Analysis of copy number variation in men with non-obstructive azoospermia.

BACKGROUND: Recent findings demonstrate that single nucleotide variants can cause non-obstructive azoospermia (NOA). In contrast, copy number variants (CNVs) were only analysed in few studies in infertile men. Some have reported a higher prevalence of CNVs in infertile versus fertile men. OBJECTIVES: This study aimed to elucidate if CNVs are associated with NOA. MATERIALS AND METHODS: We performed array-based comparative genomic hybridisation (aCGH) in 37 men with meiotic arrest, 194 men with Sertoli cell-only phenotype, and 21 control men. We filtered our data for deletions affecting genes and prioritised the affected genes according to the literature search. Prevalence of CNVs was compared between all groups. Exome data of 2,030 men were screened to detect further genetic variants in prioritised genes. Modelling was performed for the protein encoded by the novel candidate gene TEKT5 and we stained for TEKT5 in human testicular tissue. RESULTS: We determined the cause of infertility in two individuals with homozygous deletions of SYCE1 and in one individual with a heterozygous deletion of SYCE1 combined with a likely pathogenic missense variant on the second allele. We detected heterozygous deletions affecting MLH3, EIF2B2, SLX4, CLPP and TEKT5, in one subject each. CNVs were not detected more frequently in infertile men compared with controls. DISCUSSION: While SYCE1 and MLH3 encode known meiosis-specific proteins, much less is known about the proteins encoded by the other identified candidate genes, warranting further analyses. We were able to identify the cause of infertility in one out of the 231 infertile men by aCGH and in two men by using exome sequencing data. CONCLUSION: As aCGH and exome sequencing are both expensive methods, combining both in a clinical routine is not an effective strategy. Instead, using CNV calling from exome data has recently become more precise, potentially making aCGH dispensable.

Serum concentrations of dihydrotestosterone are associated with symptoms of hypogonadism in biochemically eugonadal men.

PURPOSE: Symptoms of hypogonadism are often reported by subjects with normal serum testosterone (T) levels. We aimed to assess the association between clinical symptoms in andrological outpatients and sex steroids levels. METHODS: This is a retrospective cross-sectional cohort study in an Academic clinic and research unit. International Index of Erectile Function (IIEF, EF domain) and Aging Males Symptoms scale (AMS) questionnaires were completed by 635 and 574 men, respectively (mean age: 47.3 ± 13.9 and 47.4 ± 13.8 years, p = 0.829), free of interfering medications with complaints possibly related to hypogonadism. RESULTS: Serum total/free T as well as dihydro-T (DHT) was associated with IIEF-EF and AMS scores in the overall population using univariate analyses. Multivariate approaches revealed DHT concentrations in subjects with normal T levels (n = 416, Total T > 12 nmol/L) to be significant predictors of AMS scores. A 0.1 nmol/l serum DHT increase within the eugonadal range was associated with a 4.67% decrease in odds of having worse symptoms (p = 0.011). In men with biochemical hypogonadism (Total T < 12 nmol/L), total and free T rather than DHT were associated with AMS results. This association was not found for IIEF-EF scores. Indirect effects of age and BMI were seen for relations with hormone concentrations but not questionnaire scores. CONCLUSION: DHT can be associated with symptoms of hypogonadism in biochemically eugonadal men. Serum DHT measurement might be helpful once the diagnosis of hypogonadism has been ruled out but should not be routinely included in the primary diagnostic process.

A novel xeno-organoid approach: exploring the crosstalk between human iPSC-derived PGC-like and rat testicular cells.

Specification of germ cell-like cells from induced pluripotent stem cells has become a clinically relevant tool for research. Research on initial embryonic processes is often limited by the access to foetal tissue, and in humans, the molecular events resulting in primordial germ cell (PGC) specification and sex determination remain to be elucidated. A deeper understanding of the underlying processes is crucial to describe pathomechanisms leading to impaired reproductive function. Several protocols have been established for the specification of human pluripotent stem cell towards early PGC-like cells (PGCLC), currently representing the best model to mimic early human germline developmental processes in vitro. Further sex determination towards the male lineage depends on somatic gonadal cells providing the necessary molecular cues. By establishing a culture system characterized by the re-organization of somatic cells from postnatal rat testes into cord-like structures and optimizing efficient PGCLC specification protocols, we facilitated the co-culture of human germ cell-like cells within a surrogate testicular microenvironment. Specified conditions allowed the survival of rat somatic testicular and human PGCLCs for 14 days. Human cells maintained the characteristic expression of octamer-binding transcription factor 4, SRY-box transcription factor 17, and transcription factor AP-2 gamma and were recovered from the xeno-organoids by cell sorting. This novel xeno-organoid approach will allow the in vitro exploration of early sex determination of human PGCLCs.

Challenging human somatic testicular cell reassembly by protein kinase inhibition -setting up a functional in vitro test system.

Signalling pathways and cellular interactions defining initial processes of testis morphogenesis, i.e. cord formation, are poorly understood. In vitro cell-based systems modelling cord formation can be utilised as platforms to interrogate processes of tubulogenesis. We aimed at testing our established cord formation in vitro model using adult human testicular cells as a quantitative assay that can facilitate future studies on cord morphogenesis. We challenged the responsiveness of our system with a broad-spectrum protein kinase inhibitor, K252a. Cultured testicular cells were treated with various K252a concentrations under constant exposure and compound withdrawal. To quantify cell reaggregation changes, we performed computer-assisted phase-contrast image analysis of aggregate size and number. Cell reaggregation was analysed in detail by categorisation of aggregates into size groups and accounting for changes in aggregate number per size category. We found a dose-related disturbance of testicular cell reaggregation. K252a decreased aggregate size (IC50 of 203.3 nM) and reduced the large aggregate numbers. Video recordings revealed that treatment with K252a at a concentration above IC50 interfered with aggregate coalescence into cords. Short-term exposure and compound wash-out induced irreversible decrease in large aggregates. We propose our in vitro model as a functional platform to quantitatively investigate seminiferous tubulogenesis under pharmacological impact.

The initial maturation status of marmoset testicular tissues has an impact on germ cell maintenance and somatic cell response in tissue fragment culture.

Successful in vitro spermatogenesis was reported using immature mouse testicular tissues in a fragment culture approach, raising hopes that this method could also be applied for fertility preservation in humans. Although maintaining immature human testicular tissue fragments in culture is feasible for an extended period, it remains unknown whether germ cell survival and the somatic cell response depend on the differentiation status of tissue. Employing the marmoset monkey (Callithrix jacchus), we aimed to assess whether the maturation status of prepubertal and peri-/pubertal testicular tissues influence the outcome of testis fragment culture. Testicular tissue fragments from 4- and 8-month-old (n = 3, each) marmosets were cultured and evaluated after 0, 7, 14, 28 and 42 days. Immunohistochemistry was performed for identification and quantification of germ cells (melanoma-associated antigen 4) and Sertoli cell maturation status (anti-Müllerian hormone: AMH). During testis fragment culture, spermatogonial numbers were significantly reduced (P < 0.05) in the 4- but not 8-month-old monkeys, at Day 0 versus Day 42 of culture. Moreover, while Sertoli cells from 4-month-old monkeys maintained an immature phenotype (i.e. AMH expression) during culture, AMH expression was regained in two of the 8-month-old monkeys. Interestingly, progression of differentiation to later meiotic stage was solely observed in one 8-month-old marmoset, which was at an intermediate state regarding germ cell content, with gonocytes as well as spermatocytes present, as well as Sertoli cell maturation status. Although species-specific differences might influence the outcome of testis fragment experiments in vitro, our study demonstrated that the developmental status of the testicular tissues needs to be considered as it seems to be decisive for germ cell maintenance, somatic cell response and possibly the differentiation potential.

Immunohistochemical characterization of the anti-Müllerian hormone receptor type 2 (AMHR-2) in human testes.

PURPOSE: In males, AMH is secreted by immature Sertoli cells; following exposure to endogenous androgens, Sertoli cells undergo a process of maturation which ultimately inhibits AMH expression to undetectable levels in the serum. However, expression of AMH receptor (AMHR-2) has never been studied in human testes, and high intratubular concentrations of AMH have been reported in recent literature. We therefore assessed expression of AMHR-2 in several testicular tissue samples by immunohistochemistry (IHC). METHODS: The IHC method was first validated on tissue samples from healthy human testis (n = 2) and from marmoset ovary (n = 1). The same method was then used for assessment on testicular histopathology specimens from patients with mixed atrophy (MA, n = 2), spermatogenetic arrest (SA, n = 2), Sertoli cell-only syndrome (SCO, n = 1), Klinefelter syndrome (KS, n = 1), and nonseminomatous germ cell tumors (NSGCT, n = 1). Tissue samples from two subjects at different pubertal stages (AndroProtect (AP), aged 5 and 14 years) with hematological malignancies were also retrieved. RESULTS: In adult men, AMHR-2 was expressed on peritubular mesenchymal cells, with patterns closely mirroring α-smooth muscle actin expression. Similar patterns were preserved in almost all conditions; however, in nonseminomatous germ cell tumors the tissue architecture was lost, including AMHR-2 expression. More positive and diffuse staining was observed in tissue samples from prepubertal testes. CONCLUSIONS: In specimens from both healthy and affected testes, AMHR-2 expression appears weaker in adult than in prepubertal tissue sections. The persistence of AMHR-2 expression seemingly hints at a possible effect of intratesticular AMH on the tubular walls.

Differences in blastomere totipotency in 2-cell mouse embryos are a maternal trait mediated by asymmetric mRNA distribution.

It is widely held that the first two blastomeres of mammalian embryos are equally totipotent and that this totipotency belongs to the group of regulative properties. However, this interpretation neglects an important aspect: evidence only came from successful monozygotic twins which can speak only for those pairs of half-embryos that are able to regulate in the first place. Are the frequently occurring incomplete pairs simply an artefact, or do they represent a real difference, be it in the imperfect blastomere's ability to regulate growth or in the distribution of any compound X that constrains regulation? Using the model system of mouse embryos bisected at the 2-cell stage after fertilization, we present evidence that the interblastomere differences evade regulation by external factors and are already latent in oocytes. Specifically, an interblastomere imbalance of epiblast production persists under the most diverse culture conditions and applies to the same extent in parthenogenetic counterparts. As a result, cases in which twin blastocysts continued to develop in only one member account for 65 and 57% of zygotic and parthenogenetic pairs, respectively. The interblastomere imbalance is related to the subcellular distribution of gene products, as documented for the epiblast-related gene Cops3, using mRNA FISH in super-resolution mode confocal microscopy. Blastomere patterns of Cops3 mRNA distribution are α-amanitin-resistant. Thus, the imbalance originates not from de novo transcription, but from influences which are effective before fertilisation. These data expose previously unrecognized limits of regulative capacities of 2-cell stage blastomeres and point to aspects of cytoplasmic organization of the mouse oocyte that segregate unequally to blastomeres during cleavage.

Ageing in men with normal spermatogenesis alters spermatogonial dynamics and nuclear morphology in Sertoli cells.

BACKGROUND: Ageing in men is believed to be associated with fertility decline and elevated risk of congenital disorders for the offspring. The previous studies also reported reduced germ and Sertoli cell numbers in older men. However, it is not clear whether ageing in men with normal spermatogenesis affects the testis and germ cell population dynamics in a way sufficient for transmitting adverse age effects to the offspring. OBJECTIVES: We examined men with normal spermatogenesis at different ages concerning effects on persisting testicular cell types, that is the germ line and Sertoli cells, as these cell populations are prone to be exposed to age effects. MATERIAL AND METHODS: Ageing was assessed in testicular biopsies of 32 patients assigned to three age groups: (i) 28.8 ± 2.7 years; (ii) 48.1 ± 1 years; and (iii) 70.9 ± 6.2 years, n = 8 each, with normal spermatogenesis according to the Bergmann-Kliesch score, and in a group of meiotic arrest patients (29.9 ± 3.8 years, n = 8) to decipher potential links between different germ cell types. Besides morphometry of seminiferous tubules and Sertoli cell nuclei, we investigated spermatogenic output/efficiency, and dynamics of spermatogonial populations via immunohistochemistry for MAGE A4, PCNA, CREM and quantified A-pale/A-dark spermatogonia. RESULTS: We found a constant spermatogenic output (CREM-positive round spermatids) in all age groups studied. In men beyond their mid-40s (group 2), we detected increased nuclear and nucleolar size in Sertoli cells, indirectly indicating an elevated protein turnover. From the 7th decade (group 3) of life onwards, testes showed increased proliferation of undifferentiated spermatogonia, decreased spermatogenic efficiency and elevated numbers of proliferating A-dark spermatogonia. DISCUSSION AND CONCLUSION: Maintaining normal sperm output seems to be an intrinsic determinant of spermatogenesis. Ageing appears to affect this output and might provoke compensatory proliferation increase in A spermatogonia which, in turn, might hamper germ cell integrity.

Complete spermatogenesis in intratesticular testis tissue xenotransplants from immature non-human primate.

STUDY QUESTION: Can full spermatogenesis be achieved after xenotransplantation of prepubertal primate testis tissue to the mouse, in testis or subcutaneously? SUMMARY ANSWER: Intratesticular xenotransplantation supported the differentiation of immature germ cells from marmoset (Callithrix jacchus) into spermatids and spermatozoa at 4 and 9 months post-transplantation, while in subcutaneous transplants, spermatogenic arrest was observed at 4 months and none of the transplants survived at 9 months. WHAT IS KNOWN ALREADY: Auto-transplantation of cryopreserved immature testis tissue (ITT) could be a potential fertility restoration strategy for patients with complete loss of germ cells due to chemo- and/or radiotherapy at a young age. Before ITT transplantation can be used for clinical application, it is a prerequisite to demonstrate the feasibility of the technique and identify the conditions required for establishing spermatogenesis in primate ITT transplants. Although xenotransplantation of ITT from several species has resulted in complete spermatogenesis, in human and marmoset, ITT has not been successful. STUDY DESIGN, SIZE, DURATION: In this study, we used marmoset as a pre-clinical animal model. ITT was obtained from two 6-month-old co-twin marmosets. A total of 147 testis tissue pieces (~0.8-1.0 mm3 each) were transplanted into the testicular parenchyma (intratesticular; n = 40) or under the dorsal skin (ectopic; n = 107) of 4-week-old immunodeficient Swiss Nu/Nu mice (n = 20). Each mouse received one single marmoset testis tissue piece in each testis and 4-6 pieces subcutaneously. Xenotransplants were retrieved at 4 and 9 months post-transplantation and evaluations were performed with regards to transplant survival, spermatogonial quantity and germ cell differentiation. PARTICIPANTS/MATERIALS, SETTING, METHODS: Transplant survival was histologically evaluated by haematoxylin-periodic acid Schiff (H/PAS) staining. Spermatogonia were identified by MAGE-A4 via immunohistochemistry. Germ cell differentiation was assessed by morphological identification of different germ cell types on H/PAS stained sections. Meiotically active germ cells were identified by BOLL expression. CREM immunohistochemistry was performed to confirm the presence of post-meiotic germ cells and ACROSIN was used to determine the presence of round, elongating and elongated spermatids. MAIN RESULTS AND THE ROLE OF CHANCE: Four months post-transplantation, 50% of the intratesticular transplants and 21% of the ectopic transplants were recovered (P = 0.019). The number of spermatogonia per tubule did not show any variation. In 33% of the recovered intratesticular transplants, complete spermatogenesis was established. Overall, 78% of the intratesticular transplants showed post-meiotic differentiation (round spermatids, elongating/elongated spermatids and spermatozoa). However, during the same period, spermatocytes (early meiotic germ cells) were the most advanced germ cell type present in the ectopic transplants. Nine months post-transplantation, 50% of the intratesticular transplants survived, whilst none of the ectopic transplants was recovered (P < 0.0001). Transplants contained more spermatogonia per tubule (P = 0.018) than at 4 months. Complete spermatogenesis was observed in all recovered transplants (100%), indicating a progressive spermatogenic development in intratesticular transplants between the two time-points. Nine months post-transplantation, transplants contained more seminiferous tubules with post-meiotic germ cells (37 vs. 5%; P < 0.001) and fewer tubules without germ cells (2 vs. 8%; P = 0.014) compared to 4 months post-transplantation. LARGE SCALE DATA: N/A. LIMITATIONS, REASONS FOR CAUTION: Although xenotransplantation of marmoset ITT was successful, it does not fully reflect all aspects of a future clinical setting. Furthermore, due to ethical restrictions, we were not able to prove the functionality of the spermatozoa produced in the marmoset transplants. WIDER IMPLICATIONS OF THE FINDINGS: In this pre-clinical study, we demonstrated that testicular parenchyma provides the required microenvironment for germ cell differentiation and long-term survival of immature marmoset testis tissue, likely due to the favourable temperature regulation, growth factors and hormonal support. These results encourage the design of new experiments on human ITT xenotransplantation and show that intratesticular transplantation is likely to be superior to ectopic transplantation for fertility restoration following gonadotoxic treatment in childhood. STUDY FUNDING/COMPETING INTEREST(S): This project was funded by the ITN Marie Curie Programme 'Growsperm' (EU-FP7-PEOPLE-2013-ITN 603568) and the scientific Fund Willy Gepts from the UZ Brussel (ADSI677). D.V.S. is a post-doctoral fellow of the Fonds Wetenschappelijk Onderzoek (FWO; 12M2815N). No conflict of interest is declared.

AMH and INSL3 in testicular and extragonadal pathophysiology: what do we know?

BACKGROUND: It is commonly accepted that testicular function is prevalently regulated by the hypothalamic-pituitary-gonadal axis: The pulsatile secretion of GnRH by the hypothalamus induces pituitary expression of the two gonadotropins FSH and LH, which then stimulate Sertoli and Leydig cells, respectively, therefore regulating steroidogenesis and spermatogenesis. However, a growing body of evidence has recently suggested that other hormones act on the reproductive tract since the early phases of fetal development. Anti-Müllerian hormone and INSL3 are still largely used only for research purposes despite being increasingly recognized as markers of Sertoli and Leydig cells function, respectively. OBJECTIVES: Provide an up-to-date review of the role of anti-Müllerian hormone and INSL3 in human pathophysiology according to current evidence. MATERIALS AND METHODS: A thorough literature review was performed on PubMed, OVID MEDLINE/EMBASE and Google Scholar for papers concerning anti-Müllerian hormone and INSL3 in human males. RESULTS: INSL3 is not acutely regulated by the hypothalamic-pituitary axis but is constitutively secreted by Leydig cells, therefore representing a valid marker for their number and status. Anti-Müllerian hormone expression, on the other hand, is downregulated by androgens, therefore occurring mostly at the early stages of testicular differentiation and before the onset of puberty. Several conditions affecting testicular development, such as male hypogonadotropic hypogonadism, and their treatment have been associated to specific pattern of INSL3 and anti-Müllerian hormone expression, proving a role for both hormones in the diagnostic and therapeutic management. Recent reports suggest a role for both anti-Müllerian hormone and INSL3 in extra gonadal physiology, such as cardiovascular and bone health. CONCLUSION: Anti-Müllerian hormone and INSL3 are markers of Sertoli and Leydig cells maturation, respectively, usually involved in the pathogenesis of disorders of sexual differentiation. However, their role in testicular pathology has only been hinted at in the last decades. Recent evidence supports an involvement of both anti-Müllerian hormone and INSL3 in extragonadal pathophysiology as well.

Topographic protein profiling of the age-related proteome in the retinal pigment epithelium of Callithrix jacchus with respect to macular degeneration.

In the retinal pigment epithelium (RPE) several factors within the macular compared to peripheral regions cause differences in physiological aging. The molecular mechanisms during aging in the context of topography are not well known. The proteome of RPE of different aged macular-bearing primates Callithrix jacchus was thus analysed with ion mobility mass spectrometry. Macular and periphery of neonate RPE were well differentiated from aged tissues as demonstrated by principal component analysis. This finding was mainly due to proteins involved in major developmental processes and the visual cycle. The distinction of adult from senile tissue and macular from periphery was more subtle. The hypotheses of inflammation increasing with age was supported. High expression levels of proteins related to oxidative stress (e.g., cathepsin B) and chaperones (e.g., HSP90) were detected in aged RPE as confirmed by Western blot and immunohistochemical analysis. Decreased levels of proteins participating in angiostatic properties (e.g., thrombospondin 1) and the integrity of tissue basement membranes with age (e.g., nidogen 1) were in agreement with neovascularization. This study presents targets for further investigations of the mechanisms of the aging process with the aim to elucidate predictive factors for the conversion of physiological aging into pathological conditions. SIGNIFICANCE: The current study characterized the different protein profiles of the retinal pigment epithelium (RPE) of the macula-bearing, non-human primate Callithrix jacchus during life-time. In addition, the subproteomes of macular and peripheral RPE were investigated. Differently expressed proteins described developmental processes in neonate tissue and destructive mechanisms in aged samples. Insights into the physiological aging process of the RPE and its conversion into pathophysiological conditions were gained. They assist in designing therapeutical approaches to counteract age-related diseases of the retina.

Retrospective analysis: reproducibility of interblastomere differences of mRNA expression in 2-cell stage mouse embryos is remarkably poor due to combinatorial mechanisms of blastomere diversification.

STUDY QUESTION: What is the prevalence, reproducibility and biological significance of transcriptomic differences between sister blastomeres of the mouse 2-cell embryo? SUMMARY ANSWER: Sister 2-cell stage blastomeres are distinguishable from each other by mRNA analysis, attesting to the fact that differentiation starts mostly early in the mouse embryo; however, the interblastomere differences are poorly reproducible and invoke the combinatorial effects of known and new mechanisms of blastomere diversification. WHAT IS KNOWN ALREADY: Transcriptomic datasets for single blastomeres in mice have been available for years but have never been systematically analysed together, although such an analysis may shed light onto some unclarified topics of early mammalian development. Two unknowns that remain are at which stage embryonic blastomeres start to diversify from each other and what is the molecular origin of that difference. At the earliest postzygotic stage, the 2-cell stage, opinions differ regarding the answer to these questions; one group claims that the first zygotic division yields two equal blastomeres capable of forming a full organism (totipotency) and another group claims evidence for interblastomere differences reminiscent of the prepatterning found in embryos of lower taxa. Regarding the molecular origin of interblastomere differences, there are four prevalent models which invoke (1) oocyte anisotropy, (2) sperm entry point, (3) partition errors of the transcript pool and (4) asynchronous embryonic genome activation in the two blastomeres. STUDY DESIGN, SIZE, DURATION: Seven transcriptomic studies published between 2011 and 2017 were eligible for retrospective analysis, since both blastomeres of the mouse 2-cell embryo had been analysed individually regarding the original pair associations and since the datasets were made available in public repositories. Five of these studies, encompassing a total of 43 pairs of sister blastomeres, were selected for further analyses based on high interblastomere correlations of mRNA levels. A double cut-off was used to select mRNAs that had robust interblastomere differences both within and between embryos (hits). The hits of each study were compared and contrasted with the hits of the other studies using Venn diagrams. The hits shared by at least four of five studies were analysed further by bioinformatics. PARTICIPANTS/MATERIALS, SETTING, METHODS: PubMed was systematically examined for mRNA expression profiles of single 2-cell stage blastomeres in addition to publicly available microarray datasets (GEO, ArrayExpress). Based on the original normalizations, data from seven studies were screened for pairwise sample correlation at the gene level (Spearman), and the top five datasets with the highest correlation were subjected to hierarchical cluster analysis. Interblastomere differences of gene expression were expressed as a ratio of the higher to the lower mRNA level for each pair of blastomeres. A double cut-off was used to make the call of interblastomere difference, accepting genes with mRNA ratios above 2 when observed in at least 50% of the pairs, and discarding the other genes. The proportion of interblastomere differences common to at least four of the five datasets was calculated. Finally, the corresponding gene, pathway and enrichment analyses were performed utilizing PANTHER and GORILLA platforms. MAIN RESULTS AND THE ROLE OF CHANCE: An average of 17% of genes within the datasets are differently expressed between sister blastomeres, a proportion which falls to 1% when considering the differences that are common to at least four of the five studies. Housekeeping mRNAs were not included in the 17% and 1% gene lists, suggesting that the interblastomere differences do not occur simply by chance. The 1% of shared interblastomere differences comprise 100 genes, of which 35 are consistent with at least one of the four prevalent models of sister blastomere diversification. Bioinformatics analysis of the remaining 65 genes that are not consistent with the four models suggests that at least one more mechanism is at play, potentially related to the endomembrane system. Although there are many dimensions to the issue of reproducibility (biological, experimental, analytical), we consider that the sister blastomeres are poised to escape high interblastomere correlations of mRNA levels, because at least five sources of diversity superimpose on each other, accounting for at least 25 = 32 different states. As a result, interblastomere mRNA differences of a given 2-cell embryo are necessarily difficult to reproduce in another 2-cell embryo. LARGE SCALE DATA: Data were as provided by the original studies (GSE21688, GSE22182, GSE27396, GSE45719, GSE57249, E-MTAB-3321, GSE94050). LIMITATIONS, REASONS FOR CAUTION: The original studies present similarities (e.g. fertilization in vivo after ovarian stimulation) as well as differences (e.g. mouse strains, method and timing of blastomere separation). We identified robust mRNA differences between the sister blastomeres, but these differences are underestimated because our double cut-off method works with thresholds and affords more protection against false positives than false negatives. Regarding the false negatives, transcriptome analysis may have captured only part of the interblastomere differences due to: (1) the 2-fold cut-off not being sensitive enough to detect the remaining part of the interblastomere differences, (2) the detection limit of the transcriptomic methods not being sufficient, or (3) interblastomere differences being oblivious to transcriptomic identification because transcriptional changes are oscillatory or because differences are mediated non-transcriptionally or post-transcriptionally. Regarding the false positives, it seems unlikely that a difference was found just by chance for the same group of transcripts due to the same technical error, given that different laboratories produced the data. WIDER IMPLICATIONS OF THE FINDINGS: It is clear that the sister blastomeres are distinguishable from each other by mRNA analysis even at the 2-cell stage; however, efforts to identify large stable patterns may be in vain. This elicits thoughts about the wisdom of adding new transcriptomic datasets to the ones that already exist; if all transcriptomic datasets produced so far show a reproducibility of 1%, then any future study would probably face the same issue again. Possibly, a solid identification of the 'large stable pattern that should be there but was not found' requires an even larger dataset than the sum of the seven datasets considered here. Conversely, small stable patterns may be easier to identify, but their biological relevance is less obvious. Alternatively, interblastomere differences may not be mediated by nucleic acids but by other cellular components. STUDY FUNDING/COMPETING INTEREST(S): This study was supported by the Deutsche Forschungsgemeinschaft (grant DFG BO 2540-4-3 to M.B. and grant NO 413/3-3 to V.N.). The authors declare that they have no competing financial interests.

Raman micro-spectroscopy analysis of different sperm regions: a species comparison.

STUDY QUESTION: Is Raman micro-spectroscopy a valid approach to assess the biochemical hallmarks of sperm regions (head, midpiece and tail) in four different species? SUMMARY ANSWER: Non-invasive Raman micro-spectroscopy provides spectral patterns enabling the biochemical characterization of the three sperm regions in the four species, revealing however high similarities for each region among species. WHAT IS KNOWN ALREADY: Raman micro-spectroscopy has been described as an innovative method to assess sperm features having the potential to be used as a non-invasive selection tool. However, except for nuclear DNA, the identification and assignment of spectral bands in Raman-profiles to the different sperm regions is scarce and controversial. STUDY DESIGN SIZE, DURATION: Raman spectra from head, midpiece and tail of four different species were obtained. Sperm samples were collected and smeared on microscope slides. Air dried samples were subjected to Raman analysis using previously standardized procedures. PARTICIPANTS/MATERIALS, SETTING, METHODS: Sperm samples from (i) two donors attending the infertility clinic at the Centre of Reproductive Medicine and Andrology; (ii) two C57BL/6 -TgN (ACTbEGFP) 1Osb adult mice; (iii) two adult Cynomolgus monkeys (Macaca fascicularis) and (iv) two sea urchins (Arbacia punctulata) were used to characterize and compare their spectral profiles. Differences and similarities were confirmed by principal component analysis (PCA). MAIN RESULTS AND THE ROLE OF CHANCE: Several novel region-specific peaks were identified. The three regions could be differentiated by distinctive Raman patterns irrespective of the species. However, regardless of the specie, their main spectral pattern remains mostly unchanged. These results were corroborated by the PCA analysis and suggest that the basic constituents of spermatozoa are biochemically similar among species. LIMITATIONS REASONS FOR CAUTION: Further research should be performed in live sperm to validate the detected spectral bands and their use as markers of distinctive regions. WIDER IMPLICATIONS OF THE FINDINGS: Raman peaks that have never been described in the sperm cell were detected. Particularly important are those that are unique to the midpiece as they might be a reference to the identification of sperm mitochondria, whose function is highly correlated with that of sperm. In the future, Raman micro-spectroscopy has the potential to be applied in assessment of male fertility. LARGE SCALE DATA: N/A. STUDY FUNDING AND COMPETING INTEREST(S): This work was supported by BMBF project 'Sperm Ident' (FKZ:13N13024) and the DAAD-CRUP bilateral exchange program (AI A06/16-57213087). S.A. is a recipient of a fellowship from the Portuguese foundation for science and technology (FCT-SFRH/BPD/110160/2015) and R.DC. is a recipient of a DAAD PhD stipend (91590556). There is no competing interest.

A diagnostic germ cell score for immature testicular tissue at risk of germ cell loss.

STUDY QUESTION: Can a systematic scoring procedure provide crucial information on the status of highly heterogeneous immature human testicular tissues in the context of cryopreservation for fertility preservation? SUMMARY ANSWER: We developed a systematic histological score as a novel diagnostic tool which differentiates the patient cohort according to the status of germ cell differentiation and number of spermatogonia (normal, diminished and absent), and which could be relevant in the fertility clinic. WHAT IS KNOWN ALREADY: Cryopreservation of testicular tissue of immature boys is currently considered the option for future fertility restoration. However, experimental techniques for the derivation of sperm as well as valid diagnostic scoring of these immature testis tissues are not yet reported. STUDY DESIGN, SIZE, DURATION: Testicular tissues of 39 patients (aged 2-20 years) who attended our clinic for cryopreservation between 2010 and 2015 were analyzed to determine the variability of testicular tissue composition, germ cell numbers and differentiation status. PARTICIPANTS/MATERIALS, SETTING, METHODS: Human testicular tissue samples were divided into three groups. Group NT included patients suffering from diseases which do not directly affect the testes (n = 6; aged 6-14 years), group AT included patients suffering from diseases that directly affect the testes (n = 14; 2-17 years), and group KS (Klinefelter patients, n = 19; 12-20 years). Based on immunohistochemical stainings for MAGEA4, the differentiation status as well as the numbers of gonocytes, spermatogonia and spermatocytes were determined. MAIN RESULTS AND THE ROLE OF CHANCE: Testicular tissue samples from the NT group contained a mean of 100.3 spermatogonia/mm3 (×103). Highly heterogeneous and significantly lower mean numbers of spermatogonia were scored in testes from boys after cytotoxic exposures or with pre-existing disease (AT group: 35.7 spermatogonia/mm3 (×103); KS group: 1.8 spermatogonia/mm3 (×103)). In addition, the germ cell differentiation status was determined and revealed tissues with either spermatogonia and gonocytes, only spermatogonia, spermatogonia and spermatocytes, or all three germ cell types were present. Based on spermatogonial numbers and differentiation status, we developed a germ cell score which we applied to each individual patient sample. LIMITATIONS REASONS FOR CAUTION: Normal human testicular tissue samples are difficult to obtain for ethical reasons and the sample numbers were small. However, six such samples provide a valid baseline for the normal situation. WIDER IMPLICATIONS OF THE FINDINGS: Fertility preservation of immature male tissues is an emerging field and is currently offered in many specialized centers worldwide. Our diagnostic germ cell score delivers an easily applicable tool, facilitating patient counseling and thus ensuring comparability between the centers with regard to future studies. STUDY FUNDING/COMPETING INTEREST(S): This study was supported by the Funding Initiative: Translational Research, Ministry of Innovation, Science and Research, Federal State of North Rhine Westphalia (z1403ts006). The authors declare that they do not have competing financial interests.

Reassembly of adult human testicular cells: can testis cord-like structures be created in vitro?

STUDY QUESTION: Can enzymatically dispersed testicular cells from adult men reassemble into seminiferous cord-like structures in vitro? SUMMARY ANSWER: Adult human testicular somatic cells reassembled into testicular cord-like structures via dynamic interactions of Sertoli and peritubular cells. WHAT IS KNOWN ALREADY: In vitro approaches using dispersed single cell suspensions of human testes to generate seminiferous tubule structures and to initiate their functionality have as yet shown only limited success. STUDY DESIGN, SIZE, DURATION: Testes from 15 adult gender dysphoria patients (mean ± standard deviation age 35 ± 9.3 years) showing spermatogonial arrest became available for this study after sex-reassignment surgery. In vitro primary testicular somatic cell cultures were generated to explore the self-organizing ability of testicular somatic cells to form testis cords over a 2-week period. Morphological phenotype, protein marker expression and temporal dynamics of cell reassembly were analyzed. PARTICIPANTS/MATERIALS, SETTING, METHODS: Cell suspensions obtained by two-step enzymatic digestion were plated onto glass coverslips in 24-well plates. To obtain adherent somatic cells, the supernatant was discarded on Day 2. The culture of the attached cell population was continued. Reassembly into cord-like structures was analyzed daily by microscopic observations. Endpoints were qualitative changes in morphology. Cell types were characterized by phase-contrast microscopy and immunohistochemistry. Dynamics of cord formation were recorded by time-lapse microscopy. MAIN RESULTS AND THE ROLE OF CHANCE: Primary adult human testicular cells underwent sequential morphological changes including compaction and reaggregation resulting in round or elongated cord-like structures. Time-lapse video recordings within the first 4 days of culture revealed highly dynamic processes of migration and coalescence of reaggregated cells. The cellular movements were mediated by peritubular cells. Immunohistochemical analysis showed that both SRY-related high mobility box 9-positive Sertoli and α-smooth muscle actin-positive peritubular myoid cells interacted and contributed to cord-like structure formation. LARGE SCALE DATA: Not applicable. LIMITATIONS, REASONS FOR CAUTION: Owing to scarcity of normal human testicular tissue, testes from gender dysphoria patients were used in the study. The regressed status might influence the experimental responses of primary cells. We observed basic morphological features resembling in vivo testicular cords, however, the proof of functionality (e.g. support of germ cells) will need further studies. WIDER IMPLICATIONS OF THE FINDINGS: The proposed in vitro culture system may open opportunities for examination of testicular cell interactions during testicular tubulogenesis. Further refinement of our approach may enable initiation of ex vivo spermatogenesis. STUDY FUNDING/COMPETING INTERESTS: The work was supported by EU-FP7-PEOPLE-2013-ITN 603568: 'Growsperm'. No conflict of interests is declared.

Andrology of male-to-female transsexuals: influence of cross-sex hormone therapy on testicular function.

Patients with gender dysphoria are offered cross-sex hormone therapy and sex reassignment surgery to achieve the transition between the sex assigned at birth and gender identity. According to international guidelines, cross-sex hormone therapy in trans-women should lead to a psychologically and physiologically healthy body with feminized serum hormone levels, resulting in suppression of spermatogenesis. However, in a recently published multi-center study, we discovered a high proportion of patients with male serum hormone levels and qualitatively intact spermatogenesis on the day of sex reassignment surgery. The objective of this study was to review the content of 11 publications that focus on the influence of cross-sex hormone therapy on testicular morphology. These publications were identified based on a PubMed search for the key words transgender/transsexual/gender dysphoria in male-to-female persons, cross-sex hormone therapy, and testicular tissues. Whereas three publications described a marked reduction of the spermatogenic level in all patients examined, eight publications reported inconsistent results. Histological analyses showed highly variable outcomes from qualitatively normal spermatogenesis and undisturbed Leydig/Sertoli cell morphology to full testicular regression with severe cellular damage and hyalinization. Explanations for these heterogeneous findings include insufficient cross-sex hormone therapy regarding dosage or duration. As complete spermatogenesis is associated with virilized serum hormone levels, these patients may face challenges especially after sex reassignment surgery in adjusting to the abruptly established hypogonadal state following removal of the testes. These findings also suggest that contraception should be discussed, and fertility preservation should be offered during/prior to cross-sex hormone therapy. There is a need for more individualized and better-controlled cross-sex hormone therapy and post-treatment regimens. Evidence-based guidelines for attending clinicians need to be established in order to deliver the most appropriate care.

Totipotency segregates between the sister blastomeres of two-cell stage mouse embryos.

Following fertilization in mammals, it is generally accepted that totipotent cells are exclusive to the zygote and to each of the two blastomeres originating from the first mitotic division. This model of totipotency was inferred from a minority of cases in which blastomeres produced monozygotic twins in mice. Was this due to experimental limitation or biological constraint? Here we removed experimental obstacles and achieved reliable quantification of the prevalence of dual totipotency among mouse two-cell stage blastomeres. We separated the blastomeres of 1,252 two-cell embryos, preserving 1,210 of the pairs. Two classes of monozygotic twins became apparent at the blastocyst stage: 27% formed a functional epiblast in both members (concordant), and 73% did so in only one member of the pair (discordant) - a partition that proved insensitive to oocyte quality, sperm-entry point, culture environment and pattern of cleavage. In intact two-cell embryos, the ability of sister blastomeres to generate epiblast was also skewed. Class discovery clustering of the individual blastomeres' and blastocysts' transcriptomes points to an innate origin of concordance and discordance rather than developmental acquisition. Our data place constraints on the commonly accepted idea that totipotency is allocated equally between the two-cell stage blastomeres in mice.

Irradiation affects germ and somatic cells in prepubertal monkey testis xenografts.

Study question: Does irradiation evoke adverse effects in germ and somatic cells in testis xenografts from prepubertal monkeys? Summary answer: In addition to the expected depletion of germ cells, a dose-dependent effect of irradiation was observed at the mRNA and protein level in Sertoli and peritubular myoid cells. What is known already: Testicular irradiation studies in monkeys have focused on the dose-dependent effects on germ cells. Previous studies using intact animals or xenografts reported that germ cells are highly sensitive to irradiation. Their depletion was demonstrated by morphometric and histological analyses. The effect of irradiation on expression of Sertoli and peritubular myoid cell markers, however, has not yet been described. Study design, size, duration: The testes of two prepubertal macaques (Macaca fascicularis) were dissected into testicular fragments. Fragments were randomly exposed in vitro to one of the following three doses of irradiation: 0 Gy, n = 60; 1 Gy, n = 54; 4 Gy, n = 72. Non-irradiated control fragments (0 Gy) were placed into the Faxitron for 6.6 min without irradiation. For 1 Gy and 4 Gy irradiation was applied for 1.7 and 6.6 min, respectively. Grafts were then either immediately analyzed or subcutaneously implanted under the back skin of 39 nude mice and analyzed after 6.5 months. Participants/materials setting methods: Post grafting, 133 testicular xenografts were retrieved. The body weight, serum testosterone level and seminal vesical weight of the host mice as well as the number and weight of retrieved grafts were determined. Larger grafts were used to evaluate both mRNA expression profiles and protein expression patterns. In total, 71 testicular fragments were used for morphometric and histological analysis while 68 fragments were analyzed for gene expression. For PCR arrays, M. fascicularis-specific primer sequences were employed. Irradiation-induced changes in the transcript levels of 34 marker genes were determined for each testicular graft. The effects of irradiation on peritubular myoid cells and Sertoli cells were confirmed by immunohistochemical analysis of chemokine (C-X-C motif) ligand type 11 (CXCL11), alpha smooth muscle actin (SMA) and chemokine (C-X-C motif) ligand type 12 (CXCL12). Main results and the role of chance: The four testes gave rise to 106 xenografts, which were individually analyzed, limiting the role of chance despite using only two monkeys in the study. Prior to grafting, the two donors displayed spermatogonia as the most advanced germ cell type in 95% and 70% of seminiferous tubules, respectively, while remaining tubules contained SCO. No spermatocytes were encountered prior to grafting in either monkey. After 6.5 months, non-irradiated grafts displayed spermatocytes in 15.4% and 1.8% of seminiferous tubules indicating an induction of meiosis. Irradiation resulted in a complete absence of spermatocytes. The percentage of seminiferous tubules containing spermatogonia declined in a dose-dependent manner. In non-irradiated xenografts, ~40% of tubules contained spermatogonia. This proportion was reduced to 3.4% and 4.3% in the 1 Gy treated group and to 1.3% and 0.2% in 4 Gy irradiated grafts. A dose-dependent decline in mRNA levels of selected germ cell marker genes supported the morphologically detected loss of germ cells. Irradiation had no effect on CXCL12 transcript levels. At the protein level, CXCL12-positive Sertoli cells were most abundant in the 1 Gy group compared to the 4 Gy group (P < 0.05), indicating a potential role of CXCL12 during recovery of primate spermatogenesis. The most prominent radiation-evoked changes were for CXCL11, which was localized to smooth muscle cells of blood vessels and seminiferous tubules. Transcript levels declined in a dose-dependent manner in grafts from both monkeys (MM687: P < 0.01 (0 Gy versus 4 Gy), MM627: P < 0.05 (0 Gy versus 4 Gy), P < 0.001 (1 Gy versus 4 Gy)). CXCL11 patterns of protein expression revealed irradiation-dependent changes as well. That peritubular cells are affected by X-irradiation was substantiated by changes at the transcript level between 1 and 4 Gy exposed groups (P < 0.01) and at the protein level of SMA (P < 0.05, 0 Gy versus 4 Gy). Large scale data: n/a. Limitations, reasons for caution: The spermatogonial stem cell system in primates is remarkably different from rodents. Therefore, data from a non-human primate may be more relevant to man. However, species-specific differences amongst primates cannot be fully excluded and the use of only two donors may raise concerns toward the generalization of the findings. There may also be important differences across the prepubertal period (e.g. infancy, early childhood) that are not represented by the ages included in the present study. Wider implications of the findings: This study is the first to indicate relevant testicular somatic cell responses following irradiation of prepubertal primate tissue. In addition to the well-known depletion of germ cells, the changes in Sertoli, and in particular peritubular myoid, cells may have important consequences for spermatogenic recovery. These novel findings should be taken into consideration when irradiation effects are assessed in tumor survivors. Study funding and competing interest(s): Interdisciplinary Center for Clinical Research (IZKF) Münster (Schl2/001/13) and the Excellence Cluster 'Cells in Motion' at the University Münster. There are no conflicts of interest to declare.

Single-cell gene expression analysis reveals diversity among human spermatogonia.

STUDY QUESTION: Is the molecular profile of human spermatogonia homogeneous or heterogeneous when analysed at the single-cell level? SUMMARY ANSWER: Heterogeneous expression profiles may be a key characteristic of human spermatogonia, supporting the existence of a heterogeneous stem cell population. WHAT IS KNOWN ALREADY: Despite the fact that many studies have sought to identify specific markers for human spermatogonia, the molecular fingerprint of these cells remains hitherto unknown. STUDY DESIGN, SIZE, DURATION: Testicular tissues from patients with spermatogonial arrest (arrest, n = 1) and with qualitatively normal spermatogenesis (normal, n = 7) were selected from a pool of 179 consecutively obtained biopsies. Gene expression analyses of cell populations and single-cells (n = 105) were performed. Two OCT4-positive individual cells were selected for global transcriptional capture using shallow RNA-seq. Finally, expression of four candidate markers was assessed by immunohistochemistry. PARTICIPANTS/MATERIALS, SETTING, METHODS: Histological analysis and blood hormone measurements for LH, FSH and testosterone were performed prior to testicular sample selection. Following enzymatic digestion of testicular tissues, differential plating and subsequent micromanipulation of individual cells was employed to enrich and isolate human spermatogonia, respectively. Endpoint analyses were qPCR analysis of cell populations and individual cells, shallow RNA-seq and immunohistochemical analyses. MAIN RESULTS AND THE ROLE OF CHANCE: Unexpectedly, single-cell expression data from the arrest patient (20 cells) showed heterogeneous expression profiles. Also, from patients with normal spermatogenesis, heterogeneous expression patterns of undifferentiated (OCT4, UTF1 and MAGE A4) and differentiated marker genes (BOLL and PRM2) were obtained within each spermatogonia cluster (13 clusters with 85 cells). Shallow RNA-seq analysis of individual human spermatogonia was validated, and a spermatogonia-specific heterogeneous protein expression of selected candidate markers (DDX5, TSPY1, EEF1A1 and NGN3) was demonstrated. LIMITATIONS, REASONS FOR CAUTION: The heterogeneity of human spermatogonia at the RNA and protein levels is a snapshot. To further assess the functional meaning of this heterogeneity and the dynamics of stem cell populations, approaches need to be developed to facilitate the repeated analysis of individual cells. WIDER IMPLICATIONS OF THE FINDINGS: Our data suggest that heterogeneous expression profiles may be a key characteristic of human spermatogonia, supporting the model of a heterogeneous stem cell population. Future studies will assess the dynamics of spermatogonial populations in fertile and infertile patients. LARGE SCALE DATA: RNA-seq data is published in the GEO database: GSE91063. STUDY FUNDING/COMPETING INTEREST(S): This work was supported by the Max Planck Society and the Deutsche Forschungsgemeinschaft DFG-Research Unit FOR 1041 Germ Cell Potential (grant numbers SCHO 340/7-1, SCHL394/11-2). The authors declare that there is no conflict of interest.

[Cell biology and physiology of male reproduction]. / Zellbiologie und Physiologie der männlichen Fortpflanzung.

BACKGROUND: An adequate and undisturbed generation of fertile sperm is a prerequisite for fatherhood. Therefore, spermatogenesis is of central importance for male fertility. The testes, however, not only hold the germinal epithelium as the sperm-generating organ but also acts as a gland releasing androgens to control male reproductive function. This dual testicular function provides options to couple and coordinate spermatogenesis and steroidogenesis. METHODS: The regulation of both processes via the hypothalamus-pituitary-gonadal axis is arranged via feedback loops, which are interconnected but also enable separate modulation of germ cell production and endocrine activity. Many parameters of gonadal function can be determined and provide information about physiological or pathological changes of testis function. OBJECTIVES: This article introduces the physiological basics of testis function and presents the repertoire of endpoints determined in clinical andrology to facilitate a deeper understanding for clinical diagnostics of male fertility.