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.

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.

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.

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.

Throughout in vitro first spermatogenic wave: Next-generation sequencing gene expression patterns of fresh and cryopreserved prepubertal mice testicular tissue explants.

Introduction: Suitable cryopreservation procedures of pre-pubertal testicular tissue associated with efficient culture conditions are crucial in the fields of fertility preservation and restoration. In vitro spermatogenesis remains a challenging technical procedure to undergo a complete spermatogenesis.The number of haploid cells and more specifically the spermatic yield produced in vitro in mice is still extremely low compared to age-matched in vivo controls and this procedure has never yet been successfully transferred to humans. Methods: To evaluate the impact of in vitro culture and freezing procedure, pre-pubertal testicular mice testes were directly cultured until day 4 (D4), D16 and D30 or cryopreserved by controlled slow freezing then cultured until D30. Testes composed of a panel of 6.5 dpp (days postpartum), 10.5 dpp, 22.5 dpp, and 36.5 dpp mice were used as in vivo controls. Testicular tissues were assessed by histological (HES) and immunofluorescence (stimulated by retinoic acid gene 8, STRA8) analyses. Moreover, a detailed transcriptome evaluation study has been carried out to study the gene expression patterns throughout the first in vitro spermatogenic wave. Results: Transcriptomic analyses reveal that cultured tissues expression profiles are almost comparable between D16 and D30; highlighting an abnormal kinetic throughout the second half of the first spermatogenesis during in vitro cultures. In addition, testicular explants have shown dysregulation of their transcriptomic profile compared to controls with genes related to inflammation response, insulin-like growth factor and genes involved in steroidogenesis. Discussion: The present work first shows that cryopreservation had very little impact on gene expression in testicular tissue, either directly after thawing or after 30 days in culture. Transcriptomic analysis of testis tissue samples is highly informative due to the large number of expressed genes and identified isoforms. This study provides a very valuable basis for future studies concerning in vitro spermatogenesis in mice.

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.

Experience, and gynaecological and reproductive health follow-up of young adult women who have undergone ovarian tissue cryopreservation.

RESEARCH QUESTION: What are the experience, and gynaecological and reproductive health outcomes in young adult women who have undergone ovarian tissue cryopreservation (OTC)? DESIGN: A retrospective observational study was conducted at a single institution between May 2019 and February 2021 including 87 women aged over 18 years undergoing OTC. Medical characteristics and questionnaire data collected more than 18 months after OTC were analysed. RESULTS: Close to 74% (n = 64/87) of women had a follow-up consultation and completed the questionnaire. Most women found the information provided on the OTC technique and the strategies proposed to restore fertility with ovarian tissue understandable and useful. The majority of patients thought that OTC had a positive impact on their well-being during disease treatment. Anti-Müllerian hormone serum concentration decreased significantly after treatment (P < 0.0001) and was significantly lower when patients received chemotherapy before OTC (P = 0.0039). The total cyclophosphamide equivalent dose was significantly higher in women with FSH concentrations above 25 IU/l after treatment (P = 0.0004). More than 70% of women who planned a pregnancy after the end of treatment succeeded, with a natural pregnancy rate close to 53%. Only nine patients (8.0%) underwent ovarian tissue transplantation for fertility restoration and six of them became pregnant and delivered at least once. CONCLUSION: Young adult women expressed a good satisfaction rate with OTC and that their experience had been beneficial. The usage rate of cryopreserved ovarian tissue remains low. The gynaecological and reproductive health follow-up consultation should be included in the supportive care provided following OTC.

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.

Achievement of complete in vitro spermatogenesis in testicular tissues from prepubertal mice exposed to mono- or polychemotherapy.

The assessment of the impact of chemotherapies on in vitro spermatogenesis in experimental models is required before considering the application of this fertility restoration strategy to prepubertal boys who received these treatments before testicular tissue cryopreservation. The present work investigated the effects of exposure of prepubertal mice to mono- (vincristine or cyclophosphamide) and polychemotherapy (a combination of vincristine and cyclophosphamide) on the first wave of in vitro spermatogenesis. When testicular tissue exposed to monochemotherapy was preserved, polychemotherapy led to severe alterations of the seminiferous epithelium and increased apoptosis in prepubertal testes prior in vitro maturation, suggesting a potential additive gonadotoxic effect. These alterations were also found in the testicular tissues of polychemotherapy-treated mice after 30 days of organotypic culture and were associated with a reduction in the germ cell/Sertoli cell ratio. The different treatments neither altered the ability of spermatogonia to differentiate in vitro into spermatozoa nor the yield of in vitro spermatogenesis. However, more spermatozoa with morphological abnormalities and fragmented DNA were produced after administration of polychemotherapy. This work therefore shows for the first time the possibility to achieve a complete in vitro spermatogenesis after an in vivo exposure of mice to a mono- or polychemotherapy before meiotic entry.

Protocol for developing a core outcome set for male infertility research: an international consensus development study.

STUDY QUESTION: We aim to develop, disseminate and implement a minimum data set, known as a core outcome set, for future male infertility research. WHAT IS KNOWN ALREADY: Research into male infertility can be challenging to design, conduct and report. Evidence from randomized trials can be difficult to interpret and of limited ability to inform clinical practice for numerous reasons. These may include complex issues, such as variation in outcome measures and outcome reporting bias, as well as failure to consider the perspectives of men and their partners with lived experience of fertility problems. Previously, the Core Outcome Measure for Infertility Trials (COMMIT) initiative, an international consortium of researchers, healthcare professionals and people with fertility problems, has developed a core outcome set for general infertility research. Now, a bespoke core outcome set for male infertility is required to address the unique challenges pertinent to male infertility research. STUDY DESIGN SIZE DURATION: Stakeholders, including healthcare professionals, allied healthcare professionals, scientists, researchers and people with fertility problems, will be invited to participate. Formal consensus science methods will be used, including the modified Delphi method, modified Nominal Group Technique and the National Institutes of Health's consensus development conference. PARTICIPANTS/MATERIALS SETTING METHODS: An international steering group, including the relevant stakeholders outlined above, has been established to guide the development of this core outcome set. Possible core outcomes will be identified by undertaking a systematic review of randomized controlled trials evaluating potential treatments for male factor infertility. These outcomes will be entered into a modified Delphi method. Repeated reflection and re-scoring should promote convergence towards consensus outcomes, which will be prioritized during a consensus development meeting to identify a final core outcome set. We will establish standardized definitions and recommend high-quality measurement instruments for individual core outcomes. STUDY FUNDING/COMPETING INTERESTS: This work has been supported by the Urology Foundation small project award, 2021. C.L.R.B. is the recipient of a BMGF grant and received consultancy fees from Exscentia and Exceed sperm testing, paid to the University of Dundee and speaking fees or honoraria paid personally by Ferring, Copper Surgical and RBMO. S.B. received royalties from Cambridge University Press, Speaker honoraria for Obstetrical and Gynaecological Society of Singapore, Merk SMART Masterclass and Merk FERRING Forum, paid to the University of Aberdeen. Payment for leadership roles within NHS Grampian, previously paid to self, now paid to University of Aberdeen. An Honorarium is received as Editor in Chief of Human Reproduction Open. M.L.E. is an advisor to the companies Hannah and Ro. B.W.M. received an investigator grant from the NHMRC, No: GNT1176437 is a paid consultant for ObsEva and has received research funding from Ferring and Merck. R.R.H. received royalties from Elsevier for a book, consultancy fees from Glyciome, and presentation fees from GryNumber Health and Aytu Bioscience. Aytu Bioscience also funded MiOXYS systems and sensors. Attendance at Fertility 2020 and Roadshow South Africa by Ralf Henkel was funded by LogixX Pharma Ltd. R.R.H. is also Editor in Chief of Andrologia and has been an employee of LogixX Pharma Ltd. since 2020. M.S.K. is an associate editor with Human Reproduction Open. K.Mc.E. received an honoraria for lectures from Bayer and Pharmasure in 2019 and payment for an ESHRE grant review in 2019. His attendance at ESHRE 2019 and AUA 2019 was sponsored by Pharmasure and Bayer, respectively. The remaining authors declare no competing interests. TRIAL REGISTRATION NUMBER: Core Outcome Measures in Effectiveness Trials (COMET) initiative registration No: 1586. Available at www.comet-initiative.org/Studies/Details/1586. TRIAL REGISTRATION DATE: N/A. DATE OF FIRST PATIENT'S ENROLMENT: N/A.

Sperm Chromatin Condensation Defect Accelerates the Kinetics of Early Embryonic Development but Does Not Modify ICSI Outcome.

The origin and quality of gametes are likely to influence the kinetics of embryonic development. The purpose of the study was to assess the impact of sperm nuclear quality, and in particular sperm chromatin condensation, on the kinetics of early embryo development after intracytoplasmic sperm injection (ICSI). Our study included 157 couples who benefitted from ICSI for male factor infertility. Chromatin condensation and DNA fragmentation were assessed in spermatozoa prior to ICSI. Above the 20% threshold of sperm condensation defect, patients were included in the abnormal sperm chromatin condensation (ASCC) group; below the 20% threshold, patients were included in the normal sperm chromatin condensation (NSCC) group. After ICSI, the oocytes were placed in the time-lapse incubator. The kinetics of the cohort's embryonic development have been modeled. The fading times of pronuclei and the time to two blastomeres (t2, first cleavage) and four blastomeres (t4, third cleavage) differed significantly between the NSCC and ASCC groups, with earlier events occurring in the ASCC group. On the other hand, the state of sperm chromatin condensation did not seem to have an impact on live birth rates or the occurrence of miscarriages. The kinetics of early embryonic development was accelerated in males with a sperm chromatin condensation defect without compromising the chances of pregnancy or promoting miscarriage. However, our study highlights the paternal contribution to early embryonic events and potentially to the future health of the conceptus.

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.

Medical biology in France: evolution and issues / Rapport des Académies nationales de médecine et de pharmacie. La biologie médicale en France : évolutions et enjeux

Medical biology is an essential part of patient care, both for the diagnosis and monitoring of diseases and for certain therapeutic advances. However, in recent years, it has been confronted with fundamental questions concerning its future. This report is the follow-up to the one published in 2018 by the National Academies of Medicine and Pharmacy and unfortunately only confirms a strong deterioration at all levels. The public authorities do not assume their role of regulator, thus allowing the excessive financialization of Medical Biology to grow considerably and lead to disproportionate groupings of Medical Biology Laboratories (MBL), destructive and sources of health risks. The result is that the Medical Biology Laboratories in towns, which are already known to be poorly distributed, are gradually becoming simple sampling sites, with patients finding themselves alone, often anxious, with their results sent to them by Internet without interpretation. Moreover, although progress in the field of Medical Biology is incredible and should constitute a major pole of attraction for young people, the disaffection of the discipline is total and worrying. Finally, innovation, in the context of current technological progress: connected devices, artificial intelligence and big data, represents a major challenge for the future. Here again, little or nothing is being done, even though the challenges are immense. After these alarming observations, the report will end with a series of recommendations aimed at optimizing the entry of MBL into a new era.

La biologie médicale est un maillon essentiel de la prise en charge des patients, tant pour le diagnostic et le suivi des maladies que pour certaines avancées thérapeutiques. Elle est toutefois, depuis quelques années, confrontée à des questions fondamentales concernant son avenir. Le présent rapport s'inscrit dans le prolongement de celui publié en 2018 par les Académies nationales de médecine et de pharmacie et ne fait malheureusement que conforter une forte dégradation à tous les niveaux. Les pouvoirs publics n'assument pas leur rôle de régulateur, permettant ainsi que la financiarisation à outrance de la biologie médicale s'amplifie considérablement et conduise à des regroupements démesurés des laboratoires de biologie médicale (LBM), destructeurs et sources de risques sanitaires. Le résultat est que les LBM de ville, dont on connaît déjà la mauvaise répartition territoriale, deviennent progressivement de simples sites de prélèvements, les patients se retrouvant alors seuls, souvent angoissés, avec leurs résultats transmis par Internet sans interprétation. Par ailleurs, bien que les progrès dans le domaine de la biologie médicale soient incroyables et devraient constituer un pôle d'attractivité majeur pour les jeunes, la désaffection de la discipline est totale et inquiétante. Enfin, l'innovation, dans le cadre des progrès technologiques actuels : dispositifs connectés, intelligence artificielle et mégadonnées (big data), représente un enjeu majeur pour l'avenir. Là encore rien n'est fait, ou presque, alors que les chantiers sont immenses. Après ces constatations alarmantes, le rapport se terminera par une série de recommandations visant à optimiser l'entrée des LBM dans une nouvelle ère.