ITGA6+ Human Testicular Cell Populations Acquire a Mesenchymal Rather than Germ Cell Transcriptional Signature during Long-Term Culture.

Autologous spermatogonial stem cell transplantation is an experimental technique aimed at restoring fertility in infertile men. Although effective in animal models, in vitro propagation of human spermatogonia prior to transplantation has proven to be difficult. A major limiting factor is endogenous somatic testicular cell overgrowth during long-term culture. This makes the culture both inefficient and necessitates highly specific cell sorting strategies in order to enrich cultured germ cell fractions prior to transplantation. Here, we employed RNA-Seq to determine cell type composition in sorted integrin alpha-6 (ITGA6+) primary human testicular cells (n = 4 donors) cultured for up to two months, using differential gene expression and cell deconvolution analyses. Our data and analyses reveal that long-term cultured ITGA6+ testicular cells are composed mainly of cells expressing markers of peritubular myoid cells, (progenitor) Leydig cells, fibroblasts and mesenchymal stromal cells and only a limited percentage of spermatogonial cells as compared to their uncultured counterparts. These findings provide valuable insights into the cell type composition of cultured human ITGA6+ testicular cells during in vitro propagation and may serve as a basis for optimizing future cell sorting strategies as well as optimizing the current human testicular cell culture system for clinical use.

Comparing genome-scale DNA methylation and CNV marks between adult human cultured ITGA6+ testicular cells and seminomas to assess in vitro genomic stability.

Autologous transplantation of spermatogonial stem cells is a promising new avenue to restore fertility in infertile recipients. Expansion of the initial spermatogonial stem cell pool through cell culturing is a necessary step to obtain enough cells for effective repopulation of the testis after transplantation. Since in vitro propagation can lead to (epi-)genetic mutations and possibly malignant transformation of the starting cell population, we set out to investigate genome-wide DNA methylation status in uncultured and cultured primary testicular ITGA6+ sorted cells and compare them with germ cell tumor samples of the seminoma subtype. Seminomas displayed a severely global hypomethylated profile, including loss of genomic imprinting, which we did not detect in cultured primary testicular ITGA6+ cells. Differential methylation analysis revealed altered regulation of gamete formation and meiotic processes in cultured primary testicular ITGA6+ cells but not in seminomas. The pivotal POU5F1 marker was hypomethylated in seminomas but not in uncultured or cultured primary testicular ITGA6+ cells, which is reflected in the POU5F1 mRNA expression levels. Lastly, seminomas displayed a number of characteristic copy number variations that were not detectable in primary testicular ITGA6+ cells, either before or after culture. Together, the data show a distinct DNA methylation patterns in cultured primary testicular ITGA6+ cells that does not resemble the pattern found in seminomas, but also highlight the need for more sensitive methods to fully exclude the presence of malignant cells after culture and to further study the epigenetic events that take place during in vitro culture.

Comparison of DNA methylation patterns of parentally imprinted genes in placenta derived from IVF conceptions in two different culture media.

STUDY QUESTION: Is there a difference in DNA methylation status of imprinted genes in placentas derived from IVF conceptions where embryo culture was performed in human tubal fluid (HTF) versus G5 culture medium? SUMMARY ANSWER: We found no statistically significant differences in the mean DNA methylation status of differentially methylated regions (DMRs) associated with parentally imprinted genes in placentas derived from IVF conceptions cultured in HTF versus G5 culture medium. WHAT IS KNOWN ALREADY: Animal studies indicate that the embryo culture environment affects the DNA methylation status of the embryo. In humans, birthweight is known to be affected by the type of embryo culture medium used. The effect of embryo culture media on pregnancy, birth and child development may thus be mediated by differential methylation of parentally imprinted genes in the placenta. STUDY DESIGN, SIZE, DURATION: To identify differential DNA methylation of imprinted genes in human placenta derived from IVF conceptions exposed to HTF or G5 embryo culture medium, placenta samples (n = 43 for HTF, n = 54 for G5) were collected between 2010 and 2012 s as part of a multi-center randomized controlled trial in the Netherlands comparing these embryo culture media. Placenta samples from 69 naturally conceived (NC) live births were collected during 2008-2013 in the Netherlands as reference material. PARTICIPANTS/MATERIALS, SETTING, METHODS: To identify differential DNA methylation of imprinted genes, we opted for an amplicon-based sequencing strategy on an Illumina MiSeq sequencing platform. DNA was isolated and 34 DMRs associated with well-defined parentally imprinted genes were amplified in a two-step PCR before sequencing using MiSeq technology. Sequencing data were analyzed in a multivariate fashion to eliminate possible confounding effects. MAIN RESULTS AND THE ROLE OF CHANCE: We found no statistically significant differences in the mean DNA methylation status of any of the imprinted DMRs in placentas derived from IVF conceptions cultured in HTF or G5 culture medium. We also did not observe any differences in the mean methylation status per amplicon nor in the variance in methylation per amplicon between the two culture medium.groups. A separate surrogate variable analysis also demonstrated that the IVF culture medium was not associated with the DNA methylation status of these DMRs. The mean methylation level and variance per CpG was equal between HTF and G5 placenta. Additional comparison of DNA methylation status of NC placenta samples revealed no statistically significant differences in mean amplicon and CpG methylation between G5, HTF and NC placenta; however, the number of placenta samples exhibiting outlier methylation levels was higher in IVF placenta compared to NC (P < 0.00001). Also, we were able to identify 37 CpG sites that uniquely displayed outlier methylation in G5 placentas and 32 CpG sites that uniquely displayed outlier methylation in HTF. In 8/37 (G5) and 4/32 (HTF) unique outliers CpGs, a medium-specific unique outlier could be directly correlated to outlier methylation of the entire amplicon. LIMITATIONS, REASONS FOR CAUTION: Due to practical reasons, not all placentas were collected during the trial, and we collected the placentas from natural conceptions from a different cohort, potentially creating bias. We limited ourselves to the DNA methylation status of 34 imprinted DMRs, and we studied only the placenta and no other embryo-derived tissues. WIDER IMPLICATIONS OF THE FINDINGS: It has often been postulated, but has yet to be rigorously tested, that imprinting mediates the effects of embryo culture conditions on pregnancy, birth and child development in humans. Since we did not detect any statistically significant effects of embryo culture conditions on methylation status of imprinted genes in the placenta, this suggests that other unexplored mechanisms may underlie these effects. The biological and clinical relevance of detected outliers with respect to methylation levels of CpGs and DMR require additional analysis in a larger sample size as well. Given the importance and the growing number of children born through IVF, research into these molecular mechanisms is urgently needed. STUDY FUNDING/COMPETING INTEREST(S): This study was funded by the March of Dimes grant number #6-FY13-153. The authors have no conflicts of interest. TRIAL REGISTRATION NUMBER: Placental biopsies were obtained under Netherlands Trial Registry number 1979 and 1298.

Spermatogonial stem cell autotransplantation and germline genomic editing: a future cure for spermatogenic failure and prevention of transmission of genomic diseases.

BACKGROUND: Subfertility affects approximately 15% of all couples, and a severe male factor is identified in 17% of these couples. While the etiology of a severe male factor remains largely unknown, prior gonadotoxic treatment and genomic aberrations have been associated with this type of subfertility. Couples with a severe male factor can resort to ICSI, with either ejaculated spermatozoa (in case of oligozoospermia) or surgically retrieved testicular spermatozoa (in case of azoospermia) to generate their own biological children. Currently there is no direct treatment for azoospermia or oligozoospermia. Spermatogonial stem cell (SSC) autotransplantation (SSCT) is a promising novel clinical application currently under development to restore fertility in sterile childhood cancer survivors. Meanwhile, recent advances in genomic editing, especially the clustered regulatory interspaced short palindromic repeats-associated protein 9 (CRISPR-Cas9) system, are likely to enable genomic rectification of human SSCs in the near future. OBJECTIVE AND RATIONALE: The objective of this review is to provide insights into the prospects of the potential clinical application of SSCT with or without genomic editing to cure spermatogenic failure and to prevent transmission of genetic diseases. SEARCH METHODS: We performed a narrative review using the literature available on PubMed not restricted to any publishing year on topics of subfertility, fertility treatments, (molecular regulation of) spermatogenesis and SSCT, inherited (genetic) disorders, prenatal screening methods, genomic editing and germline editing. For germline editing, we focussed on the novel CRISPR-Cas9 system. We included papers written in English only. OUTCOMES: Current techniques allow propagation of human SSCs in vitro, which is indispensable to successful transplantation. This technique is currently being developed in a preclinical setting for childhood cancer survivors who have stored a testis biopsy prior to cancer treatment. Similarly, SSCT could be used to restore fertility in sterile adult cancer survivors. In vitro propagation of SSCs might also be employed to enhance spermatogenesis in oligozoospermic men and in azoospermic men who still have functional SSCs albeit in insufficient numbers. The combination of SSCT with genomic editing techniques could potentially rectify defects in spermatogenesis caused by genomic mutations or, more broadly, prevent transmission of genomic diseases to the offspring. In spite of the promising prospects, SSCT and germline genomic editing are not yet clinically applicable and both techniques require optimization at various levels. WIDER IMPLICATIONS: SSCT with or without genomic editing could potentially be used to restore fertility in cancer survivors to treat couples with a severe male factor and to prevent the paternal transmission of diseases. This will potentially allow these couples to have their own biological children. Technical development is progressing rapidly, and ethical reflection and societal debate on the use of SSCT with or without genomic editing is pressing.

The histone H4 lysine 16 acetyltransferase hMOF regulates the outcome of autophagy.

Autophagy is an evolutionarily conserved catabolic process involved in several physiological and pathological processes. Although primarily cytoprotective, autophagy can also contribute to cell death; it is thus important to understand what distinguishes the life or death decision in autophagic cells. Here we report that induction of autophagy is coupled to reduction of histone H4 lysine 16 acetylation (H4K16ac) through downregulation of the histone acetyltransferase hMOF (also called KAT8 or MYST1), and demonstrate that this histone modification regulates the outcome of autophagy. At a genome-wide level, we find that H4K16 deacetylation is associated predominantly with the downregulation of autophagy-related genes. Antagonizing H4K16ac downregulation upon autophagy induction results in the promotion of cell death. Our findings establish that alteration in a specific histone post-translational modification during autophagy affects the transcriptional regulation of autophagy-related genes and initiates a regulatory feedback loop, which serves as a key determinant of survival versus death responses upon autophagy induction.

Restoring fertility in sterile childhood cancer survivors by autotransplanting spermatogonial stem cells: are we there yet?

Current cancer treatment regimens do not only target tumor cells, but can also have devastating effects on the spermatogonial stem cell pool, resulting in a lack of functional gametes and hence sterility. In adult men, fertility can be preserved prior to cancer treatment by cryopreservation of ejaculated or surgically retrieved spermatozoa, but this is not an option for prepubertal boys since spermatogenesis does not commence until puberty. Cryopreservation of a testicular biopsy taken before initiation of cancer treatment, followed by in vitro propagation of spermatogonial stem cells and subsequent autotransplantation of these stem cells after cancer treatment, has been suggested as a way to preserve and restore fertility in childhood cancer survivors. This strategy, known as spermatogonial stem cell transplantation, has been successful in mice and other model systems, but has not yet been applied in humans. Although recent progress has brought clinical application of spermatogonial stem cell autotransplantation in closer range, there are still a number of important issues to address. In this paper, we describe the state of the art of spermatogonial stem cell transplantation and outline the hurdles that need to be overcome before clinical implementation.