these questions, growth factors and hormonal changes because of their effects on follicular function appear to be more important during ovarian tissue transplantation. This review attempts to address hormones and growth factor functions with the specifics of ovarian cryopreservation and auto-transplantation

Quantitative expression of developmental genes, Pou5f1 [Oct4] and Mest [Peg1], in vitrified mouse embryos

Embryo cryopreservation is the process that water is removed from the cell by cryoprotectant materials, and embryos are stored at temperature below zero. This process may affect the viability and developmental potential of embryos. In this study, the effect of the vitrification cryotop method on the expression level of Oct4 and Mest developmental genes in mouse blastocysts was examined. The collected 2-cell embryos of superovulated mouse by oviduct flushing were divided into non-vitrified and vitrified groups. These embryos were cultured to the blastocyst stage directly in the non-vitrified group and in the vitrified group, these embryos were cultured to 4-8 cell embryos, vitrified with cryotop in these stages and after 2-6 months, warmed and cultured to blastocyst embryos. Quantitative expression of two developmental genes, namely Oct4 and Mest, were performed in these groups, using RNA purification and Real-time RT-PCR. Quantitative PCR analysis showed that the expression level of both genes, Oct4 and Mest, was reduced significantly in the vitrified-warmed group relative to the control group [p=0.046 and p=0.001]. This study revealed that morphologically normal embryos show a reduced amount of Oct4 and Mest transcripts which indicate that the vitrification method negatively effects the expression level of these two developmental genes

[Quantitative expression of BAG1, BAX and BCL-2 genes in human embryos with different fragmentation grades derived from ART]

The purpose of this study was to evaluate the quantitative expressions of BAG1, BAX and BCL-2 in human embryos with different fragmentation grades as derived from assisted reproduction technology [ART]. Fragmented and normal human 8-cell embryos were scored according to the degree of fragmentation with an inverted microscope and divided into four grades [grade I: no or minimal fragmentation [<5%], grade II: embryos with <25% fragmentation, grade III: embryos with >25% fragmentation and grade IV: apoptotic induced embryos with actinomycin D]. In this study, TUNEL labeling was initially used to detect apoptosis, and then revers transcription polymerase chain reaction [RT-PCR] and quantitative PCR were used to define the quantitative expressions of experimental genes in human embryos with different fragmentation grades. The results of TUNEL labeling showed that embryos with higher fragmentation had a high number of apoptotic bodies. The results of RT-PCR and q-PCR analyses showed a significantly decreased amount of BAGI transcript expression from group I to group IV. The highest expression of BAX gene was observed in group II, however, the transcript of BCL-2 gene was not observed in any of the experimental groups. The effect of actinomycin D on transcript expression amounts of experimental genes in apoptotic induced embryos [group IV] compared to control embryos [group I] showed a significant decrease. mRNA expression of BAG1 gene can be used as a good marker to detect apoptosis in human embryos. However, the transcript of BCL-2 gene does not play a role in the detection of apoptosis in human embryos at the 8-cell stage