A 10-year experience in testicular tissue cryopreservation for boys under 18 years of age: What can be learned from 350 cases?

BACKGROUND: A growing number of centers worldwide are preserving testicular tissue (TT) of young boys at risk of fertility loss to preserve their fertility. Data in this regard are scarce and experience sharing is essential to the optimization of the process. OBJECTIVES: This report of our 10-year activity of pediatric fertility preservation (FP) has the objective to (1) improve knowledge regarding the feasibility, acceptability, safety, and potential usefulness of the procedure; (2) analyze the impact of chemotherapy on spermatogonia in the cryopreserved TT. MATERIALS AND METHODS: For this retrospective study of data prospectively recorded, we included all boys under 18 years of age referred to the FP consultation of our academic network between October 2009 and December 2019. Characteristics of patients and cryopreservation of testicular tissue (CTT) were extracted from the clinical database. Univariate and multivariate analyses were used to assess factors associated with the risk of absence of spermatogonia in the TT. RESULTS: Three hundred and sixty-nine patients (7.2 years; 0.5-17.0) were referred to the FP consultation for malignant (70%) or non-malignant (30%) disease, of whom 88% were candidates for CTT, after a previous chemotherapy exposure (78%). The rate of recorded immediate adverse events was 3.5%, with painful episodes dominating. Spermatogonia were detected in the majority of TTs: 91.1% of those exposed to chemotherapy and 92.3% of those not exposed (p = 0.962). In multivariate analysis, the risk of absence of spermatogonia was almost three-fold higher in boys > 10 years of age ([OR] 2.74, 95% CI 1.09-7.26, p = 0.035) and four-fold higher in boys exposed to alkylating agents prior to CTT ([OR] 4.09, 95% CI 1.32-17.94, p = 0.028). DISCUSSION/CONCLUSION: This large series of pediatric FP shows that this procedure is well accepted, feasible, and safe in the short term, strengthening its place in the clinical care pathway of young patients requiring a highly gonadotoxic treatment. Our results demonstrate that CTT post-chemotherapy does not impair the chance to preserve spermatogonia in the TT except when the treatment includes alkylating agents. More data on post-CTT follow-up are still required to ensure the long-term safety and usefulness of the procedure.

Plastic used in in vitro fertilization procedures induces massive placental gene expression alterations.

BACKGROUND: The exposure to plastic derivatives during human life is deleterious. Infants conceived using ART (IVF or ICSI) have twice as many risks of major birth defects compared to naturally conceived infants. Could plastic ware used during ART trigger defects in the fetal development? METHODS: Three groups of blastocysts were transferred to pseudopregnant mice. One was obtained after IVF and embryo development in plastic ware, the second in glass ware. The third, was obtained in vivo by natural mating. On day 16.5 of pregnancy, females were sacrificed and fetal organs collected for gene expression analysis. Fetal sex was determined by RT-PCR. RNA was extracted from a pool of five placental or brain samples coming from at least two litters from the same group and analyzed by hybridisation onto the mouse Affymetrix 430.2.0 GeneChips, confirmed by RT-qPCR for 22 genes. FINDINGS: This study highlights a major impact of plastic ware on placental gene expression (1121 significantly deregulated genes), while glassware was much closer to in vivo offspring (only 200 significantly deregulated genes). Gene Ontology indicated that the modified placental genes were mostly involved in stress, inflammation and detoxification. A sex specific analysis revealed in addition a more drastic effect on female than male placentas. In the brains, whatever the comparison, less than 50 genes were found deregulated. INTERPRETATION: Embryos incubated in plastic ware resulted in pregnancy with massive alterations of placental gene expression profile in concerted biological functions. There were no obvious effects on the brains. Besides other effects, this suggests that plastic ware in ART could be a cause of the increased level of pregnancy disorders observed recurrently in ART pregnancies. FUNDING: This study was funded by two grants from the Agence de la Biomedecine in 2017 and 2019.

An interpretable and versatile machine learning approach for oocyte phenotyping.

Meiotic maturation is a crucial step of oocyte formation, allowing its potential fertilization and embryo development. Elucidating this process is important for both fundamental research and assisted reproductive technology. However, few computational tools based on non-invasive measurements are available to characterize oocyte meiotic maturation. Here, we develop a computational framework to phenotype oocytes based on images acquired in transmitted light. We trained neural networks to segment the contour of oocytes and their zona pellucida using oocytes from diverse species. We defined a comprehensive set of morphological features to describe an oocyte. These steps were implemented in an open-source Fiji plugin. We present a feature-based machine learning pipeline to recognize oocyte populations and determine morphological differences between them. We first demonstrate its potential to screen oocytes from different strains and automatically identify their morphological characteristics. Its second application is to predict and characterize the maturation potential of oocytes. We identify the texture of the zona pellucida and cytoplasmic particle size as features to assess mouse oocyte maturation potential and tested whether these features were applicable to the developmental potential of human oocytes. This article has an associated First Person interview with the first author of the paper.

A fertilin-derived peptide improves in vitro maturation and ploidy of human oocytes.

OBJECTIVE: To analyze the effect of a cyclic fertilin-derived peptide (cFEE) on in vitro maturation of human oocytes. DESIGN: Randomized study. SETTING: Fertility center in an academic hospital. PATIENT(S): Not applicable. INTERVENTION(S): Human immature germinal vesicle-stage oocytes (n = 1,629) donated for research according to French bioethics laws were randomly allocated to groups treated with 1 or 100 µM of cFEE or to a control group. They were incubated at 37 °C in 6% CO2 and 5% O2, and their maturation was assessed using time-lapse microscopy over 24 hours. In vitro maturated metaphase II oocytes were analyzed for chromosomal content using microarray comparative genomic hybridization, and their transcriptomes were analyzed using Affymetrix Clariom D microarrays. MAIN OUTCOME MEASURE(S): The percentage of oocytes undergoing maturation in vitro was observed. Aneuploidy and euploidy were assessed for all chromosomes, and differential gene expression was analyzed in oocytes treated with cFEE compared with the control to obtain insights into its mechanism of action. RESULT(S): cFEE significantly increased the percentage of oocytes that matured in vitro and improved euploidy in meiosis II oocytes by the up-regulation of FMN1 and FLNA genes, both of which encode proteins involved in spindle structure. CONCLUSION(S): cFEE improves human oocyte maturation in vitro and reduces aneuploidy. It may prove useful for treating oocytes before fertilization in assisted reproductive technology and for in vitro maturation in fertility preservation programs to improve oocyte quality and the chances for infertile couples to conceive.

Cyclic fertilin-derived peptide stimulates in vitro human embryo development.

OBJECTIVE: To study the cyclic fertilin peptide effects on preimplantation human embryogenesis. Cyclic fertilin peptide reproduces the structure of the binding site of the sperm Fertilin ß (also named A Disintegrin and Metalloprotease 2: ADAM2) disintegrin domain. It binds to the oocyte membrane and increases sperm-oocyte fusion index in human and fertilization rate in mouse, providing healthy pups. It also improves human oocyte maturation and chromosome segregation in meiosis I and binds to human embryo blastomeres, suggesting that it has a membrane receptor. DESIGN: Thawed human embryos at the 3 to 4 cells stage were randomly included in a dose-response study with cyclic fertilin peptide. Inner cell mass (ICM), trophectoderm (TE), and total cell numbers were evaluated in top- and good-quality blastocysts. SETTING: The study was performed in an academic hospital and research laboratory. PATIENT(S): Human embryos donated for research. This project was approved by the French "Agence de la Biomédecine." INTERVENTION(S): Immunofluorescence and tissue-specific gene expression analysis, using Clariom D microarrays, were performed to study its mechanism of action. MAIN OUTCOME MEASURE(S): Cyclic fertilin peptide improves blastocyst formation by almost 20%, the concentration of 1 µM being the lowest most efficient concentration. It significantly increases twice the TE cell number, without modifying the ICM. It increases the in vitro hatching rate from 14% to 45%. RESULT(S): Cyclic fertilin peptide stimulates TE growth. In the ICM, it induces transcriptional activation of intracellular protein and vesicle-mediated transport. CONCLUSION(S): Cyclic fertilin peptide dramatically improves human embryo development potential. It could be used to supplement culture medium and improve the in vitro human embryo development. Starting supplementation immediately after fertilization, instead of day 2, could significantly upgrade assisted reproductive technology outcome.

Transcriptional profiling of ß-2M-SPα-6+THY1+ spermatogonial stem cells in human spermatogenesis.

Male infertility is responsible for approximately half of all cases of reproductive issues. Spermatogenesis originates in a small pool of spermatogonial stem cells (SSCs), which are of interest for therapy of infertility but remain not well defined in humans. Using multiparametric analysis of the side population (SP) phenotype and the α-6 integrin, THY1, and ß-2 microglobulin cell markers, we identified a population of human primitive undifferentiated spermatogonia with the phenotype ß-2 microglobulin (ß-2M)-SPα-6+THY1+, which is highly enriched in stem cells. By analyzing the expression signatures of this SSC-enriched population along with other germinal progenitors, we established an exhaustive transcriptome of human spermatogenesis. Transcriptome profiling of the human ß-2M-SPα-6+THY1+ population and comparison with the profile of mouse undifferentiated spermatogonia provide insights into the molecular networks and key transcriptional regulators regulating human SSCs, including the basic-helix-loop-helix (bHLH) transcriptional repressor HES1, which we show to be implicated in maintenance of SSCs in vitro.

Prospective analysis over time of semen parameters in spinal cord-injured patients: Results of a pilot study.

BACKGROUND: Spinal cord injury often results in erectile dysfunction and an ejaculation along with impaired semen parameters. Fertility is a major concern in spinal cord injury adult males and some fear that the delay post-spinal cord injury may negatively affect sperm quality. OBJECTIVES: We aimed to (i) assess semen parameters over time in SCI patients according to age at spinal cord injury, time post-spinal cord injury, and the spinal cord injury level and completeness and (ii) measure markers in semen for inflammation and marker of oxidative stress to investigate their impact on sperm parameters. MATERIALS AND METHODS: The study is a prospective, longitudinal, pilot study over 18 months. Thirty-five men with spinal cord injury from 18 to 60 years of age were enrolled. Their mean age was 29.4 ± 6.4 years. Semen retrieval was scheduled every 6 months, allowing analysis of four ejaculates, in association with measurement of granulocyte and seminal plasma elastase concentrations to assess markers in semen for inflammation and spermatozoa DNA fragmentation to assess oxidative stress. RESULTS: Based on reference limits, a normal total sperm number, decreased motility and vitality of the spermatozoa, and increased morphological abnormalities were found. Mean round cell and granulocyte concentrations were elevated in the semen. Markers in semen for inflammation and marker of oxidative stress were elevated in several semen samples, compared to reference limits. However, neither the presence of markers in semen for inflammation or oxidative stress, the completeness or the level of the spinal cord lesion, the age or the time post-spinal cord injury had a negative impact on the semen quality over time. DISCUSSION: There was no significant decline in semen quality in spinal cord injury patients over time within the limitations of this pilot study. Moreover, a chronic genital inflammatory status was not associated with impairment of semen quality. CONCLUSION: The present findings are reassuring for men with spinal cord injury and could guide the management of their reproductive ability. According to these preliminary data, not all spinal cord injury patients who are able to ejaculate require systematic freezing of their spermatozoa.

Case Report: Abnormalities of sperm motility and morphology in a patient with Leber hereditary optic neuropathy: Improvement after idebenone therapy.

Case: We report the sperm characteristics of a male patient who developed, when he was 18 years old, a Leber hereditary optic neuropathy, a hereditary optic neuropathy due to mtDNA mutation as well as variants in the nuclear DNA. At the age of 30 years-old, he complained of infertility lasting for 2 years. Semen analyses showed low motility spermatozoa and a high percentage of morphological or ultrastructural abnormalities. Levels of epididymal markers were strongly atypical. Idebenone was prescribed as treatment of his Leber hereditary optic neuropathy in order to improve his visual acuity. After 5 months of this treatment, motility of spermatozoa increased, and their vitality improved. A natural conception occurred. Outcome: This case is the first description of an anomaly of spermatozoas and of the epididymis epithelium in a patient with Leber hereditary optic neuropathy. It draws attention to sperm pathologies in patients with mitochondrial disorders. The role of the mtDNA mutations must be suspected since it plays an important role in the development and motility of spermatozoa. In addition, idebenone can by-pass the complex I and transfer electrons to complex III. It has been suspected to have a favorable effect on spermatogenesis. Conclusion: This case confirms the possibility of sperm dysfunction in Leber hereditary optic neuropathy and the interest of idebenone as a treatment for infertility due to mtDNA mutations in human.

Cyclic FEE Peptide Improves Human Sperm Movement Parameters without Modification of Their Energy Metabolism.

Cyclic fertilin peptide (cFEE: phenylalanine, glutamic acid; glutamic acid) improves gamete interaction in humans. We investigate whether it could be via improvement of sperm movement parameters and their mitochondrial ATP production. Sperm movement parameters were studied using computer-assisted sperm analysis (CASA) in sperm samples from 38 patients with normal sperm in medium supplemented with cyclic fertilin against a control group. Sperm mitochondrial functions were studied using donor's sperm, incubated or not with cFEE. It was evaluated by the measurement of their ATP production using bioluminescence, their respiration by high resolution oxygraphy, and of mitochondrial membrane potential (MMP) using potentiometric dyes and flow cytometry. cFEE significantly improved sperm movement parameters and percentage of hyperactivated sperm. Impact of inhibitors showed OXPHOS as the predominant energy source for sperm movement. However, cFEE had no significant impact on any of the analyzed mitochondrial bioenergetic parameters, suggesting that it could act via a more efficient use of its energy resources.

Characteristics and intentions of heterosexual couples comprising a transgender man awaiting sperm donation to conceive a child.

BACKGROUND: More than half of transgender (TG) men wish to have children. Until recently, TG people in France were rarely offered gamete donation, mainly because the Bioethics Law allows the use of assisted reproductive technologies (ART) only in infertile couples. The only option currently available for heterosexual couples with a TG man is ART with sperm donation. The Center for Study and Preservation of Eggs and Sperm (CECOS) of the Cochin Hospital is the first French center to propose sperm donation to such couples, and has done so since 1999. OBJECTIVES: To determine the main characteristics and intentions of 43 couples, including a TG man and his cisgender female partner awaiting sperm donation. MATERIALS AND METHODS: A retrospective analysis was carried out on the records from October 2010 to December 2019, of 43 couples with a TG man who applied for sperm donation at the CECOS of the Cochin Hospital (Paris, France). RESULTS: The mean age of TG men and cisgender women was 32 ± 6.6 and 29.7 ± 4.6 years, respectively. In 77% of cases, the couple met before the man's transition. Eighty-one percent of the couples were in a stable relationship for at least 3 years, and 94% wished to have a child for no more than 5 years. Almost all of the couples (95%) intended to inform their child of their conception by sperm donation and the father's transidentity (92%). DISCUSSION: Due to restrictive French legislation, the profile of our couples probably does not reflect that of all couples consisting of a TG man and a cisgender woman. The study took place over a long period of time and the characteristics of the couples could probably change over time. CONCLUSION: The couples often met before the man's transition, cohabited for several years, intended to inform their child of sperm donation and the father's transidentity.

Partial Sperm beta1 Integrin Subunit Deletion Proves its Involvement in Mouse Gamete Adhesion/Fusion.

We have previously shown, using antibodies, that the sperm alpha6beta1 integrin is involved in mouse gamete fusion in vitro. Here we report the conditional knockdown of the sperm Itgb1 gene. It induced a drastic failure of sperm fusogenic ability with sperm accumulation in the perivitelline space of in vitro inseminated oocytes deleted or not for the Itgb1 gene. These data demonstrate that sperm, but not oocyte, beta1 integrin subunit is involved in gamete adhesion/fusion. Curiously, knockdown males were fertile in vivo probably because of the incomplete Cre-mediated deletion of the sperm Itgb1 floxed gene. Indeed, this was shown by Western blot analysis and confirmed by both the viability and litter size of pups obtained by mating partially sperm Itgb1 deleted males with females producing completely deleted Itgb1 oocytes. Because of the total peri-implantation lethality of Itgb1 deletion in mice, we assume that sperm that escaped the Itgb1 excision seemed to be preferentially used to fertilize in vivo. Here, we showed for the first time that the deletion, even partial, of the sperm Itgb1 gene makes the sperm unable to normally fertilize oocytes. However, to elucidate the question of the essentiality of its role during fertilization, further investigations using a mouse expressing a recombinase more effective in male germ cells are necessary.

Transgender fathering: Children's psychological and family outcomes.

Medical advances in assisted reproductive technology have created new ways for transgender persons to become parents outside the context of adoption. The limited empirical data does not support the idea that trans-parenthood negatively impacts children's development. However, the question has led to lively societal debates making the need for evidence-based studies urgent. We aimed to compare cognitive development, mental health, gender identity, quality of life and family dynamics using standardized instruments and experimental protocols in 32 children who were conceived by donor sperm insemination (DSI) in French couples with a cisgender woman and a transgender man, the transition occurring before conception. We constituted two control groups matched for age, gender and family status. We found no significant difference between groups regarding cognitive development, mental health, and gender identity, meaning that neither the transgender fatherhood nor the use of DSI had any impact on these characteristics. The results of the descriptive analysis showed positive psycho-emotional development. Additionally, when we asked raters to differentiate the family drawings of the group of children of trans-fathers from those who were naturally conceived, no rater was able to differentiate the groups above chance levels, meaning that what children expressed through family drawing did not indicate cues related to trans-fatherhood. However, when we assessed mothers and fathers with the Five-Minute Speech Sample, we found that the emotions expressed by transgender fathers were higher than those of cisgender fathers who conceived by sex or by DSI. We conclude that the first empirical data regarding child development in the context of trans-parenthood are reassuring. We believe that this research will also improve transgender couple care and that of their children in a society where access to care remains difficult in this population. However, further research is needed with adolescents and young adults.

Two frequent loss-of-function mutations in Aurora Kinase C gene in Algerian infertile men with macrozoospermia.

Macrozoospermia is associated with severe male infertility. To date, the only gene implicated in this phenotype is the Aurora Kinase C gene. We report in this work the genetic screening of AURKC mutations in 34 patients with macrozoospermia among 3,536 Algerian infertile men. Nineteen patients (56%) were homozygotes for the c.144delC mutation, eight (23.52%) homozygotes for the c.744C>G (p.Y248*) mutation and two (5.88%) compound heterozygotes. No AURKC mutation was identified in five patients (14.7%). Interestingly and although it is generally accepted that nearly all positive mutated AURKC patients have close to 100% large-head spermatozoa, our results showed that 11 patients with AURKC mutations (32.35%) had large-headed spermatozoa lower than 70% (7 with c.144delC and 4 with p.Y248*), and no mutation was found in 2 patients who had >70% of macrocephalic spermatozoa. Twenty ICSI attempts were performed before genetic screening resulting in 39 embryos but no pregnancy was obtained. The sequencing of AURKC exons 3 and 6 is appropriate as a first-line genetic exploration in these patients to avoid unsuccessful ICSI attempts. A percentage of large head spermatozoa beyond 25% and a percentage of multiflagellar spermatozoa beyond 10% are predictive of a positive mutation diagnosis.

The follicular fluid metabolome differs according to the endometriosis phenotype.

RESEARCH QUESTION: Is there a follicular fluid-specific metabolic profile in deep infiltrating endometriosis (DIE) depending on the presence of an associated ovarian endometrioma (OMA) that could lead to the identification of biomarkers for diagnosis and prognosis of the disease? DESIGN: In this prospective cohort study, proton nuclear magnetic resonance (1H-NMR) experiments were carried out on 50 follicular fluid samples from patients presenting with DIE, associated or not associated with an OMA, and 29 follicular fluid samples from patients with infertility caused by a tubal obstruction. RESULTS: Concentrations of glucose, citrate, creatine and amino acids such as tyrosine and alanine were lower in women with DIE than control participants, whereas concentrations of lactate, pyruvate, lipids and ketone bodies were higher. Metabolic analysis revealed enhanced concentrations of glycerol and ketone bodies in patients with OMA, indicative of an activation of lipolysis followed by beta-oxidation. Concentrations of lactate and pyruvate were increased in patients without OMA, whereas the concentration of glucose was decreased, highlighting activation of the anaerobic glycolysis pathway. Differences in concentrations of amino acids such as threonine and glutamine were also statistically relevant in discriminating between the presence or absence of OMA. CONCLUSIONS: Results indicate a mitochondrial dysregulation in endometriosis phenotypes, with a modified balance between anaerobic glycolysis and beta-oxidation in OMA phenotypes that could affect the fertility of women with endometriosis. As the composition of the follicular fluid has been shown to be correlated with oocyte development and outcome of implantation after fertilization, these findings may help explain the high level of infertility in these patients.

Sperm SPACA6 protein is required for mammalian Sperm-Egg Adhesion/Fusion.

Three genes are known to be essential for gamete adhesion/fusion (Cd9, Izumo1 and Juno). Here, we confirmed that Spaca6 null males are infertile and showed that their sperm accumulate in the perivitelline space but are unable to fuse with oocyte. Like IZUMO1, SPACA6 which is expressed by human sperm, is remained on the equatorial segment after acrosomal reaction and is involved in human fertilization since an anti-SPACA6 antibody inhibited it. Despite the similarity of the phenotypes caused by Spaca6 and Izumo1 knockouts, these are not redundant and the essential relocation of IZUMO1 is not affected by the lack of SPACA6. We propose a model in which IZUMO1 and SPACA6 would be part of a molecular complex necessary for gamete fusion and that their concomitant presence would be required for the recruitment of another essential molecular actor, such as a fusogen, for the fusion to take place.

Mutations in TTC29, Encoding an Evolutionarily Conserved Axonemal Protein, Result in Asthenozoospermia and Male Infertility.

In humans, structural or functional defects of the sperm flagellum induce asthenozoospermia, which accounts for the main sperm defect encountered in infertile men. Herein we focused on morphological abnormalities of the sperm flagellum (MMAF), a phenotype also termed "short tails," which constitutes one of the most severe sperm morphological defects resulting in asthenozoospermia. In previous work based on whole-exome sequencing of a cohort of 167 MMAF-affected individuals, we identified bi-allelic loss-of-function mutations in more than 30% of the tested subjects. In this study, we further analyzed this cohort and identified five individuals with homozygous truncating variants in TTC29, a gene preferentially and highly expressed in the testis, and encoding a tetratricopeptide repeat-containing protein related to the intraflagellar transport (IFT). One individual carried a frameshift variant, another one carried a homozygous stop-gain variant, and three carried the same splicing variant affecting a consensus donor site. The deleterious effect of this last variant was confirmed on the corresponding transcript and protein product. In addition, we produced and analyzed TTC29 loss-of-function models in the flagellated protist T. brucei and in M. musculus. Both models confirmed the importance of TTC29 for flagellar beating. We showed that in T. brucei the TPR structural motifs, highly conserved between the studied orthologs, are critical for TTC29 axonemal localization and flagellar beating. Overall our work demonstrates that TTC29 is a conserved axonemal protein required for flagellar structure and beating and that TTC29 mutations are a cause of male sterility due to MMAF.

Disorder-to-Order Markers of a Cyclic Hexapeptide Inspired from the Binding Site of Fertilin ß Involved in Fertilization Process.

Synthetic peptides mimicking the binding site of fertilin ß to its receptor, integrin α6ß1, were shown to inhibit sperm-egg fusion when added to in vitro media. In contrast, the synthetic cyclic hexapeptide, cyclo(Cys1-Ser2-Phe3-Glu4-Glu5-Cys6), named as cFEE, proved to stimulate gamete fusion. Owing to its biological specificity, this hexapeptide could help improve the in vitro fertilization pregnancy rate in human. In an attempt to establish the structure-activity relationship of cFEE, its structural dynamics was herein analyzed by means of ultraviolet circular dichroism (UV-CD) and Raman scattering. The low concentration CD profile in water, containing mainly a deep minimum at ∼202 nm, is consistent with a rather unordered chain. However, an ordering trend of the peptide loop has been observed in a less polar solvent such as methanol, where the UV-CD signal shape is formed by a double negative marker at ∼202/215 nm, indicating the presence of a type-II' ß-turn. Raman spectra recorded in aqueous samples upon a 100-fold concentration increase, still showed an important population (∼30%) of the disordered structure. The structural flexibility of the disulfide bridge was confirmed by the Raman markers arising from the Cys1-Cys6 disulfide bond-stretch motions. Density functional theory calculations highlighted the formation of the type-II' ß-turn on the four central residues of cFEE (i.e., -Ser2-Phe3-Glu4-Glu5-) either with a left- or with a right-handed disulfide. The structure with a left-handed S-S bond, however, appears to be more stable.

Contribution of Single-Cell Transcriptomics to the Characterization of Human Spermatogonial Stem Cells: Toward an Application in Male Fertility Regenerative Medicine?

Ongoing progress in genomic technologies offers exciting tools that can help to resolve transcriptome and genome-wide DNA modifications at single-cell resolution. These methods can be used to characterize individual cells within complex tissue organizations and to highlight various molecular interactions. Here, we will discuss recent advances in the definition of spermatogonial stem cells (SSC) and their progenitors in humans using the single-cell transcriptome sequencing (scRNAseq) approach. Exploration of gene expression patterns allows one to investigate stem cell heterogeneity. It leads to tracing the spermatogenic developmental process and its underlying biology, which is highly influenced by the microenvironment. scRNAseq already represents a new diagnostic tool for the personalized investigation of male infertility. One may hope that a better understanding of SSC biology could facilitate the use of these cells in the context of fertility preservation of prepubertal children, as a key component of regenerative medicine.