Over the past few years, extensive efforts have been made to generate in-vitro pancreatic micro-tissue
,
for disease modeling or cell replacement approaches in pancreatic related diseases such as ...diabetes mellitus. To obtain these goals, a closer look at the diverse cells participating in pancreatic development is necessary. Five major non-epithelial pancreatic (pN-Epi) cell populations namely, pancreatic endothelium, mesothelium, neural crests, pericytes, and stellate cells exist in pancreas throughout its development, and they are hypothesized to be endogenous inducers of the development. In this review, we discuss different pN-Epi cells migrating to and existing within the pancreas and their diverse effects on pancreatic epithelium during organ development mediated via associated signaling pathways, soluble factors or mechanical cell–cell interactions. In-vivo and in-vitro experiments, with a focus on N-Epi cells’ impact on pancreas endocrine development, have also been considered. Pluripotent stem cell technology and multicellular three-dimensional organoids as new approaches to generate pancreatic micro-tissues have also been discussed. Main challenges for reaching a detailed understanding of the role of pN-Epi cells in pancreas development in utilizing for in-vitro recapitulation have been summarized. Finally, various novel and innovative large-scale bioengineering approaches which may help to recapitulate cell–cell interactions and are crucial for generation of large-scale in-vitro multicellular pancreatic micro-tissues, are discussed.
Cryopreservation and transplantation of ovarian tissue is the only fertility preservation option used for prepubertal girls and women who don’t have a chance for embryo or oocyte vitrification. For ...women with aggressive cancer, hormone-responsive malignancies, autoimmune diseases, etc. ovary transplantation cannot be performed so an alternative technology called in-vitro follicle activation is thinkable. In this method, dormant primordial follicles are activated from the resting primordial pool by in-vitro culture and enter their growth phase. Different in-vitro culture media and supplements in addition to various culturing methods have been conducted for activating these dormant follicles. Furthermore, several signaling pathways such as Hippo, phosphatidylinositol-3-kinase, and mTOR influence follicle activation. Therefore, the addition of different activators of these signaling pathways can beneficially regulate this culture system. This review summarizes the findings on different aspects of human ovarian tissue culture strategies for in-vitro follicular activation, their medium, and different factors involved in this activation. Afterward, signaling pathways important for follicle activation and their clinical applications towards improving activation in culture are also reviewed.
Purpose
The aim of the present study was to improve the in vitro maturation (IVM) procedure using oocytes from surplus ovarian tissue after fertility preservation.
Methods
Twenty-five patients aged ...17–37 years were included in the study. Maturation was compared between oocytes collected in HEPES-buffered medium or saline, and we determined whether transport on ice prior to oocyte collection affected maturation. Two different IVM media were used that were supplemented with and without recombinant human midkine. Mature oocytes were assessed for aneuploidy using next-generation sequencing (NGS).
Results
On average, 36 immature oocytes were collected from each patient (range 7–90,
N
= 895). Oocytes recovered from HEPES-buffered medium matured at a higher rate than oocytes recovered from saline (36% vs 26%,
p
< 0.01). Ovarian transportation on ice prior to the procedure negatively affected maturation compared with non-transported samples (42% vs 27%,
p
< 0.01). The addition of midkine improved maturation rate (34% vs 27%,
p
< 0.05). On average, 11 MII oocytes were obtained per patient (range 1–30). NGS of 53 MII oocytes and their first polar bodies indicated that 64% were euploid.
Conclusions
The study demonstrated unexpectedly high number of immature oocytes collected from surplus ovarian tissue without any stimulation. The overall MII rate was one in three, resulting in a total number of MII oocytes that was similar to the number obtained after ovarian stimulation. If these MII oocytes prove suitable for IVF, they will provide a substantial improvement in fertility preservation for patients and advance IVM as an interesting platform for further improvements in assisted reproduction.
Adipose tissue-derived mesenchymal stem cells (AD-MSCs) are an appropriate source for regenerative medicine and transplantation therapy due to their high proliferation rate in vitro. ...Bromodeoxyuridine (BrdU) marker is widely used for evaluating cell proliferation. BrdU cell labeling method has some limitations including its effects on cell proliferation and cellular mutations followed by an abnormality in the cell. On the other hand, the city of Kiel-67 (Ki-67) nuclear protein is expressed in dividing cells throughout the mitotic process. Our main objective was to compare the effects of BrdU and Ki-67 markers on self-renewal levels in AD-MSCs. In this study, the AD-MSCs were extracted from subcutaneous adipose tissue of adult rats and cultured in α-MEM supplemented with 10% fetal bovine serum (FBS). Fourth passage AD-MSCs after characterization were evaluated by immunocytochemical assay for BrdU and Ki-67 markers based on growth and proliferation rates of AD-MSCs at 24, 48, 72, and 96 h after culture. Our results show that the growth rate of AD-MSCs significantly changed at different times for both markers, although the growth and proliferation patterns were initially different in them. Unlike the BrdU, the Ki-67 endogenous marker had no side effect and interfered with cellular proliferation. It is opined that evaluating cell proliferation rate using the Ki-67 marker is more effective than the BrdU assay.
Purpose
To investigate the effect of different FSH concentrations on human oocyte maturation in vitro and its impact on gene expression of key factors in the surrounding cumulus cells.
Methods
The ...study included 32 patients who underwent unilateral oophorectomy for ovarian tissue cryopreservation (OTC) (aged 28 years on average). Immature oocytes were collected from surplus medulla tissue. A total of 587 immature oocytes were divided into three categories according to the size of the cumulus mass: large (L-COCs), small (S-COCs), and naked oocytes (NOs), and submitted to 44-h IVM with one of the following concentrations of recombinant FSH: 0 IU/L, 20 IU/L, 40 IU/L, 70 IU/L, or 250 IU/L. After IVM, oocyte nuclear maturation stage and diameter were recorded. The relative gene expression of
FSHR
,
LHCGR
, and
CYP19A1
in cumulus cells before (day 0; D0) and after IVM were evaluated.
Results
Addition of 70 or 250 IU/L FSH to the IVM medium improved oocyte nuclear maturation compared to 0, 20, and 40 IU/L FSH by upregulating
LHCGR
and downregulating
FSHR
in the cumulus cells.
Conclusion
FSH improved oocyte nuclear maturation at concentrations above 70 IU/L suggesting a threshold for FSH during IVM of ex vivo collected human oocytes from small antral follicles. Moreover, current results for the first time highlight that FSH function in vitro is mediated via cumulus cells by downregulating
FSHR
and upregulating
LHCGR
, which was also observed when the immature oocytes progressed in meiosis from the GV to the MII stage.
An increasing body of data implicates the Septin family in the pathology of several diseases, including male fertility. The objective of this study was to evaluate the gene and protein expression ...pattern of Septin 14 in the testis tissue of azoospermic men. In addition, Septin 14 localization was also assessed in the sperm. Testicular tissues were obtained from biopsies of non-obstrutive azoospermic men who underwent diagnostic testicular biopsy in Royan institute and were divided into two groups: TESE + with positive result in testicular sperm extraction (with hypospermatogenesis pathology) and TESE- with negative result (included patients with Sertoli cell only syndrome and maturation arrest pathologies). Total RNA and protein was extracted using trizol reagent. Septin 14 gene and protein expression level were assessed by real-time reverse transcription polymerase chain reaction (RT-PCR) and Western blot techniques, respectively. The localization of Septin 14 protein was also studied by Immunocytochemistry. The expression of Septin 14 was significantly lower (p < 0. 05) in TESE- group than TESE + in both mRNA and protein levels. The localization of Septin 14 protein was detected in the head to tail of normal sperms with high localization in front of the acrosome and the neck. This is a novel localization report on Septin 14 in sperm. Regarding the presence of this protein in the sperm acrosome and neck, it can be concluded that decreasing of Septin 14 protein expression may be associated with the pathogenesis of male infertility and therefore Septin 14 expression level maybe critical for human spermatogenesis.
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.
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).
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.
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.
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.
•The proportion of growing follicles significantly increase in agar cultured group.•Human ovarian tissue cultured on agar scaffold secrates higher estardiol levels.•The expression of apototic genes ...downregulates in both agar and insert groups.•Ki67 expression increases in both cultured groups, higher for the agar group.
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.
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.
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.
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.
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.
•KL and bFGF promote the proliferation and steroidogenesis of granulosa cells.•Combination of KL and bFGF increase the expression of Ki67 in the ovarian tissue.•Ovarian cell proliferation increases by the addition of KL and bFGF combination.•Supplementation of KL and bFGF have no beneficial effect on follicular activation.
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