CAG repeat polymorphism in androgen receptor and infertility: A case-control study

Background: Androgens play a role in the development of male phenotype and spermatogenesis during puberty, the function of which is regulated by the androgen receptor (AR) gene. There is a polymorphism site in exon 1 of the gene encoding this receptor that can have different frequencies of CAG trinucleotide repeats and leads to the formation of polyglutamine chains of different lengths in the N-terminal domain of the AR protein and reduced sperm production by affecting spermatogenesis. Objective: To investigate whether the cause of a group of unexplained infertilities could be the increased frequency of CAG repeats in the AR gene of patients with oligozoospermia and azoospermia. Materials and Methods: In this case-control study, 84 men including 42 with unexplained infertility As a case group and 42 fertile men as a control group were selected. The frequency of CAG repeats was determined by the polymerase chain reaction method and then the difference in the frequency of these repeats was determined based on the difference in band size on the agarose gel. Results: The mean CAG repeat length in the azoospermia and oligozoospermia group was 17.5 ± 0.63 and in the fertile group it was 16.11 ± 0.75 (p = 0.46). In addition, most men (88.1% in the case group and 71.41% in the control group) had 13-23 repeats. Conclusion: No significant correlation was found between CAG repeat length and the risk of male factor infertility in an ethnically defined population of Iranian men. The role of regulatory factors and epigenetic changes should be taken into account too. Key words: Infertility, Azoospermia, Androgens, X chromosome, Spermatogenesis

Established Yazd human foreskin fibroblast lines (#8, #17, and #18) displaying similar characteristics to mesenchymal stromal cells: A lab resources report

Background: Fibroblasts from different parts of the human body have been used in cell biology, drug discovery and cell therapy studies. One of the most available sources of human fibroblasts is neonatal foreskin. Not only do these cells have woundhealing applications, but they are also the most popular source for pluripotent stem cell biotechnology. Moreover, several studies have indicated that different sources of fibroblasts display similar features to mesenchymal stem cells. Objective: Generation and establishment of new human foreskin fibroblast cell lines called Yazd human foreskin fibroblasts (YhFFs). Materials and Methods: In this lab resources study, the production of 3 YhFF cell lines (YhFF#8, YhFF#17, and YhFF#18) is reported. Their biological features were characterized using immunofluorescence, polymerase chain reaction, and flowcytometry for mesenchymal markers such as fibronectin, vimentin, CD44, CD73, CD90, CD105, and hematopoietic markers CD34 and CD45. Results: The YhFF cell lines were passaged more than 40 times and their normal karyotype was checked using G-binding. Similarly to previous reports, the flow cytometry analysis revealed that the YhFF cell lines displayed mesenchymal stromal cell characteristics. Conclusion: This study will contribute to the development of clinical-grade cell-based products such as micro-vesicles and exosomes for future therapeutic applications in regenerative medicine. Key words: Cell therapy, Fibroblasts, Mesenchymal stem/stromal cells, Human embryonic stem cells, Micro-vesicles

Characteristics of the human endometrial regeneration cells as a potential source for future stem cell-based therapies: A lab resources study

Background: Human endometrium with consecutive regeneration capability undergoes monthly hormonal changes for probable implantation, which confirms the presence of the cells in the basalis layer known as stem cell. Objective: Previously, we reported the isolation and culture of the mesenchymal-like cells from human endometrium. In this study, we evaluated the biological and stemness characteristics of these cells. Materials and Methods: The characterization of Yazd human endometrialderived mesenchymal stem/stromal cells (YhEnMSCs) was assessed using immunofluorescence (IF) staining for CD105, VIMENTIN, and FIBRONECTIN as markers and RT-PCR for CD166, CD10, CD105, VIMENTIN, FIBRONECTIN, MHCI, CD14, and MHCII genes. Flow cytometry (FACS) was performed for CD44, CD73, CD90, and CD105 markers. Moreover, the differentiation capacity of the YhEnMSCs to the osteoblast and adipocytes was confirmed by Alizarin Red and Oil Red staining. Results: YhEnMSCs expressed CD105, VIMENTIN, FIBRONECTIN, CD44, CD73, and CD90 markers and CD166, CD10, CD105, VIMENTIN, FIBRONECTIN, and MHCI, but, did not express CD14, MHCII. Conclusion: Our data confirm previous reports by other groups indicating the application of endometrial cells as an available source of MSCs with self-renewal and differentiation capacity. Accordingly, YhEnMSCs can be used as a suitable source for cell-based therapies. Key words: Cell-based therapy, Endometrium, Mesenchymal stem/stromal cells, Regenerative medicine, Stem cells, Uterus

Does combination of estradiol and sesame oil improve the oocyte quality, embryo development and expressions of Zp3, E-cad, and Ctnnb1 genes in mice? An experimental study

Background: Aging may reduce oocyte maturation, embryo quality, and fertility potential. Objective: To compare the effect of estradiol (E2) and sesame oil on oocyte and embryo quality between young and old mice. Materials and Methods: Sixty old and young female mice were divided in to two groups (30 mice/group, grouped by age). Each group was divided into three subgroups of mice treated with sesame oil, E2 + sesame oil, and normal saline as control group. After ovulation induction, some oocytes were considered for in vitro fertilization and the rest were assessed for morphological status. After obtaining the two-cell embryos, the embryos were collected to determine the expression of zona pellucida (ZP) glycoprotein 3, E-cadherin, and β-catenin genes and some of them followed until the blastocysts stage to evaluate the viability. Results: The findings showed that the mean ZP and perivitelline space thickness increased in the old mice that received the E2 + sesame oil treatment. The number of 2-cell embryos, blastocysts, and live cells were significantly higher in the old group treated with sesame oil respectively (p = 0.018, 0.002, and < 0.0001, respectively). The normal ZP shape and refractile body numbers increased in the old mice that were treated with sesame oil, respectively. The E-cadherin gene was downregulated in the treatment groups compared to the controls. Conclusion: Sesame oil showed a better response in the old mice, because aging is associated with an increased rate of reactive oxygen species, causing deficiencies in both oocyte and embryo qualities. Key words: Estradiol, Sesame oil, Gene expression, Oocyte, Mouse

Biological and physiological characteristics of human cumulus cell in adherent culture condition

Background: Cumulus cells, as oocyte nurse cells, provide a suitable microenvironment with growth factors and cellular interactions required for oocyte maturation. Thus, these cells may serve as a natural niche for in vitro studies of female germ cell development. Cumulus cells may help attain a better understanding of the causes of infertility in women and eventually improve the outcomes of cases that respond poorly to standard infertility treatment. Objective: The aim of this study was to isolate, culture, and investigate the biological characteristics of human cumulus cells. Materials and Methods: In this experimental study, cumulus cells were isolated, cultured, and characterized using reverse transcription-polymerase chain reaction analyses of specific genes including FOXL2, CYP19A1, FSHR, AMHR, and LHR. The presence of vimentin, a structural protein, was examined via immunofluorescent staining. Moreover, levels of anti-mullerian hormone (AMH) and progesterone secretion by cumulus cells were measured with ELISA after 2, 4, 12, 24, and 48 hr of culture. Results: In adherent culture, human cumulus cells expressed specific genes and markers as well as secreted AMH and progesterone into the medium. Conclusion: Cumulus cells secrete AMH and progesterone in an adherent culture and might be applicable for in vitro maturation (IVM) and in vitro gametogenesis (IVG) studies. Key words: Cumulus cells, Conditioned medium, In vitro maturation, In vitro gametogenesis, Niche

Xeno-free generation of new Yazd human embryonic stem cell lines (Yazd4-7) as a prior stage toward good manufacturing practice of clinical-grade raw materials from discarded embryos: A lab resources report

Background: Xeno-free generation of human embryonic stem cells (hESCs) is important to prevent potential animal contaminations in culture for advanced cellbased therapeutic applications. Xeno-free production of hESCs is the first step for manufacturing clinical-grade hESC lines. Objective: To produce new hESC lines in xeno-free condition. Materials and Methods: This lab resources report was conducted at Stem Cell Biology Research Center, Yazd, Iran from 2019-2022. 4 new hESC lines from 11 (10 fresh and 1 frozen) donated surplus discarded human embryos were established. In this study, we report the xeno-free derivation of new Yazd hESC lines (Yazd4-7), without using immunosurgery, by culturing intact zona-free blastocysts obtained from discarded embryos onto the YhFF#8 cells as a feeder layer in a microdrop culture system. The pluripotency gene expression profile of the cell lines was assessed by reverse transcription polymerase chain reaction and the expression of specific surface markers was detected using immunofluorescent staining. In vitro differentiation was induced using embryoid body formation and gene expression profile of 3 germ layers and germ cells. Reverse transcriptase polymerase chain reaction was investigated to prove their pluripotent capacity. Results: In sum, we have been able to generate 4 new hESC lines (Yazd4-7) from 11 discarded embryos in xeno-free culture conditions using a micro drop culture system and YhFF#8 as a human source feeder layer. Conclusion: The outcome of this work can be the foundation for the future allogeneic cell-based therapeutic application using clinical grade good manufacturing practicederived hESC derivatives. Key words: Derivation, Human embryonic stem cells, Human foreskin fibroblasts, Xeno-free, Good manufacturing practice, Mouse embryonic fibroblasts

Evaluation of CAG repeat length in the androgen receptor gene and polycystic ovary syndrome risk in Iranian women: A case-control study

Background: Polycystic ovary syndrome (PCOS) is a heterogeneous disorder, which affects about 15-20% of women of reproductive age. The most important etiopathogenesis factor in its incidence is hyperandrogenism; over 70 candidate genes are known to be associated with this syndrome, such as the androgen receptor (AR) gene which encodes a steroid receptor and is located on the Xq11-12 chromosome. The N-terminus of exon 1 of AR contains a polymorphic trinucleotide repeat (CAG)n region that encodes glutamine tract. There are some studies showing that shorter AR CAG repeats are significantly related to enhanced AR sensitivity. Objective: This study investigated the frequency of the polymorphic expansion of the trinucleotide CAG repeats of AR in PCOS. Materials and Methods: 160 Iranian women aged 17-40 yr participated in this casecontrol study: 80 women as PCOS patients and 80 women as healthy controls according to the Rotterdam criteria. Other similar phenotype factors such as hyperandrogenism were not considered as PCOS. The frequency of polymorphic expansion of CAG trinucleotide repeats in PCOS patients was compared with the frequency in non-PCOS controls in using two primer sets for nested polymerase chain reaction. The polymerase chain reaction products were visualized on polyacrylamide gel and then were confirmed by a sequencing process. Results: The results did not show a significant correlation between the frequency of CAG repeats in AR and PCOS incidence. Conclusion: In contrast to some previous reports, the present data showed that the CAG length in PCOS cases did not significantly differ from that of controls. So, the AR (CAG)n does not appear to be a major factor for PCOS in Iranian women. Key words: Androgen receptor, (CAG)n repeats, Polycystic ovary syndrome

Derivation of new human embryonic stem cell lines (Yazd1-3) and their vitrification using Cryotech and Cryowin tools: A lab resources report

Background: Cell banking initial outgrowths from newly derived human embryonic stem cells (hESCs) requires an efficient freezing method. Vitrification is used for the preservation of gametes and early embryos in assisted reproduction techniques (ART). Moreover, vitrification was applied for cryopreservation of hESCs using open pulled straws. Objective: To derive and characterize new hESC lines and then use Cryotech and Cryowin tools for their vitrification. Materials and Methods: Human ESC lines were generated in a microdrop culture system using mouse embryonic fibroblasts (MEFs) as the feeder layer; this was later scaled up using both MEFs and Yazd human foreskin fibroblasts batch 8 (YhFF#8). To bank the cell lines, master cell banks of 100 Cryotech and Cryowin tools were produced for each individual cell line using the vitrification method; flasks of hESC lines were also cryopreserved using a conventional slow-freezing method. Results: The pluripotency of cell lines was assessed by their expression of pluripotency-associated genes (OCT4/POU5F1, NANOG, and SOX2) and markers such as SSEA4, TRA-1-60, and TRA-2-49. Their in vitro capacity to differentiate into germ layers and germ cells using embryoid body (EB) formation and monolayer culture was assessed by screening the expression of differentiation-associated genes. The chromosomal constitution of each hESC line was assessed by G-banding karyotyping. Conclusion: Cryotech and Cryowin tools used to vitrify new hESCs at an early stage of derivation is an efficient means of preserving hESCs. Key words: Derivation, Human embryonic stem cells, Human foreskin fibroblast, Microdrop, Vitrification

The genetically modified human foreskin fibroblast cell line (YhFF#8) stably expressing Cas9 gene: A lab resource report

Background: Stable Cas9 (CRISPR-associated protein 9)-expressing cell lines have emerged as valuable tools in genetic research, enhancing the efficiency of the CRISPR/Cas9 system and streamlining gene editing procedures. These cell lines enable simultaneous editing of multiple genes and reduce the overall editing time. Objective: This study aimed to develop a stable human fibroblast cell line capable of genetic conversion into a mutant form, serving as a cellular model for a specific genetic disease. The established cell line facilitates investigation of disease mechanisms, testing of potential treatments, and gaining insights into underlying molecular processes. Materials and Methods: Human embryonic kidney 293LTV cells were used to produce pseudo-virus particles, while Yazd human foreskin fibroblasts batch 8 (YhFF#8) cells were targeted for genetic modification. Transfection of human embryonic kidney 293LTV cells with pCDH-Cas9 plasmid DNA generated pseudo-viral particles. YhFF#8 cells were transduced and selected using antibiotics. Green fluorescent protein (GFP) detection confirmed successful transduction and selection. Relative expression levels of the Cas9 gene were determined by quantitative polymerase chain reaction. Results: The study validated the fidelity of the Cas9 gene cassette sequence and its transcriptional activity. Transduced YhFF#8 cells exhibited green fluorescence, with antibiotic selection resulting in nearly 100% transduced cells. A reporter GFP gene enabled real-time monitoring of YhFF#8-Cas9-GFP-PuroR cells using fluorescence microscopy. Conclusion: YhFF#8-Cas9-GFP-PuroR cells, labeled and susceptible to genomic editing, provide an optimal source for generating induced pluripotent stem cell lines for future biomedical research. Key words: Fibroblasts, Cell line, Genetic transduction, CRISPR-Cas9