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 cell-based 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 practice-derived hESC derivatives.

The exact synchronization timing between the cleavage embryo stage and duration of progesterone therapy-improved pregnancy rates in frozen embryo transfer cycles: A cross-sectional study.

BACKGROUND: Synchronization between the embryonic stage and the uterine endometrial lining is important in the outcomes of the vitrified-warmed embryo transfer (ET) cycles. OBJECTIVE: The aim was to investigate the effect of the exact synchronization between the cleavage stage of embryos and the duration of progesterone administration on the improvement of clinical outcomes in frozen embryo transfer (FET) cycles. MATERIALS AND METHODS: 312 FET cycles were categorized into two groups: (A) day-3 ET after three days of progesterone administration (n = 177) and (B) day-2 or -4 ET after three days of progesterone administration (n = 135). Group B was further divided into two subgroups: B1: day-2 ET cycles, that the stage of embryos were less than the administrated progesterone and B2: day-4 ET cycles, that the stage of embryos were more than the administrated progesterone. The clinical outcome measures were compared between the groups. RESULTS: The pregnancy outcomes between groups A and B showed a significant differences in the chemical (40.1% vs 27.4%; p = 0.010) and clinical pregnancies (32.8% vs 22.2%; p = 0.040), respectively. The rate of miscarriage tended to be higher and live birth rate tended to be lower in group B than in group A. Also, significantly higher rates were noted in chemical pregnancy, clinical pregnancy, and live birth in group A when compared with subgroup B2. CONCLUSION: Higher rates of pregnancy and live birth were achieved in day-3 ET after three days of progesterone administration in FET cycles.

Mesenchymal Stem/Stromal-Like Cells from Diploid and Triploid Human Embryonic Stem Cells Display Different Gene Expression Profiles.

Background: Human embryonic stem cell-mesenchymal stem/stromal cell (hESCs-MSCs) open a new insight into future cell therapy applications, due to their unique characteristics, including immunomodulatory features, proliferation, and differentiation. Methods: Herein, hESCs-MSCs were characterized by immunofluorescence technique with CD105 and FIBRONECTIN as markers and FIBRONECTIN, VIMENTIN, CD10, CD105, and CD14 genes using reverse transcription-polymerase chain reaction technique. Fluorescence-activated cell sorting was performed for CD44, CD73, CD90, and CD105 markers. Moreover, these fibroblast-like cells, due to multipotent characteristics, differentiated to the osteoblast. Results: MSCs were derived from diploid and triploid hESC lines using sequential three dimensional and two dimensional cultures and characterized with the specific markers. Immunofluorescence showed the expression of FIBRONECTIN and CD105 in hESCs-MSCs. Flow cytometry data indicated no significant difference in the expression of MSC markers after 6 and 13 passages. Interestingly, gene expression profiles revealed slight differences between MSCs from diploid and triploid hESCs. hESCs-MSCs displayed osteogenic differentiation capacity, which was confirmed by Alizarin red staining. Cobnclusion: Our findings reveal that both diploid and triploid hESC lines are capable of forming MSCs; however, there are some differences in their gene expression profiles. Generation of MSCs from hESCs, as a non-invasive procedure in large scale, will lend itself for the future cell-based therapeutic applications.

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 endometrial-derived 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.

Effect of Human Testicular Cells Conditioned Medium on In Vitro Maturation and Morphology of Mouse Oocytes.

BACKGROUND: Testicular cell conditioned medium (TCCM) has been shown to induce female germ cell development In Vitro from embryonic stem cells (ESCs). Testicular cells (TCs) secrete a variety of growth factors such as growth differentiation factor-9 (GDF-9), bone morphogenetic protein 4 (BMP-4), stem cell factor (SCF), leukemia inhibitory factor (LIF), and other, that could improve oocyte maturation. Here we have investigated the effect of human TCCM (hTCCM) on in vitro maturation (IVM) and morphology of mouse oocytes. MATERIALS AND METHODS: In this experimental study, 360 germinal vesicle (GV) oocytes were obtained from NMRI mice, aged 4-6 weeks that had received 5 IU pregnant mare's serum gonadotropin (PMSG) 48 hours before. GV oocytes were subjected to IVM. 120 GV oocytes were cultured in each medium; hTCCM as the test group, DMEM + 20%FBS as the control group and Ham's F10 + HFF medium as the sham group. The rates of the IVM and perivitelline space (PVS) changes were recorded at 8, 16 and 24 hours after culture. The metaphase II (MII) oocytes were subjected for in vitro fertilization (IVF) and the fertilization rate was evaluated after 1, 2, and 3 days. RESULTS: There was a significant difference between the maturation rates in hTCCM (31.67% MII) and the control [0% MII, P<0.05, (7.5% MI, 52.5% deg. and 40%GV)] groups but there was not a significant difference between the maturation rates in hTCCM and the sham group (53.33% MII, P>0.05). IVF success rate for MII oocytes obtained from IVM in the hTCCM group was 28.94% (n=11). Our data showed that hTCCM is an effective medium for GV oocyte growth and maturation compared to the control medium. CONCLUSION: Our findings show that TCCM supports oocyte IVM in mice and affect oocyte morphology.

Does Overnight Culture of Cleaved Embryos Improve Pregnancy Rate in Vitrified-Warmed Embryo Transfer Programme?

BACKGROUND: Vitrification is a routine procedure in assisted reproductive technique (ART) lab. However, there is widespread variability between protocols of different centres. The aim of this study was to compare the chemical pregnancy, clinical pregnancy and live birth rates between one-day embryo culture and immediate transfer for frozen-thawed embryo transfer (FET) cycles. METHODS: In this cohort retrospective study, 366 FET cycles were divided into two groups: Group A, the embryos were warmed one day before transfer, and were cultured overnight; Group B, the embryos were warmed on the same day of transfer, at least were cultured 1 h before embryo transfer (ET). Chemical and clinical pregnancy and live birth rates were compared between two groups. RESULTS: The chemical pregnancy was higher in group A than B (37.9% versus 28.9%), but this difference was not significant (P = 0.07). Clinical pregnancy (30.8% versus 24.1%) and live birth (19.8% versus 22.05%) were similar in group A and B, (P = 0.15), and (P = 0.8). Conclusion: In conclusion, overnight culture and confirmation of mitosis resumption was not essential for FET cycles in vitrification method.

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 of 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 method of preserving hESCs.

Human embryonic stem cells and good manufacturing practice: Report of a 1- day workshop held at Stem Cell Biology Research Center, Yazd, 27th April 2017.

This report explains briefly the minutes of a 1-day workshop entitled; "human embryonic stem cells (hESCs) and good manufacturing practice (GMP)" held by Stem Cell Biology Research Center based in Yazd Reproductive Sciences Institute at Shahid Sadoughi University of Medical Sciences, Yazd, Iran on 27th April 2017. In this workshop, in addition to the practical sessions, Prof. Harry D. Moore from Centre for Stem Cell Biology, University of Sheffield, UK presented the challenges and the importance of the biotechnology of clinical-grade human embryonic stem cells from first derivation to robust defined culture for therapeutic applications.

Etiologies of sperm oxidative stress.

Sperm is particularly susceptible to reactive oxygen species (ROS) during critical phases of spermiogenesis. However, the level of seminal ROS is restricted by seminal antioxidants which have beneficial effects on sperm parameters and developmental potentials. Mitochondria and sperm plasma membrane are two major sites of ROS generation in sperm cells. Besides, leukocytes including polymer phonuclear (PMN) leukocytes and macrophages produce broad category of molecules including oxygen free radicals, non-radical species and reactive nitrogen species. Physiological role of ROS increase the intracellular cAMP which then activate protein kinase in male reproductive system. This indicates that spermatozoa need small amounts of ROS to acquire the ability of nuclear maturation regulation and condensation to fertilize the oocyte. There is a long list of intrinsic and extrinsic factors which can induce oxidative stress to interact with lipids, proteins and DNA molecules. As a result, we have lipid peroxidation, DNA fragmentation, axonemal damage, denaturation of the enzymes, over generation of superoxide in the mitochondria, lower antioxidant activity and finally abnormal spermatogenesis. If oxidative stress is considered as one of the main cause of DNA damage in the germ cells, then there should be good reason for antioxidant therapy in these conditions.

Pluripotency and differentiation of cells from human testicular sperm extraction: An investigation of cell stemness.

Human male germ-line stem cells (hmGSCs) and human testis-derived embryonic stem cell-like (htESC-like) cells are claimed to be in vitro pluripotent counterparts of spermatogonial stem cells (SSCs), but the origin and pluripotency of human testis-derived cell cultures are still under debate. The aim of this study was to generate putative pluripotent stem cells in vitro from human testicular sperm-extracted (TESE) samples of infertile men, and to assess their pluripotency and capacity to differentiate. TESE samples were minced, enzymatically disaggregated and dispersed into single-cell or cluster suspensions, and then cultured. Initially, cell clusters resembled those described for hmGSCs and htESC-like cells, and were positive for markers such as OCT4/POU5F1, NANOG, and TRA-2-54. Prolonged propagation of cell clusters expressing pluripotency markers did not thrive; instead, the cells that emerged possessed characteristics of mesenchymal stromal cells (MSCs) such as STRO-1, CD105/EGLN1, CD13/ANPEP, SOX9, vimentin, and fibronectin. KIT, SOX2, and CD44 were not expressed by these MSCs. The multipotential differentiation capacity of these cells was confirmed using Oil Red-O and Alizarin Red staining after induction with specific culture conditions. It is therefore concluded that pluripotent stem cells could not be derived using the conditions previously reported to be successful for TESE samples.