Infertility has become a serious disease since it affects 10%-15% of couples worldwide, and male infertility contributes to about 50% of the cases. Notably, a significant decrease occurs in the newborn population by 7.82 million in 2020 compared to 2016 in China. As such, it is essential to explore the effective methods of obtaining functional male gametes for restoring male fertility. Stem cells, including embryonic stem cells (ESCs), induced pluripotent stem cells (iPSCs), spermatogonial stem cells (SSCs), and mesenchymal stem cells (MSCs), possess the abilities of both self-renewal and differentiation into germ cells. Significantly, much progress has recently been achieved in the generation of male germ cells in vitro from various kinds of stem cells under the specified conditions, e.g., the coculturing with Sertoli cells, three-dimensional culture system, the addition of growth factors and cytokines, and/or the overexpression of germ cell-related genes. In this review, we address the current advance in the derivation of male germ cells in vitro from stem cells based on the studies of the peers and us, and we highlight the perspectives and potential application of stem cell-derived male gametes in reproductive medicine.
Humans ; Infant, Newborn ; Male ; Germ Cells ; Embryonic Stem Cells ; Cell Differentiation ; Infertility, Male ; Induced Pluripotent Stem Cells

Understanding the regulatory networks for germ cell fate specification is necessary to developing strategies for improving the efficiency of germ cell production in vitro. In this study, we developed a coupled screening strategy that took advantage of an arrayed bi-molecular fluorescence complementation (BiFC) platform for protein-protein interaction screens and epiblast-like cell (EpiLC)-induction assays using reporter mouse embryonic stem cells (mESCs). Investigation of candidate interaction partners of core human pluripotent factors OCT4, NANOG, KLF4 and SOX2 in EpiLC differentiation assays identified novel primordial germ cell (PGC)-inducing factors including BEN-domain (BEND/Bend) family members. Through RNA-seq, ChIP-seq, and ATAC-seq analyses, we showed that Bend5 worked together with Bend4 and helped mark chromatin boundaries to promote EpiLC induction in vitro. Our findings suggest that BEND/Bend proteins represent a new family of transcriptional modulators and chromatin boundary factors that participate in gene expression regulation during early germline development.
Animals ; Cell Differentiation/genetics* ; Chromatin/metabolism* ; Embryonic Stem Cells ; Germ Cells/metabolism* ; Germ Layers/metabolism* ; Mice

OBJECTIVE: As paternal age increases, the quality of sperm decreases due to increased DNA fragmentation and aneuploidy. Higher levels of structural chromosomal aberrations in the gametes ultimately decrease both the morphologic quality of embryos and the pregnancy rate. In this study, we investigated whether paternal age affected the euploidy rate. METHODS: This study was performed using the medical records of patients who underwent in vitro fertilization (IVF) procedures with preimplantation genetic screening (PGS) from January 2016 to August 2017 at a single center. Based on their morphological grade, embryos were categorized as good- or poor-quality blastocysts. The effects of paternal age were elucidated by adjusting for maternal age. RESULTS: Among the 571 total blastocysts, 219 euploid blastocysts were analyzed by PGS (38.4%). When the study population was divided into four groups according to both maternal and paternal age, significant differences were only noted between groups that differed by maternal age (group 1 vs. 3, p=0.031; group 2 vs. 4, p=0.027). Further analysis revealed no significant differences in the euploidy rate among the groups according to the morphological grade of the embryos. CONCLUSION: Paternal age did not have a significant impact on euploidy rates when PGS was performed. An additional study with a larger sample size is needed to clarify the effects of advanced paternal age on IVF outcomes.
Aneuploidy ; Blastocyst ; Chromosome Aberrations ; DNA Fragmentation ; Embryonic Development ; Embryonic Structures ; Female ; Fertilization in Vitro ; Genetic Testing ; Germ Cells ; Humans ; In Vitro Techniques ; Maternal Age ; Medical Records ; Paternal Age ; Pregnancy ; Pregnancy Rate ; Sample Size ; Spermatozoa

The survival rates of boys and men with cancer have increased due to advances in cancer treatments; however, maintenance of quality of life, including fertility preservation, remains a major issue. Fertile male patients who receive radiation and/or chemotherapy face temporary, long-term, or permanent gonadal damage, particularly with exposure to alkylating agents and whole-body irradiation, which sometimes induce critical germ cell damage. These cytotoxic treatments have a significant impact on a patient's ability to have their own biological offspring, which is of particular concern to cancer patients of reproductive age. Therefore, various strategies are needed in order to preserve male fertility. Sperm cryopreservation is an effective method for preserving spermatozoa. Advances have also been achieved in pre-pubertal germ cell storage and research to generate differentiated male germ cells from various types of stem cells, including embryonic stem cells, induced pluripotent stem cells, and spermatogonial stem cells. These approaches offer hope to many patients in whom germ cell loss is associated with sterility, but are still experimental and preliminary. This review examines the current understanding of the effects of chemotherapy and radiation on male fertility.
Alkylating Agents ; Cryopreservation ; Drug Therapy ; Embryonic Stem Cells ; Fertility ; Fertility Preservation ; Germ Cells ; Gonads ; Hope ; Humans ; Induced Pluripotent Stem Cells ; Infertility ; Infertility, Male ; Male ; Methods ; Quality of Life ; Radiotherapy ; Spermatogenesis ; Spermatozoa ; Stem Cells ; Survival Rate ; Whole-Body Irradiation

OBJECTIVE: To determine the localization, expression, and function of Toll-like receptors (TLRs) in fallopian tube epithelial cells. METHODS: The localization of TLRs in fallopian tube epithelial cells was investigated by immunostaining. Surprisingly, the intensity of staining was not equal in the secretory and ciliated cells. After primary cell culture of fallopian tube epithelial cells, ring cloning was used to isolate colonies of ciliated epithelial cells, distinct from non-ciliated epithelial cells. The expression of TLRs 1–10 was examined by quantitative real-time polymerase chain reaction, and protein localization was confirmed by immunostaining. The function of the TLRs was determined by interleukin (IL)-6 and IL-8 production in response to TLR2, TLR3, TLR5, TLR7, and TLR9 ligands. RESULTS: Fallopian tube epithelial cells expressed TLRs 1–10 in a cell-type-specific manner. Exposing fallopian tube epithelial cells to TLR2, TLR3, TLR5, TLR7, and TLR9 agonists induced the secretion of proinflammatory cytokines such as IL-6 and IL-8. CONCLUSION: Our findings suggest that TLR expression in the fallopian tubes is cell-type-specific. According to our results, ciliated cells may play more effective role than non-ciliated cells in the innate immune defense of the fallopian tubes, and in interactions with gametes and embryos.
Clone Cells ; Cloning, Organism ; Cytokines ; Embryonic Structures ; Epithelial Cells* ; Fallopian Tubes* ; Female ; Germ Cells ; Humans* ; Interleukin-6 ; Interleukin-8 ; Interleukins ; Ligands ; Primary Cell Culture ; Real-Time Polymerase Chain Reaction ; Toll-Like Receptors*

It is not fully clear why there is a higher contribution of pluripotent stem cells (PSCs) to the chimera produced by injection of PSCs into 4-cell or 8-cell stage embryos compared with blastocyst injection. Here, we show that not only embryonic stem cells (ESCs) but also induced pluripotent stem cells (iPSCs) can generate F0 nearly 100% donor cell-derived mice by 4-cell stage embryo injection, and the approach has a "dose effect". Through an analysis of the PSC-secreted proteins, Activin A was found to impede epiblast (EPI) lineage development while promoting trophectoderm (TE) differentiation, resulting in replacement of the EPI lineage of host embryos with PSCs. Interestingly, the injection of ESCs into blastocysts cultured with Activin A (cultured from 4-cell stage to early blastocyst at E3.5) could increase the contribution of ESCs to the chimera. The results indicated that PSCs secrete protein Activin A to improve their EPI competency after injection into recipient embryos through influencing the development of mouse early embryos. This result is useful for optimizing the chimera production system and for a deep understanding of PSCs effects on early embryo development.
Activins ; metabolism ; Animals ; Cells, Cultured ; Embryonic Development ; Germ Layers ; metabolism ; Mice ; Pluripotent Stem Cells ; cytology ; metabolism

Artificial insemination by donor is an important means of improving the likelihood of pregnancy in couples affected by male factor infertility, but it poses medical, legal, and ethical issues due to the involvement of third parties, such as the sperm donor. In Korea, the Bioethics and Safety Act was enacted for the purpose of preventing and eliminating unethical research on germ cells, and such research was limited to matters related to the use of assisted reproductive technologies, centering on embryos, oocytes, and protecting the health of oocyte donors. However, this law is incomplete in terms of specific standards or regulations relating to the donation and receipt of sperm. In Korea, artificial insemination by donor has been carried out without a standard operating protocol for donation and receipt of sperm, which would include testing sperm donors for diseases, limiting the number of donor offspring, compensation for donations, and the role of anonymity and non-anonymity. The diversity of policies worldwide shows that each country has its unique set of guidelines tailored for its own specific needs and practical considerations. Herein, I present a standard operating protocol of medical, legal, and ethical principles for artificial insemination by donor that is suitable for domestic circumstances, along with a comparison of recommendations and guidelines of other countries concerning sperm donation issues.
Anonyms and Pseudonyms ; Bioethics ; Compensation and Redress ; Embryonic Structures ; Ethics ; Family Characteristics ; Germ Cells ; Humans ; Infertility ; Infertility, Male ; Insemination, Artificial ; Jurisprudence ; Korea ; Male ; Oocytes ; Pregnancy ; Reproductive Techniques, Assisted ; Social Control, Formal ; Spermatozoa ; Tissue Donors

Oxidative stress (OS) has been recognized as a significant cause of suboptimal assisted reproductive outcome. Many of the sperm preparation and manipulation procedures that are necessary in the in vitro environment can result in excessive production of reactive oxygen species (ROS) thereby exposing the gametes and growing embryos to significant oxidative damage. Antioxidants have long been utilized in the management of male subfertility as they can counterbalance the elevated levels of ROS inducing a high state of OS. Few studies have looked into the clinical effectiveness of antioxidants in patients undergoing assisted reproduction. While an overall favorable outcome has been perceived, the specific clinical indication and optimal antioxidant regimen remain unknown. The goal of our review is to explore the sources of ROS in the in vitro environment and provide a clinical scenario-based approach to identify the circumstances where antioxidant supplementation is most beneficial to enhance the outcome of assisted reproduction.
Antioxidants* ; Embryonic Structures ; Germ Cells ; Humans ; In Vitro Techniques ; Infertility, Male ; Male ; Oxidative Stress ; Reactive Oxygen Species ; Reproduction ; Reproductive Techniques, Assisted* ; Spermatozoa ; Treatment Outcome

Post-translational modifications, such as methylation, acetylation and phosphorylation, of histone proteins play important roles in regulating dynamic chromatin structure. Histone demethylation has become one of the most active research areas of epigenetics in the past decade. To date, with the exception of histone H3 lysine 79 methylation, the demethylases for all major lysine methylation sites have been discovered. These enzymes have been shown to be involved in various biological processes, with embryonic development being an exciting emerging area. This review will primarily discuss the involvement of these demethylases in the regulation of mammalian embryonic development, including their roles in embryonic stem cell pluripotency, primordial germ cell (PGC) formation and maternal-to-zygotic transition.
Acetylation ; Biological Processes ; Chromatin ; Embryonic Development* ; Embryonic Stem Cells ; Epigenomics ; Female ; Germ Cells ; Histone Demethylases* ; Histones* ; Lysine ; Methylation ; Phosphorylation ; Pregnancy ; Protein Processing, Post-Translational

OBJECTIVETo perform a genome-wide alternative polyadenylation (APA) profiling in both mouse female germline stem cells (FGSCs) and embryonic stem cells (ESCs) and explore the role of germline-specific APA in the biological behaviors of FGSCs.

METHODSWe used a high-throughput sequencing-based method 3T-Seq to profile the genome-wide 3' termini of the transcripts and delineate all the APA sites in mouse FGSCs and ESCs. The genes with altered APA sites in FGSCs compared with ESCs were analyzed with DAVID Gene Ontology tool for their biological roles.

RESULTSWe identified a total of 50243 APA sites in 16973 genes. In FGSCs, 1148 genes were shown to have alterations in 3'UTR length, among which 795 ( 66%) genes had shortened and 353 (34%) had lengthened 3'UTR. Some of the genes with shortened 3'UTR were involved in germ cell development.

CONCLUSIONSOur genome-wide APA profiling analysis reveals a cell type-specific APA alternation in FGSCs, and APA-mediated 3'UTR alteration contributes to germline-related biological process. This study provides a framework for understanding the post-transcriptional regulation mechanisms in FGSCs.

3' Untranslated Regions ; Animals ; Cell Differentiation ; Embryonic Germ Cells ; metabolism ; Embryonic Stem Cells ; metabolism ; Female ; Gene Expression Regulation ; Genome ; Mice ; Polyadenylation