A 20-year overview of fertility preservation in boys: new insights gained through a comprehensive international survey.

STUDY QUESTION: Twenty years after the inception of the first fertility preservation programme for pre-pubertal boys, what are the current international practices with regard to cryopreservation of immature testicular tissue? SUMMARY ANSWER: Worldwide, testicular tissue has been cryopreserved from over 3000 boys under the age of 18 years for a variety of malignant and non-malignant indications; there is variability in practices related to eligibility, clinical assessment, storage, and funding. WHAT IS KNOWN ALREADY: For male patients receiving gonadotoxic treatment prior to puberty, testicular tissue cryopreservation may provide a method of fertility preservation. While this technique remains experimental, an increasing number of centres worldwide are cryopreserving immature testicular tissue and are approaching clinical application of methods to use this stored tissue to restore fertility. As such, standards for quality assurance and clinical care in preserving immature testicular tissue should be established. STUDY DESIGN SIZE DURATION: A detailed survey was sent to 17 centres within the recently established ORCHID-NET consortium, which offer testicular tissue cryopreservation to patients under the age of 18 years. The study encompassed 60 questions and remained open from 1 July to 1 November 2022. PARTICIPANTS/MATERIALS SETTING METHODS: Of the 17 invited centres, 16 completed the survey, with representation from Europe, Australia, and the USA. Collectively, these centres have cryopreserved testicular tissue from patients under the age of 18 years. Data are presented using descriptive analysis. MAIN RESULTS AND THE ROLE OF CHANCE: Since the establishment of the first formal fertility preservation programme for pre-pubertal males in 2002, these 16 centres have cryopreserved tissue from 3118 patients under the age of 18 years, with both malignant (60.4%) and non-malignant (39.6%) diagnoses. All centres perform unilateral biopsies, while 6/16 sometimes perform bilateral biopsies. When cryopreserving tissue, 9/16 centres preserve fragments sized ≤5 mm3 with the remainder preserving fragments sized 6-20 mm3. Dimethylsulphoxide is commonly used as a cryoprotectant, with medium supplements varying across centres. There are variations in funding source, storage duration, and follow-up practice. Research, with consent, is conducted on stored tissue in 13/16 centres. LIMITATIONS REASONS FOR CAUTION: While this is a multi-national study, it will not encompass every centre worldwide that is cryopreserving testicular tissue from males under 18 years of age. As such, it is likely that the actual number of patients is even higher than we report. Whilst the study is likely to reflect global practice overall, it will not provide a complete picture of practices in every centre. WIDER IMPLICATIONS OF THE FINDINGS: Given the research advances, it is reasonable to suggest that cryopreserved immature testicular tissue will in the future be used clinically to restore fertility. The growing number of patients undergoing this procedure necessitates collaboration between centres to better harmonize clinical and research protocols evaluating tissue function and clinical outcomes in these patients. STUDY FUNDING/COMPETING INTERESTS: K.D. is supported by a CRUK grant (C157/A25193). R.T.M. is supported by an UK Research and Innovation (UKRI) Future Leaders Fellowship (MR/S017151/1). The MRC Centre for Reproductive Health at the University of Edinburgh is supported by MRC (MR/N022556/1). C.L.M. is funded by Kika86 and ZonMW TAS 116003002. A.M.M.v.P. is supported by ZonMW TAS 116003002. E.G. was supported by the Research Program of the Research Foundation-Flanders (G.0109.18N), Kom op tegen Kanker, the Strategic Research Program (VUB_SRP89), and the Scientific Fund Willy Gepts. J.-B.S. is supported by the Swedish Childhood Cancer Foundation (TJ2020-0026). The work of NORDFERTIL is supported by the Swedish Childhood Cancer Foundation (PR2019-0123; PR2022-0115), the Swedish Research Council (2018-03094; 2021-02107), and the Birgitta and Carl-Axel Rydbeck's Research Grant for Paediatric Research (2020-00348; 2021-00073; 2022-00317; 2023-00353). C.E is supported by the Health Department of the Basque Government (Grants 2019111068 and 2022111067) and Inocente Inocente Foundation (FII22/001). M.P.R. is funded by a Medical Research Council Centre for Reproductive Health Grant No: MR/N022556/1. A.F. and N.R. received support from a French national research grant PHRC No. 2008/071/HP obtained by the French Institute of Cancer and the French Healthcare Organization. K.E.O. is funded by the University of Pittsburgh Medical Center and the US National Institutes of Health HD100197. V.B-L is supported by the French National Institute of Cancer (Grant Seq21-026). Y.J. is supported by the Royal Children's Hospital Foundation and a Medical Research Future Fund MRFAR000308. E.G., N.N., S.S., C.L.M., A.M.M.v.P., C.E., R.T.M., K.D., M.P.R. are members of COST Action CA20119 (ANDRONET) supported by COST (European Cooperation in Science and Technology). The Danish Child Cancer Foundation is also thanked for financial support (C.Y.A.). The authors declare no competing interests. TRIAL REGISTRATION NUMBER: N/A.

A splice donor variant of GAS8 induces structural disorganization of the axoneme in sperm flagella and leads to nonsyndromic male infertility.

Motile cilia and flagella are closely related organelles structured around a highly conserved axoneme whose formation and maintenance involve proteins from hundreds of genes. Defects in many of these genes have been described to induce primary ciliary dyskinesia (PCD) mainly characterized by chronic respiratory infections, situs inversus and/or infertility. In men, cilia/flagella-related infertility is usually caused by asthenozoospermia due to multiple morphological abnormalities of the sperm flagella (MMAF). Here, we investigated a cohort of 196 infertile men displaying a typical MMAF phenotype without any other PCD symptoms. Analysis of WES data identified a single case carrying a deleterious homozygous GAS8 variant altering a splice donor consensus site. This gene, also known as DRC4, encodes a subunit of the Nexin-Dynein Regulatory Complex (N-DRC), and has been already associated to male infertility and mild PCD. Confirming the deleterious effect of the candidate variant, GAS8 staining by immunofluorescence did not evidence any signal from the patient's spermatozoa whereas a strong signal was present along the whole flagella length in control cells. Concordant with its role in the N-DRC, transmission electron microscopy evidenced peripheral microtubule doublets misalignments. We confirm here the importance of GAS8 in the N-DRC and observed that its absence induces a typical MMAF phenotype not necessarily accompanied by other PCD symptoms.

Efficient pathway for men fertility preservation in testicular cancer or lymphoma: a cross-sectional study of national 2018 data.

BACKGROUND: In 15-49 years-old men, the main cancers are testicular cancer (TC) and lymphomas (L): freezing of ejaculated sperm is primarily used for male fertility preservation (FP) before cancer treatment. Our objective was to analyze the French FP rate in 15-49 years-old men diagnosed with TC or L in 2018. We designed a national descriptive cross-sectional study of sperm banking rate in men with a diagnosis of TC, Hodgkin L (HL) or non-Hodgkin L (NHL). From the French National Cancer Institute (INCa) 2018 data, we extracted the estimated incidence of TC and L in metropolitan France. From the 2018 activity report of CECOS network (Centers for Study and Banking of Eggs and Sperm), we extracted the number of men with TC or L who banked ejaculated sperm. We estimated the proportion of 15-49 years-old men diagnosed with TC or L who banked sperm. RESULTS: Among 15-49 years-old men, INCa estimated 38,048 new cancer diagnoses in metropolitan France in 2018: 2,630 TC and 3,913 L (943 HL and 2,970 NHL). The CECOS network provided data from 26/27 metropolitan centers (96% response rate): 1,079 sperm banking for men with TC, 375 for HL and 211 for NHL. We estimated that the 2018 sperm banking rate in France was 41% for TC, 40% for HL, and 7% for NHL. CONCLUSIONS: To our knowledge, our paper is the first cross-sectional study with multicenter and national data analyzing FP rate in cancer men: it suggests an efficient pathway for men to FP before cancer treatment, compared to previously published studies. Although sperm banking rate in 15-49 years-old men could definitely be improved, further studies should evaluate the information given to patients before gonadotoxic treatments, the factors associated with the absence of sperm banking and whether this lack of referral induces a loss of chance for these men.

RéSUMé: CONTEXTE: Chez les hommes de 15 à 49 ans, les principaux cancers sont le cancer du testicule (CT) et les lymhomes (L): la congélation de spermatozoïdes éjaculés est utilisée en première intention pour leur préservation de fertilité (PF) avant traitement du cancer. Notre objectif était d'analyser le taux de PF chez les hommes de 15 à 49 ans diagnostiqués avec un CT ou un L en 2018 en France. Nous avons réalisé une étude nationale transversale descriptive du taux de congelation de spermatozoïdes chez les hommes âgés de 15 à 49 ans diagnostiqués avec un CT, un L de Hodgkin (LH) ou un L non-Hodgkinien (LNH). A partir des données de l'Institut National du Cancer (INCa) de 2018, nous avons extrait l'incidence estimée de CT et de L en France métropolitaine. A partir des données du bilan d'activité 2018 de la Federation Française des CECOS (Centre d'Etude et de Conservation des Oeufs et du Sperme), nous avons extrait le nombre d'hommes avec un CT ou un L qui ont congelé leurs spermatozoïdes. Nous avons enfin estimé la proportion d'hommes de 15 à 49 ans diagnostiqués avec un CT ou un L qui ont congelé leurs spermatozoïdes. RéSULTATS: Chez les hommes de 15 à 49 ans, l'INCa a estimé en 2018 38 048 nouveaux cas de cancers diagnostiqués en France métropolitaine en 2018: 2 630 CT et 3 913 L (943 LH et 2 970 LNH). Le réseau des CECOS a produit les résultats issus de 26/27 centres métropolitains (taux de réponse de 96%): 1 079 congélations de sperme pour des hommes atteints de CT, 375 pour LH et 211 pour LNH. Nous avons estimé que le taux de congelation de spermatozoïdes de 2018 en France était de 41% pour le CT, 40% pour le LH et 7% pour le LNH. CONCLUSIONS: A notre connaissance, notre travail est la première étude transversale multicentrique de données nationales analysant le taux de PF chez les hommes atteints de cancer: il suggère un parcours patient efficace pour la PF des hommes avant traitement d'un cancer, par rapport aux études précédemment publiées. Bien que le taux de PF chez les hommes puisse certainemen être amélioré, des études futures devraient évaluer l'information donnée aux patients avant traitement gonadotoxique, les facteurs associés à l'absence de PF et si le défaut d'adressage au CECOS induit un perte de chance pour ces hommes. MOTS-CLéS: Chimiothérapie, Radiothérapie, Oncofertiité, Azoospermia, Paternité.

Steroidogenesis and androgen/estrogen signaling pathways are altered in in vitro matured testicular tissues of prepubertal mice.

Children undergoing cancer treatments are at risk for impaired fertility. Cryopreserved prepubertal testicular biopsies could theoretically be later matured in vitro to produce spermatozoa for assisted reproductive technology. A complete in vitro spermatogenesis has been obtained from mouse prepubertal testicular tissue, although with low efficiency. Steroid hormones are essential for the progression of spermatogenesis, the aim of this study was to investigate steroidogenesis and steroid signaling in organotypic cultures. Histological, RT-qPCR, western blot analyses, and steroid hormone measurements were performed on in vitro cultured mouse prepubertal testicular tissues and age-matched in vivo controls. Despite a conserved density of Leydig cells after 30 days of culture (D30), transcript levels of adult Leydig cells and steroidogenic markers were decreased. Increased amounts of progesterone and estradiol and reduced androstenedione levels were observed at D30, together with decreased transcript levels of steroid metabolizing genes and steroid target genes. hCG was insufficient to facilitate Leydig cell differentiation, restore steroidogenesis, and improve sperm yield. In conclusion, this study reports the failure of adult Leydig cell development and altered steroid production and signaling in tissue cultures. The organotypic culture system will need to be further improved before it can be translated into clinics for childhood cancer survivors.

Results and perinatal outcomes from 189 ICSI cycles of couples with asthenozoospermic men and flagellar defects assessed by transmission electron microscopy.

RESEARCH QUESTION: Do patients presenting with flagella ultrastructural defects as assessed by electron microscopy, and defined within three phenotypes (dysplasia of the fibrous sheath [DFS], primary flagellar dyskinesia [PFD] and non-specific flagellar abnormalities [NSFA]), have decreased chances of success in intracytoplasmic sperm injection (ICSI) or adverse obstetric and neonatal outcomes? DESIGN: Retrospective analysis of 189 ICSI cycles from 80 men with spermatozoa flagellum ultrastructural defects (DFS [n = 16]; PFD [n = 14]; NSFA [n = 50] compared with a control group (n = 97). Cycles were cumulatively analysed. All fresh and frozen embryo transfers resulting from each ICSI attempt were included. The effect of transmission electron microscopy (TEM) phenotype on the main ICSI outcomes was assessed by a multivariate logistic regression combined with a generalized linear mixed model to account for the non-independence of the observations. RESULTS: No predictive value of TEM phenotype was found on the main outcomes of ICSI, namely fertilization rates, pregnancy and delivery rates, and cumulative pregnancy and delivery rates. Cumulative pregnancy rates ranged from 29.0-43.3% in the different TEM phenotype subgroups compared with 36.8% in the control group. Cumulative live birth rates ranged from 24.6-36.7% compared with 31.4% in the control group. No increase was found in miscarriages, preterm births, low birth weights or birth abnormalities. CONCLUSIONS: Data on the cumulative chances of success in ICSI of patients with ultrastructural flagellar defects, a rare cause of male infertility often associated with an underlying genetic cause, are reassuring, as are obstetrical and neonatal outcomes in this population.

Impact of low- or moderate-risk gonadotoxic chemotherapy prior to testicular tissue freezing on spermatogonia quantity in human (pre)pubertal testicular tissue.

STUDY QUESTION: What is the impact of low- or moderate-risk gonadotoxic chemotherapy received prior to testicular tissue freezing (TTF), and of the cancer itself, on spermatogonia quantity in testicular tissue from (pre)pubertal boys? SUMMARY ANSWER: Vincristine, when associated with alkylating agents, has an additional adverse effect on spermatogonia quantity, while carboplatin has no individual contribution to spermatogonia quantity, in testicular tissue of (pre)pubertal boys, when compared to patients who have received non-alkylating chemotherapy. WHAT IS KNOWN ALREADY: The improved survival rates after cancer treatment necessitate the inclusion of fertility preservation procedures as part of the comprehensive care for patients, taking into consideration their age. Sperm cryopreservation is an established procedure in post-pubertal males while the TTF proposed for (pre)pubertal boys remains experimental. Several studies exploring testicular tissue of (pre)pubertal boys after TTF have examined the tubular fertility index (TFI, percentage of seminiferous tubule cross-sections containing spermatogonia) and the number of spermatogonia per seminiferous tubule cross-section (S/T). All studies have demonstrated that TFI and S/T always decrease after the introduction of chemotherapeutic agents, especially those which carry high gonadotoxic risks such as alkylating agents. STUDY DESIGN, SIZE, DURATION: Testicular tissue samples from 79 (pre)pubertal boys diagnosed with cancer (from 6 months to 16 years of age) were cryopreserved between May 2009 and June 2014. Their medical diagnoses and previous chemotherapy exposures were recorded. We examined histological sections of (pre)pubertal testicular tissue to elucidate whether the chemotherapy or the primary diagnosis affects mainly TFI and S/T. PARTICIPANTS/MATERIALS, SETTING, METHODS: (Pre)pubertal boys with cancer diagnosis who had been offered TTF prior to conditioning treatment for hematopoietic stem cell transplantation were included in the study. All the patients had previously received chemotherapy with low- or moderate-risk for future fertility. We have selected patients for whom the information on the chemotherapy received was complete. The quantity of spermatogonia and quality of testicular tissue were assessed by both morphological and immunohistochemical analyses. MAIN RESULTS AND THE ROLE OF CHANCE: A significant reduction in the number of spermatogonia was observed in boys treated with alkylating agents. The mean S/T values in boys exposed to alkylating agents were significantly lower compared to boys exposed to non-alkylating agents (P = 0.018). In contrast, no difference was observed for patients treated with carboplatin as the sole administered alkylating agent compared to the group of patients exposed to non-alkylating agents. We observed an increase of S/T with age in the group of patients who did not receive any alkylating agent and a decrease of S/T with age when patients received alkylating agents included in the cyclophosphamide equivalent dose (CED) formula (r = 0.6166, P = 0.0434; r = -0.3759, P = 0.0036, respectively). The TFI and S/T decreased further in the group of patients who received vincristine in combination with alkylating agents (decrease of 22.4%, P = 0.0049 and P < 0.0001, respectively), but in this group the CED was also increased significantly (P < 0.0001). Multivariate analysis, after CED adjustment, showed the persistence of a decrease in TFI correlated with vincristine administration (P = 0.02). LIMITATIONS, REASONS FOR CAUTION: This is a descriptive study of testicular tissues obtained from (pre)pubertal boys who were at risk of infertility. The study population is quite heterogeneous, with a small number of patients in each sub-group. Our results are based on comparisons between patients receiving alkylating agents compared to patients receiving non-alkylating agents rather than chemotherapy-naive patients. The French national guidelines for fertility preservation in cancer patients recommend TTF before highly gonadotoxic treatment. Therefore, all the patients had received low- or moderate-risk gonadotoxic chemotherapy before TTF. Access to testicular tissue samples from chemotherapy-naive patients with comparable histological types of cancer was not possible. The functionality of spermatogonia and somatic cells could not be tested by transplantation or in vitro maturation due to limited sample sizes. WIDER IMPLICATIONS OF THE FINDINGS: This study summarizes the spermatogonial quantity of (pre)pubertal boys prior to TTF. We confirmed a negative correlation between the cumulative exposure to alkylating agents and spermatogonial quantity. In addition, the synergistic use of vincristine in combination with alkylating agents showed a cumulative deleterious effect on the TFI. For patients for whom fertility preservation is indicated, TTF should be proposed for chemotherapy with a predicted CED above 4000 mg/m2. However, the data obtained from vincristine and carboplatin use should be confirmed in a subsequent study including more patients. STUDY FUNDING/COMPETING INTEREST(S): This study had financial support from a French national research grant PHRC No. 2008/071/HP obtained by the French Institute of Cancer and the French Healthcare Organization. The sponsors played no role in the study. The authors declare no conflicts of interest. TRIAL REGISTRATION NUMBER: N/A.

[Restatement. Cancer and fertility preservation]. / Mise au point. Cancer et préservation de la fertilité.

CANCER AND FERTILITY PRESERVATION. The integration of fertility preservation into the treatment pathway is a major issue for quality of life after cancer, particularly for very young children, adolescents and young adults. Responses must be adapted to age, gender and treatment. The recommendations of the French National Cancer Institute (INCa) aim to promote information on the risks of different treatments for fertility and on the possibilities of preserving fertility, in order to allow an informed choice, and to improve the quality of the medical service rendered in order to reduce inequalities in care. Referral to a center specialized in fertility preservation is sometimes recommended, so that a technique adapted to the patient's situation can be implemented before treatment begins.

CANCER ET PRÉSERVATION DE LA FERTILITÉ. L'intégration de la préservation de la fertilité dans le parcours de soins est un enjeu majeur pour la qualité de vie après cancer, en particulier pour les très jeunes enfants, les adolescents et les jeunes adultes. Les réponses doivent être adaptées à l'âge, au sexe et au traitement. Les recommandations de l'Institut national du cancer (INCa) visent à favoriser l'information sur les risques des différents traitements vis-à-vis de la fertilité et sur les possibilités de la préserver, pour permettre un choix éclairé, et d'améliorer la qualité du service médical rendu afin de réduire les inégalités de soins. L'orientation vers un centre spécialisé dans la préservation de la fertilité est parfois préconisée, afin qu'une technique adaptée à la situation du patient puisse être mise en oeuvre avant le début des traitements.

New Mutations in DNHD1 Cause Multiple Morphological Abnormalities of the Sperm Flagella.

Male infertility is a common and complex disease and presents as a wide range of heterogeneous phenotypes. Multiple morphological abnormalities of the sperm flagellum (MMAF) phenotype is a peculiar condition of extreme morphological sperm defects characterized by a mosaic of sperm flagellum defects to a total asthenozoospermia. At this time, about 40 genes were associated with the MMAF phenotype. However, mutation prevalence for most genes remains individually low and about half of individuals remain without diagnosis, encouraging us to pursue the effort to identify new mutations and genes. In the present study, an a cohort of 167 MMAF patients was analyzed using whole-exome sequencing, and we identified three unrelated patients with new pathogenic mutations in DNHD1, a new gene recently associated with MMAF. Immunofluorescence experiments showed that DNHD1 was totally absent from sperm cells from DNHD1 patients, supporting the deleterious effect of the identified mutations. Transmission electron microscopy reveals severe flagellum abnormalities of sperm cells from one mutated patient, which appeared completely disorganized with the absence of the central pair and midpiece defects with a shortened and misshapen mitochondrial sheath. Immunostaining of IFT20 was not altered in mutated patients, suggesting that IFT may be not affected by DNHD1 mutations. Our data confirmed the importance of DNHD1 for the function and structural integrity of the sperm flagellum. Overall, this study definitively consolidated its involvement in MMAF phenotype on a second independent cohort and enriched the mutational spectrum of the DNHD1 gene.

Complete meiosis in rat prepubertal testicular tissue under in vitro sequential culture conditions.

BACKGROUND: Testicular tissue cryopreservation before gonadotoxic treatments allows fertility preservation in children suffering from cancer. Fertility restoration strategies, in particular in vitro maturation of prepubertal testicular tissue, are being developed mainly in animal models. The rat, widely used in biomedical research, including in reproductive biology, is a relevant model. OBJECTIVES: To determine whether sequential two-step culture protocols can improve the efficiency of rat in vitro spermatogenesis. MATERIALS AND METHODS: Rat prepubertal testicular tissues were cultured on agarose gels with either a one-step or two-step protocol with or without polydimethylsiloxane (PDMS) ceiling chips. The progression of spermatogenesis, germ/Sertoli cell ratio, cell proliferation, seminiferous tubule area, and intratubular cell density were assessed by histological and immunohistochemical analyses. Terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) assays and Peanut Agglutinin (PNA) lectin labeling were performed to analyze the Deoxyribonucleic Acid (DNA) integrity and differentiation step of in vitro-produced spermatids. RESULTS: Sequential two-step protocols allowed the production of spermatids with a higher efficiency compared with the one-step culture protocol. However, the efficiency was low, as less than 1.5% of tubules contained spermatids. Most of the in vitro-produced spermatids contained unfragmented DNA and were at an early step of differentiation. Rare elongating spermatids could be detected in the cultured explants. Although complete in vitro spermatogenesis could not be obtained with PDMS ceiling chips, entry into meiosis was promoted in one-step organotypic cultures. DISCUSSION AND CONCLUSION: Complete in vitro meiosis and the beginning of the elongation phase of spermiogenesis were obtained in a rat model using sequential culture methods. Because of their low efficiency, further work will be necessary to identify the culture conditions allowing the completion of spermiogenesis. These optimizations could pave the way for future applications, including the development of an in vitro fertility restoration procedure for childhood cancer survivors, which is still far from being clinically available.

What should be done in terms of fertility preservation for patients with cancer? The French 2021 guidelines.

AIM: To provide practice guidelines about fertility preservation (FP) in oncology. METHODS: We selected 400 articles after a PubMed review of the literature (1987-2019). RECOMMENDATIONS: Any child, adolescent and adult of reproductive age should be informed about the risk of treatment gonadotoxicity. In women, systematically proposed FP counselling between 15 and 38 years of age in case of treatment including bifunctional alkylating agents, above 6 g/m2 cyclophosphamide equivalent dose (CED), and for radiation doses on the ovaries ≥3 Gy. For postmenarchal patients, oocyte cryopreservation after ovarian stimulation is the first-line FP technique. Ovarian tissue cryopreservation should be discussed as a first-line approach in case of treatment with a high gonadotoxic risk, when chemotherapy has already started and in urgent cases. Ovarian transposition is to be discussed prior to pelvic radiotherapy involving a high risk of premature ovarian failure. For prepubertal girls, ovarian tissue cryopreservation should be proposed in the case of treatment with a high gonadotoxic risk. In pubertal males, sperm cryopreservation must be systematically offered to any male who is to undergo cancer treatment, regardless of toxicity. Testicular tissue cryopreservation must be proposed in males unable to cryopreserve sperm who are to undergo a treatment with intermediate or severe risk of gonadotoxicity. In prepubertal boys, testicular tissue preservation is: - recommended for chemotherapy with a CED ≥7500 mg/m2 or radiotherapy ≥3 Gy on both testicles. - proposed for chemotherapy with a CED ≥5.000 mg/m2 or radiotherapy ≥2 Gy. If several possible strategies, the ultimate choice is made by the patient.

Understanding the Underlying Molecular Mechanisms of Meiotic Arrest during In Vitro Spermatogenesis in Rat Prepubertal Testicular Tissue.

In vitro spermatogenesis appears to be a promising approach to restore the fertility of childhood cancer survivors. The rat model has proven to be challenging, since germ cell maturation is arrested in organotypic cultures. Here, we report that, despite a meiotic entry, abnormal synaptonemal complexes were found in spermatocytes, and in vitro matured rat prepubertal testicular tissues displayed an immature phenotype. RNA-sequencing analyses highlighted up to 600 differentially expressed genes between in vitro and in vivo conditions, including genes involved in blood-testis barrier (BTB) formation and steroidogenesis. BTB integrity, the expression of two steroidogenic enzymes, and androgen receptors were indeed altered in vitro. Moreover, most of the top 10 predicted upstream regulators of deregulated genes were involved in inflammatory processes or immune cell recruitment. However, none of the three anti-inflammatory molecules tested in this study promoted meiotic progression. By analysing for the first time in vitro matured rat prepubertal testicular tissues at the molecular level, we uncovered the deregulation of several genes and revealed that defective BTB function, altered steroidogenic pathway, and probably inflammation, could be at the origin of meiotic arrest.

Assessment of the architecture and integrity of frozen-thawed testicular tissue from (pre)pubertal boys with cancer.

BACKGROUND: Testicular tissue freezing is proposed for fertility preservation to (pre)pubertal boys with cancer before highly gonadotoxic treatment. Studies accurately comparing human (pre)pubertal testicular tissue quality before freezing and after thawing are exceptional. No study has reported this approach in a systematic manner and routine care. OBJECTIVES: To assess the impact of a control slow freezing protocol on testicular tissue architecture and integrity of (pre)pubertal boys after thawing. MATERIALS AND METHODS: (Pre)pubertal boys (n = 87) with cancer from 8 Reproductive Biology Laboratories of the French CECOS network benefited from testicular tissue freezing before hematopoietic stem cell transplantation. Seminiferous tubule cryodamage was determined histologically by scoring morphological alterations and by quantifying intratubular spermatogonia and the expression of DNA replication and repair marker in frozen-thawed testicular fragments. RESULTS: A significant increase in nuclear and epithelial score alterations was observed after thawing (p < 0.0001). The global lesional score remained lower than 1.5 and comparable to fresh testicular tissue. The number of intratubular spermatogonia and the expression of DNA replication and repair marker in spermatogonia and Sertoli cells did not vary significantly after thawing. These data showed the good preservation of the seminiferous tubule integrity and architecture after thawing, as previously reported in our studies performed in prepubertal mice and rats. DISCUSSION: The current study reports, for the first time, the development of a semi-quantitative analysis of cryodamage in human (pre)pubertal testicular tissue, using a rapid and useful tool that can be proposed in routine care to develop an internal and external quality control for testicular tissue freezing. This tool can also be used when changing one or several parameters of the freezing-thawing procedure. CONCLUSION: Control slow freezing protocol without seeding maintains the seminiferous tubule architecture and integrity, the concentration of spermatogonia and the expression of DNA replication and repair marker in spermatogonia and Sertoli cells after thawing.

Challenging cases in oncofertility: insights from a national specialized e-meeting for fertility preservation specialists.

PURPOSE: To determine the use of a new specialized E-Meeting for Complex Cases in Oncofertility by fertility preservation specialists (FPSs) MATERIAL AND METHODS: We present 3 years of activity of the E-Meeting for Complex Cases in Oncofertility, a new tool created in September 2016 which allows national oncofertility experts to share viewpoints about challenging cases for which they do not have experience or sufficient data in order to provide them an emergency advice within 48 h. Second, a survey was conducted to evaluate the use of this e-meeting for participating FPSs. RESULTS: One hundred and four experts have joined the e-meeting since its set-up, and 109 challenging cases have been submitted. The mean age of the patients was 22.4 ± 8.9 years, and 87.0% were female. Each submitted case received on average of 1.8 ± 1.1 different strategies for FP and the opinions of 7.1 ± 3.4 experts. Among the FPSs who submitted cases, seeking opinions from other FPSs allowed them to confirm their care plan (N = 49, 84.4%), to offer different options to their patients (N = 34, 58.6%), and to compare their practices with those of other specialists (N = 23, 39.6%). All respondents reported a self-perceived improvement in their practice of oncologic FP (n = 80, 100.0%). CONCLUSION: Specific attention should be paid to challenging cases for which the experiences of only a few individuals exist. Enhancing communication between FPSs through oncofertility networks, pooling experiences, and collecting the most complex cases is required to improve the management of these patients.

Activation of the cannabinoid receptor type 2 by the agonist JWH133 promotes the first wave of in vitro spermatogenesis.

BACKGROUND: Oncological procedures have irreversible side effects on germ cells for childhood cancer survival boys. In vitro culture of prepubertal testicular tissue has been proposed to restore fertility; however, recent data on animal models showed that meiotic and post-meiotic progression was impaired. OBJECTIVES: As potential key inducers of the mitosis-meiosis switch, type 2 cannabinoid receptor (CB2 ) has been proposed to play a central role in the meiotic entry of male germ cells. Herein, the in vitro first spermatogenesis wave in mice was used to understand the impact of CB2 activation on the differentiation of spermatogonia until elongated spermatids. MATERIALS AND METHODS: A first set of cultured testicular explants of 6.5 days post-partum (dpp) mice was performed to assess the impact of a range of JWH133 supplementation (10 nm, 100 nm, 1 µm, 10 µm). Then, the progressive development of germ cells at key timepoints of spermatogenesis was evaluated throughout (i) in vitro culture (day 2 [D2], D3, D6, D10, D18, and D30) coupled with (ii) in vivo counterparts (8.5, 9.5, 12.5, 16.5, 24.5, and 36.5 dpp). RESULTS: CB2 was detected at the plasma membrane of cells, and a successful completion of spermatogenesis was obtained in vitro. One day after the activation of CB2 by 1 µm of the agonist JWH133, percentage of zygotene spermatocyte I increased. CONCLUSION: After 30 days of culture, (i) an enrichment of haploid germ cells detected by flow cytometry, (ii) a reduced necrotic area, and (iii) an increase in the density of post-meiotic germ cells were observed. We showed that the activation of CB2 improves in vitro entry into meiosis and differentiation of spermatogonia, mimicking physiological meiotic transition.

Dynamics of epigenetic modifications in ICSI embryos from in vitro-produced spermatozoa.

BACKGROUND: In prepubertal boys with cancer, fertility preservation relies on testicular tissue freezing before treatment. In vitro maturation of frozen/thawed tissues could be one of the procedures envisaged to restore the fertility of cured patients. It is necessary to ascertain in the mouse model that in vitro-generated spermatozoa are able to ensure embryo development, without altering the epigenetic processes occurring during the pre-implantation period. OBJECTIVES: The aims of the present study were to investigate the fertilizing ability of in vitro-produced spermatozoa and explore several epigenetic marks at different stages of embryo development. MATERIALS AND METHODS: Fresh or controlled slow-frozen (CSF)/thawed testicular tissues from 6 to 7 days post-partum (dpp) mice were cultured for 30 days. Intracytoplasmic sperm injection (ICSI) experiments were performed using in vitro-produced spermatozoa. Testicular spermatozoa from 36 to 37 dpp mice were used as in vivo controls. DNA methylation/hydroxymethylation and histone post-translational modifications (H3K4me3, H3K27me3 and H3K9ac) were analysed by immunofluorescence from the zygote to the blastocyst stages. RESULTS: The spermatozoa generated in cultures of fresh or CSF testicular tissues were able to initiate embryonic development. The freezing of prepubertal testicular tissues limits the production of spermatozoa in vitro and the fertilization rate after ICSI. Similar levels of H3K4me3, H3K27me3 and H3K9ac were found in ICSI embryos derived from in vitro- and in vivo-produced spermatozoa. DNA methylation levels were increased in 4-cell embryos and morula obtained by ICSI with in vitro-produced spermatozoa. DISCUSSION AND CONCLUSION: Our study shows for the first time that the use of in vitro-produced spermatozoa alters DNA methylation/demethylation dynamics but has little impact on H3K4me3, H3K27me3 and H3K9ac levels in mouse early embryos. Further work will have to be performed to determine whether the use of these gametes is not deleterious for embryo development before considering a human application.

Improving Freezing Protocols and Organotypic Culture: A Histological Study on Rat Prepubertal Testicular Tissue.

Testicular tissue freezing before gonadotoxic treatments allows the preservation of fertility for children suffering from cancer. Recently, the testis organ culture method was presented as a relevant method to restore the fertility of these patients. However, the yield of spermatozoa production is low in the mouse model and no gamete has been obtained in vitro in the rat model. Here, we assess different cryopreservation protocols and culture conditions to improve the efficiency of in vitro maturation of rat prepubertal testes. Testes from male rats aged 5 or 8 days post-partum were cultured onto agarose gels of different percentages. After determining the best culture conditions, different cryopreservation protocols were assessed. Finally, testicular tissues were cultured with media of various compositions and analyzed at different time points. Our results show that the cryopreservation protocols allow the preservation of tissue architecture, cell proliferation, with no or moderate increase of cell death. In vitro spermatogenesis did not proceed beyond the pachytene spermatocyte stage. Only 2 of the 6 tested media allowed the survival of differentiated germ cells over the 45-day culture period. In conclusion, this study highlights the necessity to further improve the organ culture method before applying it into the clinics.

[AFU and SALF recommendations for the evaluation of male infertility]. / Recommandations de l'AFU et de la SALF concernant l'évaluation de l'homme infertile.

BACKGROUND: The aim of these Association Française d'Urologie (AFU) and Société d'Andrologie de Langue Française (SALF) common recommendations are to provide practice guidelines for the French Urological and Andrological community regarding the evaluation of infertile men. MATERIAL AND METHODS: Literature search in PubMed using the keywords "male infertility", "diagnosis", "management" and "evaluation" limited to clinical articles in English and French prior to 1/01/2020. To inform the level of evidence, the HAS grading system (2013) was applied. RESULTS: Concerning the evaluation of infertile men, the AFU and the SALF recommend : (1) a systematic interview exploring the family history, the fertility history of the man outside the couple, the patient's personal history that may have an impact on his fertility, lifestyle habits, treatments, symptoms and possible sexual difficulties of the couple; (2) a general physical examination to assess signs of hypogonadism and secondary sexual characters; (3) a scrotal physical examination performed by an urologist or andrologist to assess (i) the testes for volume and consistency, (ii) vas deferens and epididymes for total or partial absence or nodules, and (iii) presence of varicoceles; (4) Performing two semen analyses, according to World Health Organization guidelines, if the first one has at least one abnormaly; (5) a scrotal ultrasound as part of routine investigation, that can be completed with an endorectal pelvic ultrasound according to the clinic; (6) an endocrine evaluation with at least a Testosterone and FSH serum determination; (7) Karyotype analysis in infertile men with a sperm concentration ≤10 106/mL; (8) assessment of Yq microdeletions in infertile men with a sperm concentration ≤1 106/mL; (9) Cystic fibrosis transmembrane conductance regulator gene evaluation in case of suspicion for bilateral or unilateral congenital agenesis of vas deferens and seminal vesicles. The interest of tests analyzing DNA fragmentation (TUNEL, SCSA) is still under investigation. CONCLUSION: These guidelines can be applied in routine clinical practice in all infertile men.

Paradoxical risk of reduced fertility after exposure of prepubertal mice to vincristine or cyclophosphamide at low gonadotoxic doses in humans.

Cancer treatment can have long-term side effects in cured patients and infertility is one of them. Given the urgency of diagnosis in children with cancer, the toxicity of treatments on the gonad was overshadowed for a long time. In the present study, prepubertal mice were treated by vincristine or cyclophosphamide commonly used in acute leukaemia treatment. The prepubertal exposure to cyclophosphamide, at a low gonadotoxic dose in humans (< 3.5 g/m2), led to morphological alterations of prepubertal testicular tissue. An increased proportion of spermatozoa with hypocondensed chromatin and oxidized DNA associated with decreased fertility were uncovered at adulthood. Short- and long-term morphological alterations of the testicular tissue, disturbed progression of spermatogenesis along with increased proportions of isolated flagella and spermatozoa with fragmented DNA were evidenced in vincristine-treated mice. Moreover, the fertility of mice exposed to vincristine was severely affected despite being considered low-risk for fertility in humans. Paternal exposure to vincristine or cyclophosphamide before puberty had no impact on offspring development. Contrary to the current gonadotoxic risk classification, our results using a mouse model show that vincristine and cyclophosphamide (< 3.5 g/m2) present a high gonadotoxic risk when administered before the initiation of spermatogenesis.

Exposure to Chemotherapy During Childhood or Adulthood and Consequences on Spermatogenesis and Male Fertility.

Over the last decade, the number of cancer survivors has increased thanks to progress in diagnosis and treatment. Cancer treatments are often accompanied by adverse side effects depending on the age of the patient, the type of cancer, the treatment regimen, and the doses. The testicular tissue is very sensitive to chemotherapy and radiotherapy. This review will summarize the epidemiological and experimental data concerning the consequences of exposure to chemotherapy during the prepubertal period or adulthood on spermatogenic progression, sperm production, sperm nuclear quality, and the health of the offspring. Studies concerning the gonadotoxicity of anticancer drugs in adult survivors of childhood cancer are still limited compared with those concerning the effects of chemotherapy exposure during adulthood. In humans, it is difficult to evaluate exactly the toxicity of chemotherapeutic agents because cancer treatments often combine chemotherapy and radiotherapy. Thus, it is important to undertake experimental studies in animal models in order to define the mechanism involved in the drug gonadotoxicity and to assess the effects of their administration alone or in combination on immature and mature testis. These data will help to better inform cancer patients after recovery about the risks of chemotherapy for their future fertility and to propose fertility preservation options.