Sperm DNA methylation is predominantly stable in mice offspring born after transplantation of long-term cultured spermatogonial stem cells.

BACKGROUND: Spermatogonial stem cell transplantation (SSCT) is proposed as a fertility therapy for childhood cancer survivors. SSCT starts with cryopreserving a testicular biopsy prior to gonadotoxic treatments such as cancer treatments. When the childhood cancer survivor reaches adulthood and desires biological children, the biopsy is thawed and SSCs are propagated in vitro and subsequently auto-transplanted back into their testis. However, culturing stress during long-term propagation can result in epigenetic changes in the SSCs, such as DNA methylation alterations, and might be inherited by future generations born after SSCT. Therefore, SSCT requires a detailed preclinical epigenetic assessment of the derived offspring before this novel cell therapy is clinically implemented. With this aim, the DNA methylation status of sperm from SSCT-derived offspring, with in vitro propagated SSCs, was investigated in a multi-generational mouse model using reduced-representation bisulfite sequencing. RESULTS: Although there were some methylation differences, they represent less than 0.5% of the total CpGs and methylated regions, in all generations. Unsupervised clustering of all samples showed no distinct grouping based on their pattern of methylation differences. After selecting the few single genes that are significantly altered in multiple generations of SSCT offspring compared to control, we validated the results with quantitative Bisulfite Sanger sequencing and RT-qPCRin various organs. Differential methylation was confirmed only for Tal2, being hypomethylated in sperm of SSCT offspring and presenting higher gene expression in ovaries of SSCT F1 offspring compared to control F1. CONCLUSIONS: We found no major differences in DNA methylation between SSCT-derived offspring and control, both in F1 and F2 sperm. The reassuring outcomes from our study are a prerequisite for promising translation of SSCT to the human situation.

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.

A comparative analysis of human adult testicular cells expressing stem Leydig cell markers in the interstitium, vasculature, and peritubular layer.

BACKGROUND: Origin of human adult Leydig cells (ALCs) is not well understood. This might be partly due to limited data available on the identification and location of human precursor and stem Leydig cells (SLCs) which hampers the study on the development of ALCs. OBJECTIVES: The aim of the present study was to investigate whether described human (PDGFRα, NGFR) and rodent (NES, PDGFRα, THY1, NR2F2) SLC markers are expressed by a common cell population within human adult testicular interstitial cells in vivo and before and after in vitro propagation. MATERIALS AND METHODS: Immunohistochemical analyses were used to identify localization of human adult testicular interstitial cells expressing described SLC markers. Next, interstitial cells were isolated and cultured. The percentage of cells expressing one or more SLC markers was determined before and after culture using flow cytometry. RESULTS: NR2F2 and PDGFRα were present in peritubular, perivascular, and Leydig cells, while THY1 was expressed in peritubular and perivascular cells. Although NES and NGFR were expressed in endothelial cells, co-localization with PDGFRα was found for both in vitro, although for NGFR only after culture. All marker positive cells were able to undergo propagation in vitro. DISCUSSION: The partly overlap in localization and overlap in expression in human testicular cells indicate that PDGFRα, NR2F2, and THY1 are expressed within the same ALC developmental lineage from SLCs. Based on the in vitro results, this is also true for NES and after in vitro propagation for NGFR. CONCLUSION: Our results that earlier described SLC markers are expressed in overlapping human interstitial cell population opens up further research strategies aiming for a better insight in the Leydig cell lineage and will be helpful for development of strategies to cure ALC dysfunction.

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.

Strains matter: Success of murine in vitro spermatogenesis is dependent on genetic background.

The current strategy to preserve fertility of male prepubertal cancer patients consists of cryopreservation of a testicular tissue biopsy containing spermatogonial stem cells (SSCs). While in humans, fertility restoration strategies from prepubertal testicular tissues are still under investigation and have not yet resulted in complete germ cell differentiation, in mice various studies have described production of sperm and offspring through testicular organ culture and transplantation of in vitro propagated SSCs. Organ culture has shown to be successful in generating mature spermatozoa when using testicular fragments from various mouse strains, including CD1 and C57BL/6 J. Conversely, in vitro proliferation of SSCs from C57BL/6 J mice is highly inefficient when compared to other strains such as DBA2 or hybrid mice of C57BL/6 J and DBA2 with 75% C57BL/6 J background (B6D2F2). In this study, we investigated in vitro spermatogenesis by organ culture using testicular tissue from C57BL/6 J and B6D2F2 mice. Whereas spermatogenesis was initiated and completed in C57BL/6 J fragments, it could not be effectively supported in B6D2F2 testicular tissue. While maturation of Sertoli cells and Leydig cells functionality appeared to be identical between the two strains, in B6D2F2 tissue spermatogenesis did not proceed past the spermatocyte step, followed by a rapid decline of the number of all germ cells in the fragments. This suggests that the spermatogenic potential in vitro is dependent on specialized sites in the genome and therefore the organ culture conditions suboptimal for some strains of mice.

Long-term health in recipients of transplanted in vitro propagated spermatogonial stem cells.

STUDY QUESTION: Is testicular transplantation of in vitro propagated spermatogonial stem cells associated with increased cancer incidence and decreased survival rates in recipient mice? SUMMARY ANSWER: Cancer incidence was not increased and long-term survival rate was not altered after transplantation of in vitro propagated murine spermatogonial stem cells (SSCs) in busulfan-treated recipients as compared to non-transplanted busulfan-treated controls. WHAT IS KNOWN ALREADY: Spermatogonial stem cell autotransplantation (SSCT) is a promising experimental reproductive technique currently under development to restore fertility in male childhood cancer survivors. Most preclinical studies have focused on the proof-of-principle of the functionality and efficiency of this technique. The long-term health of recipients of SSCT has not been studied systematically. STUDY DESIGN, SIZE, DURATION: This study was designed as a murine equivalent of a clinical prospective study design. Long-term follow-up was performed for mice who received a busulfan treatment followed by either an intratesticular transplantation of in vitro propagated enhanced green fluorescent protein (eGFP) positive SSCs (cases, n = 34) or no transplantation (control, n = 37). Using a power calculation, we estimated that 36 animals per group would be sufficient to provide an 80% power and with a 5% level of significance to demonstrate a 25% increase in cancer incidence in the transplanted group. The survival rate and cancer incidence was investigated until the age of 18 months. PARTICIPANTS/MATERIALS, SETTING, METHODS: Neonatal male B6D2F1 actin-eGFP transgenic mouse testis were used to initiate eGFP positive germline stem (GS) cell culture, which harbor SSCs. Six-week old male C57BL/6 J mice received a single dose busulfan treatment to deplete the testis from endogenous spermatogenesis. Half of these mice received a testicular transplantation of cultured eGFP positive GS cells, while the remainder of mice served as a control group. Mice were followed up until the age of 18 months (497-517 days post-busulfan) or sacrificed earlier due to severe discomfort or illness. Survival data were collected. To evaluate cancer incidence a necropsy was performed and tissues were collected. eGFP signal in transplanted testis and in benign and malignant lesions was assessed by standard PCR. MAIN RESULTS AND THE ROLE OF CHANCE: We found 9% (95% CI: 2-25%) malignancies in the transplanted busulfan-treated animals compared to 26% (95% CI: 14-45%) in the busulfan-treated control group, indicating no statistically significant difference in incidence of malignant lesions in transplanted and control mice (OR: 0.3, 95% CI: 0.1-1.1). Furthermore, none of the malignancies that arose in the transplanted animals contained eGFP signal, suggesting that they are not derived from the in vitro propagated transplanted SSCs. Mean survival time after busulfan treatment was found to be equal, with a mean survival time for transplanted animals of 478 days and 437 days for control animals (P = 0.076). LARGE SCALE DATA: NA. LIMITATIONS, REASONS FOR CAUTION: Although we attempted to mimic the future clinical application of SSCT in humans as close as possible, the mouse model that we used might not reflect all aspects of the future clinical setting. WIDER IMPLICATIONS OF THE FINDINGS: The absence of an increase in cancer incidence and a decrease in survival of mice that received a testicular transplantation of in vitro propagated SSCs is reassuring in light of the future clinical application of SSCT in humans. STUDY FUNDING/COMPETING INTEREST(S): This study was funded by KiKa (Kika86) and ZonMw (TAS 116003002). The authors report no financial or other conflict of interest relevant to the subject of this article.