[Onco-TESE and testicular cancer]. / Onco-TESE et cancer du testicule.

INTRODUCTION: Fertility preservation is essential before cancer treatment. When ejaculated sperm preservation is not possible, testicular tissue can be surgically collected by Onco-TESE technic (Oncological Testicular Sperm Extraction) to isolate sperm. We report on our experience with Onco-TESE in testicular cancer patients at the Rouen University Hospital. MATERIAL AND METHOD: Retrospective study including all pubescent men, treated for testicular cancer, uni- or bilateral, before any carcinological therapy, who have undergone Onco-TESE at the Rouen University Hospital. Fragment weight, detection of sperm or its precursors were analysed. A histological interpretation of the testicular tumor was carried out. For each positive sample, straws were kept at the French Sperm Bank. RESULTS: Twenty-four patients had an Onco-TESE: 58.34% severe sperm alteration (SSA) and 41.36% sperm collection failure (SCF), between 1996 and 2019. The mean age was 26.6 (±5.29) years. The mean procedure and length of stay were 71minutes (±30.7) and 3.75 days (±2.83), respectively. The rate of positive testicular biopsies (TB) was 58.33% overall and 66,67% in the case of TB on tumour testis. One patient had a Clavian-Dindo III complication. The mean number of straws preserved per patient was 14.28 (±15.34) for 7.14% use. CONCLUSION: Our results seem to confirm that Onco-TESE is an effective solution for preserving fertility in men with testicular cancer in cases of SSA or SCF. LEVEL OF EVIDENCE: III.

Establishment, maintenance and functional integrity of the blood-testis barrier in organotypic cultures of fresh and frozen/thawed prepubertal mouse testes.

STUDY QUESTION: Can the spatio-temporal formation of an intact blood-testis barrier (BTB), which is essential for the progression of spermatogenesis, be reproduced in cultures of fresh or frozen/thawed prepubertal mouse testes? SUMMARY ANSWER: Organotypic cultures allow the establishment and maintenance of major BTB components and the formation of a functional BTB in mouse testicular tissues. WHAT IS KNOWN ALREADY: In vitro maturation of prepubertal testicular tissues is a promising approach to restore fertility in adult survivors of childhood cancer. Although gametes can be successfully obtained from prepubertal mouse testes in organotypic cultures, the spermatogenic yield remains low compared to in vivo controls. STUDY DESIGN, SIZE, DURATION: Mouse testicular tissues were frozen using controlled slow freezing (CSF) or solid surface vitrification (SSV) procedures. A total of 158 testes (fresh n = 58, CSF n = 58 or SSV n = 42) from 6 to 7 days postpartum (dpp) mice were cultured at 34°C in basal medium (α-MEM, 10% KnockOut Serum Replacement, 5 µg/ml gentamicin) at a gas-liquid interphase (under 20% O2), with or without 10-6 M retinol, for 9, 16 and 30 days. In addition, 32 testes from 6-7, 15-16, 22-23 and 36-37 dpp mice were used as in vivo controls. PARTICIPANTS/MATERIALS, SETTING, METHODS: The mRNA levels of BTB genes (Claudin 3, Claudin 11, Zonula occludens 1 and Connexin-43), germ cell-specific genes (Sal-like protein 4, Kit oncogene, Stimulated by retinoic acid gene 8, Synaptonemal complex protein 3, Transition protein 1 and Protamine 2), markers of Sertoli cell immaturity/maturity (anti-Mullerian hormone, androgen receptor, cyclin-dependent kinase inhibitor 1b) and the androgen-regulated gene Reproductive homeobox 5 (Rhox5) were measured by quantitative RT-PCR (RT-qPCR). The localization of BTB proteins in seminiferous tubules was studied by immunohistochemistry and spermatogenic progression was evaluated histologically. The integrity of the BTB was assessed using a biotin tracer. MAIN RESULTS AND THE ROLE OF CHANCE: Modest differences in Claudin 11 (Cldn11), Zonula occludens 1 (Zo-1), Connexin-43 (Cx43) transcript levels and in the localization of the corresponding proteins were found between in vitro cultures of fresh or frozen/thawed testes and in vivo controls (P < 0.05). However, a 32-77-fold decrease in Claudin 3 (Cldn3) mRNA levels and a lack of CLDN3 immunolabelling in 36-44% of seminiferous tubules were observed in 30-day organotypic cultures (P < 0.05). Although Sertoli cell maturation and the completion of a full spermatogenic cycle were achieved after 30 days of culture, meiotic and postmeiotic progression was altered in cultured testicular tissues (P < 0.05). Moreover, an increased BTB permeability and a decreased expression of Rhox5 were observed at the end of the culture period in comparison with in vivo controls (P < 0.05). Completion of spermatogenesis occurred in vitro in seminiferous tubules with an intact BTB, and in those expressing or lacking CLDN3. LARGE SCALE DATA: None. LIMITATIONS, REASONS FOR CAUTION: Further studies will be needed to determine whether the expression of other BTB components is altered and to decipher the reason for lower Cldn3 and Rhox5 mRNA levels in organotypic cultures. WIDER IMPLICATIONS OF THE FINDINGS: This work contributes to a better understanding of the molecular mechanisms occurring in in vitro matured prepubertal testes. The organotypic culture system will have to be developed further and optimized for human tissue, before potential clinical applications can be envisaged. STUDY FUNDING AND COMPETING INTEREST(S): This work was supported by Rouen University Hospital, Ligue contre le Cancer (to L.D.), and co-supported by European Union and Région Normandie (to A.O.). Europe gets involved in Normandie with European Régional Development Fund (ERDF). The authors declare that they have no conflict of interest.

Assessment of sperm nuclear quality after in vitro maturation of fresh or frozen/thawed mouse pre-pubertal testes.

STUDY QUESTION: Is nuclear quality of in vitro generated spermatozoa from fresh or frozen/thawed pre-pubertal mouse testes similar to that of their in vivo counterparts? SUMMARY ANSWER: The production of spermatozoa with aneuploidy, DNA fragmentation or chromatin condensation defects was not significantly increased in organotypic cultures compared to in vivo controls. WHAT IS KNOWN ALREADY: Although murine spermatozoa have been produced in vitro from pre-pubertal testes, their nuclear DNA integrity has never been investigated. STUDY DESIGN, SIZE, DURATION: Fresh and frozen/thawed testicular fragments from 6 to 7 days postpartum (dpp) mice were cultured for 30 days. Testicular tissues were frozen by controlled slow freezing (CSF) or solid surface vitrification (SSV). In total, 30 fresh, 30 CSF, 30 SSV testes were used for in vitro maturation and 6 testes from 36 to 37 dpp mice were used as in vivo controls. PARTICIPANTS/MATERIALS, SETTING, METHODS: Murine spermatozoa were extracted from pooled in vitro cultured testicular fragments and from in vivo controls. Sperm aneuploidy was analyzed by fluorescence in situ hybridization (FISH), DNA fragmentation by terminal deoxynucleotidyl transferase mediated dUTP nick end labeling, chromatin condensation by aniline blue staining, telomere length and number by quantitative FISH, DNA oxidation by immunocytochemical detection of 8-hydroxy-2'-deoxyguanosine (8-OHdG). Because of the low spermatogenic yield in cultures, a hundred spermatozoa extracted from pooled tissues were examined and compared to their in vivo counterparts. MAIN RESULTS AND THE ROLE OF CHANCE: Most of spermatozoa generated in vitro and in vivo were haploid, contained unfragmented DNA and normally condensed chromatin. A similar proportion of spermatozoa with aneuploidy, DNA fragmentation or chromatin condensation defects was found in cultures and in vivo. No significant difference in telomere length was found within the nuclei of in vitro and in vivo generated spermatozoa. However, the number of telomere spots was lower in gametes obtained from cultures of fresh, CSF and SSV testes than in their natural counterparts (P < 0.01). Moreover, the proportion of spermatozoa containing 8-OHdG was significantly increased in frozen/thawed tissues in comparison to fresh tissues and in vivo controls (P < 0.05). LARGE SCALE DATA: None. LIMITATIONS REASONS FOR CAUTION: Further studies will be needed to enhance the production of spermatozoa in organotypic cultures while preserving their quality, to investigate epigenetic modifications and embryonic development. WIDER IMPLICATIONS OF THE FINDINGS: This is the first study comparing the nuclear quality of in vitro and in vivo generated murine spermatozoa. The organotypic culture system will have to be adapted for human tissue and extensive analyses of human gamete quality will have to be performed before potential clinical applications can be envisaged. STUDY FUNDING AND COMPETING INTEREST(S): This work was supported by Rouen University Hospital, Ligue contre le Cancer, Agence de la Biomédecine, Association Laurette Fugain, France Lymphome Espoir, and co-supported by European Union and Région Normandie. Europe gets involved in Normandie with European Régional Development Fund (ERDF). The authors declare that they have no conflict of interest.

Evaluation of apoptotic- and autophagic-related protein expressions before and after IVM of fresh, slow-frozen and vitrified pre-pubertal mouse testicular tissue.

STUDY QUESTION: Do freezing and in vitro culture procedures enhance the expression of proteins involved in apoptotic or autophagic pathways in murine pre-pubertal testicular tissue? SUMMARY ANSWER: IVM strongly modified apoptosis- and autophagy-related relative protein levels in mice testicular tissue whereas the impact of cryopreservation procedures was minimal at the end of the culture. WHAT IS KNOWN ALREADY: In vitro spermatogenesis remains a challenging technical issue as it imposes to find a very close balance between survival and death of germ cell natural precursors (i.e. gonocytes and spermatogonia), which will eventually undergo a complete spermatogenesis close to in vivo conditions. The establishment of efficient culture conditions coupled with suitable cryopreservation procedures (e.g. controlled slow freezing [CSF] and solid surface vitrification [SSV]) of pre-pubertal testicular tissue is a crucial step in the fields of fertility preservation and restoration to improve the spermatic yield obtained in vitro. STUDY DESIGN, SIZE, DURATION: Here, we study cryopreservation procedures (i.e. CSF or SSV) and the impact of culture media compositions. A first set of 66 mouse pre-pubertal testes were directly cultured during 30, 36, 38 and 60 days (D) from 2.5 to 6.5-day-old CD-1 mice to evaluate the impact of time-aspect of culture and to endorse the reverse phase protein microarrays (RPPM) technique as an adapted experimental tool for the field of in vitro spermatogenesis. Ninety others fresh, slow-frozen and vitrified pre-pubertal testes were cultured during 30 days for the principal study to evaluate the impact of cryopreservation procedures before and after culture. Thirty-four testes dissected from 2.5, 6.5, 36.5, 40.5, 42.5 and 62.5 days postpartum (dpp) mice, corresponding to the time frames of spermatogenesis orchestrated in vitro, were used as in vivo controls. PARTICIPANTS/MATERIALS, SETTING, METHODS: After in vitro culture, testicular tissue samples originated from 2.5 or 6.5-day-old CD-1 male mice were analyzed using RPPM. This targeted proteomic technique allowed us to assess the expression level of 29 apoptosis- and autophagy-related factors by normalizing blank-corrected signal values. In addition, morphological analyses (e.g. HES, PAS, TRA98 and CREM) and DNA fragmentation in intra-tubular cells (i.e. terminal deoxynucleotidyl transferase dUTP nick end labeling; TUNEL) were assessed for the distinct experimental conditions tested as well as for in vivo control mouse testes. MAIN RESULTS AND THE ROLE OF CHANCE: A validation of the RPPM procedure in the field of in vitro spermatogenesis was completed with assay and array robustness before a principal study concerning the evaluation of the impact of in vitro culture and cryopreservation procedures. The proportion of elongated spermatids and the total cell number per seminiferous tubule tended to be very different between the in vivo and in vitro conditions (P < 0.05), suggesting the presence of a beneficial regulation on the first spermatogenesis wave by intrinsic apoptosis (Caspase_9) and autophagy (Atg5) factors (P < 0.0003 and r2 = 0.74). Concerning the impact of culture media compositions, a basic medium (BM) composed of αMEM plus 10% KnockOut™ serum replacement and gentamicin supplemented with retinol (Rol) and vitamin E (Vit. E) was selected as the best culture medium for fresh 6.5 dpp tissue cultured during 30D with 27.7 ± 8.10% of seminiferous tubules containing elongated spermatids. Concerning the impact of cryopreservation procedures, SSV did not have any impact on the morphological parameters evaluated after culture in comparison to fresh tissue (FT) controls. The proportion of tubules with elongated spermatids on testicular explants cultured with BMRol+Vit. E was not different between SSV (6.6 ± 1.6%) and CSF (5.3 ± 1.9%); however, round spermatids were observed more frequently for SSV (19 ± 6.2%) than CSF (3.3 ± 1.9%, P = 0.0317). Even if the proportion of TUNEL-positive cells for BMRol+Vit. E was higher at D30 after SSV (4.12 ± 0.26%) than CSF (1.86 ± 0.12%, P = 0.0022) and FT (2.69 ± 0.33%, P = 0.0108), the DNA damages observed at the end of the culture (i.e. D30) were similar to respective 6.5 dpp controls. In addition, the relative protein level expression ratio of an apoptotic factor, the phosphorylated FADD on Fas, was reduced by 64-fold in vitrified testes cultured with BMRol+Vit. E. Furthermore, we found in this study that the StemPro®-34 SFM culture medium supplemented with growth factors (e.g. EGF, bFGF, GDNF and LIF) prevented the differentiation of spermatogonial stem cells in favor of a significant proliferation with a better architectural pattern than in vivo 6.5 dpp controls with an increase of seminiferous tubules area for FT (P = 0.0357) and CSF (P = 0.0317). LIMITATIONS REASONS FOR CAUTION: Despite our promising results, the evaluation of apoptotic- and autophagic-related proteins was studied for a limited amount of proteins and on global testicular tissue. WIDER IMPLICATIONS OF THE FINDINGS: The data presented herein will help to improve apoptotic and autophagic understanding during the first spermatogenic wave. Moreover, our findings illustrate for the first time that, using finely-tuned experimental conditions, a testicular in vitro culture combined with proteomic technologies may significantly facilitate the study of cryopreservation procedures and in vitro culture evaluations. This study may also contribute to improve work on testicular tissues from pre-pubertal and adolescent cancer survivors. STUDY FUNDING/COMPETING INTEREST(S): This study was supported by a Ph.D. grant from the Rouen Normandie Université and a financial support from 'la Ligue nationale contre le cancer' (both awarded to L.D.), funding from Institute for Research and Innovation in Biomedicine (IRIB), Agence de la Biomédecine, and co-supported by European Union and Région Normandie. Europe gets involved in Normandie with European Regional Development Fund (ERDF). The authors declare that there is no conflict of interest.

[Evaluation of practices and costs of vasectomy. French monocentric experience]. / Évaluation des pratiques et des coûts de la vasectomie. Expérience monocentrique française.

INTRODUCTION: Since the law of 4 July 2001, vasectomy has been recognized as a method of male contraception. We report the experience of vasectomy practice in a hospital-university center. METHODS: A monocentric retrospective cohort study of 45 patients who benefited from a contraceptive vasectomy between July 2001 and May 2016. For each patient were studied: modalities of implementation, compliance with the recommendations of the 2001 law, costs and benefits generated by the intervention, the effectiveness of the gesture on the control spermograms, the satisfaction of the patients by a telephone questionnaire. RESULTS: The mean age was 41.3 years. The second consultation was carried out in 91 % of the cases but the reflection period was not respected in 24 % of the cases. Written consent was signed in 89 % of cases. Vasectomy was performed on an outpatient basis in 73 % of cases, under local anaesthesia in 6.7 % of cases. The average cost per patient was 660.63 euros for an average gain of 524.50 euros, a loss of 136.13 euros. On the control spermogram, 54.3 % were azoosperms but the 3-month delay was not observed in 23 % of them. No patients expressed regret after surgery. CONCLUSION: The recommendations of the 2001 law were not systematically followed. This lack of standardization of practices, potential reflection of a lack of interest, is to be highlighted with the extra cost generated. The revaluation of the act should be integrated into the reflection of improvement of male sterilization practices. LEVEL OF PROOF: 4.

Vitamin A prevents round spermatid nuclear damage and promotes the production of motile sperm during in vitro maturation of vitrified pre-pubertal mouse testicular tissue.

STUDY QUESTION: Does vitamin A (retinol, Rol) prevent round spermatid nuclear damage and increase the production of motile sperm during in vitro maturation of vitrified pre-pubertal mouse testicular tissue? SUMMARY ANSWER: The supplementation of an in vitro culture of ~0.75 mm3 testicular explants from pre-pubertal mice with Rol enhances spermatogenesis progression during the first spermatogenic wave. WHAT IS KNOWN ALREADY: The production of functional spermatozoa in vitro has only been achieved in the mouse model and remains a rare event. Establishing an efficient culture medium for vitrified pre-pubertal testicular tissue is now a crucial step to improve the spermatic yield obtained in vitro. The role of Rol in promoting the differentiation of spermatogonia and their entry into meiosis is well established; however, it has been postulated that Rol is also required to support their full development into elongated spermatids. STUDY DESIGN, SIZE, DURATION: A total of 60 testes from 6.5 days post-partum (dpp) mice were vitrified/warmed, cut into fragments and cultured for 30 days: 20 testes were used for light microscopy and histological analyses, 20 testes for DNA fragmentation assessment in round spermatids and 20 testes for induced sperm motility assessment. Overall, 16 testes of 6.5 dpp were used as in vitro fresh tissue controls and 12 testes of 36.5 dpp mice as in vivo controls. Testes were vitrified with the optimal solid surface vitrification procedure and cultured with an in vitro organ culture system until Day 30 (D30). Histological analysis, cell death, degenerating round spermatids, DNA fragmentation in round spermatids and induced sperm motility were assessed. Testosterone levels were measured in media throughout the culture by radioimmunoassay. MAIN RESULTS AND THE ROLE OF CHANCE: At D30, better tissue development together with higher differentiation of spermatogonial stem cells, and higher global cell division ability were observed for vitrified/warmed testicular fragments of ~0.75 mm3 with a culture medium supplemented with Rol compared to controls. During in vitro culture of vitrified pre-pubertal testicular tissue, Rol enhanced and maintained the entry of spermatogonia into meiosis and promoted a higher spermatic yield. Furthermore, decreased round spermatid nuclear alterations and DNA damage combined with induced sperm motility comparable to in vivo highlight the crucial role of Rol in the progression of spermatogenesis during the first wave. LIMITATIONS, REASONS FOR CAUTION: Despite our promising results, the culture media will have to be further improved and adapted within the context of a human application. WIDER IMPLICATIONS OF THE FINDINGS: The results have potential implications for the handling of human pre-pubertal testicular tissues cryopreserved for fertility preservation. However, because some alterations in round spermatids persist after in vitro culture with Rol, the procedure needs to be optimized before human application, bearing in mind that the murine and human spermatogenic processes differ in many respects. LARGE SCALE DATA: None. STUDY FUNDING AND COMPETING INTERESTS: This study was supported by a Ph.D. grant from the Normandy University and a financial support from 'la Ligue nationale contre le cancer' (both awarded to L.D.), funding from Rouen University Hospital, Institute for Research and Innovation in Biomedicine (IRIB) and Agence de la Biomédecine. The authors declare that there is no conflict of interest.

Light microscopy morphological characteristics of the sperm flagellum may be related to axonemal abnormalities.

Although electron microscopy provides a detailed analysis of ultrastructural abnormalities, this technique is not available in all laboratories. We sought to determine whether certain characteristics of the flagellum as assessed by light microscopy were related to axonemal abnormalities. Forty-one patients with an absence of outer dynein arms (type I), a lack of a central complex (type III) and an absence of peripheral doublets (type IV) were studied. Sperm morphology was scored according to David's modified classification. Flagella with an irregular thickness were classified as being of normal length, short or broken. There were correlations between missing outer dynein arms and abnormal, short or coiled flagellum. Type III patients showed the highest flagellar defects (a short (P = 0.0027) or an absent flagellum (P = 0.011)). Just over 68% of the irregular flagella were short in Type III patients, whereas this value was only 34.5% in type I and 26.4% in type IV (P = 0.002). There was a negative correlation between misassembly and spermatozoa of irregular flagella (r = -0.79; P = 0.019). It is concluded that light microscopy analysis of flagellum abnormalities may help provide a correct diagnosis, identify sperm abnormalities with fertility potentials and outcomes in assisted reproduction technologies and assess the genetic risk.

The feasibility of fertility preservation in adolescents with Klinefelter syndrome.

STUDY QUESTION: Is fertility preservation feasible after the onset of puberty in adolescents with Klinefelter syndrome (KS)? SUMMARY ANSWER: Fertility preservation counseling should be an integral part of the care of XXY adolescents. Frozen ejaculated or testicular spermatozoa and even frozen immature germ cells can give them the potential to conceive their genetic progeny. However, no biological or clinical parameters were predictive of mature or immature germ cell retrieval. WHAT IS KNOWN ALREADY: KS is the commonest sex chromosome disorder observed in azoospermic infertile males. Testicular sperm extraction success decreases with age and after testosterone therapy. Arguably, spermatozoa should be retrieved from KS males at the onset of puberty and before testosterone therapy to increase the chance of success. STUDY DESIGN, SIZE, DURATION: A retrospective study was performed in eight KS adolescents, aged between 15 and 17 years, who were referred for counseling about their future fertility to the center CECOS (Centre d'Etude et de Conservation des Oeufs et du Sperme humain) at Rouen University Hospital between October 2008 and December 2011. PARTICIPANTS/MATERIALS, SETTING, METHODS: The patients were first seen with their parents and then separately. It was proposed to them that they should provide a semen sample, if this was azoospermic, two other semen samples spaced by 3 months were collected. If azoospermia was confirmed, a bilateral testicular biopsy was proposed for sperm retrieval and testicular tissue preservation. Each adolescent met the psychologist before undergoing testicular biopsy. Paraffin-embedded testicular tissue was evaluated after staining with hematoxylin-eosin and saffron and immunostaining using vimentin, anti-Müllerian hormone, androgen receptor and MAGE-A4 antibodies. Sertoli cell maturity, germ cell identification and lamina propria alteration were assessed on seminiferous tubules. MAIN RESULTS AND THE ROLE OF CHANCE: KS adolescents were not deeply concerned about their future fertility and only became involved in the process of fertility preservation after at least three medical consultations. The parents agreed immediately that fertility preservation should be attempted. Seven non-mosaic XXY adolescents presented with azoospermia and one XXY/XY adolescent had oligozoospermia. Increased plasma levels of FSH and LH as well as bilateral testicular hypotrophy were observed in all patients. The XXY/XY adolescent banked four semen samples before testosterone replacement therapy. Two patients refused testicular biopsy. Five patients accepted a bilateral testicular biopsy. Spermatozoa were retrieved in one patient, elongated spermatids and spermatocytes I in a second patient. LIMITATIONS, REASONS FOR CAUTION: The number of patients enrolled in our study was low because the diagnosis of KS is only rarely made before or at the onset of puberty. Most XXY males are diagnosed in adulthood within the context of male infertility. WIDER IMPLICATIONS OF THE FINDINGS: Spermatozoa can be retrieved in semen sample and in testicular tissue of adolescent Klinefelter patients. Furthermore, the testis may also harbor spermatogonia and incompletely differentiated germ cells. However, the physician should discuss with the patient and his parents over a period of several months before collecting a semen sample and performing bilateral testicular biopsy. Fertility preservation might best be proposed to adolescent Klinefelter patients just after the onset of puberty when it is possible to collect a semen sample and when the patient is able to consider alternative options to achieve fatherhood and also to accept the failure of spermatozoa or immature germ cell retrieval.

Emergency IVF for embryo freezing to preserve female fertility: a French multicentre cohort study.

STUDY QUESTION: What are the outcomes of French emergency IVF procedures involving embryo freezing for fertility preservation before gonadotoxic treatment? SUMMARY ANSWER: Pregnancy rates after emergency IVF, cryopreservation of embryos, storage, thawing and embryo transfer (embryo transfer), in the specific context of the preservation of female fertility, seem to be similar to those reported for infertile couples undergoing ART. STUDY DESIGN, SIZE, DURATION: A French retrospective multicentre cohort study initiated by the GRECOT network-the French Study Group for Ovarian and Testicular Cryopreservation. We sent an e-mail survey to the 97 French centres performing the assisted reproduction technique in 2011, asking whether the centre performed emergency IVF and requesting information about the patients' characteristics, indications, IVF cycles and laboratory and follow-up data. The response rate was 53.6% (52/97). PARTICIPANTS/MATERIALS, SETTING, METHODS: Fourteen French centres reported that they performed emergency IVF (56 cycles in total) before gonadotoxic treatment, between 1999 and July 2011, in 52 patients. MAIN RESULTS AND THE ROLE OF CHANCE: The patients had a mean age of 28.9 ± 4.3 years, and a median length of relationship of 3 years (1 month-15 years). Emergency IVF was indicated for haematological cancer (42%), brain tumour (23%), sarcoma (3.8%), mesothelioma (n = 1) and bowel cancer (n = 1). Gynaecological problems accounted for 17% of indications. In 7.7% of cases, emergency IVF was performed for autoimmune diseases. Among the 52 patients concerned, 28% (n = 14) had undergone previous courses of chemotherapy before beginning controlled ovarian stimulation (COS). The initiation of gonadotoxic treatment had to be delayed in 34% of the patients (n = 19). In total, 56 cycles were initiated. The mean duration of stimulation was 11.2 ± 2.5 days, with a mean peak estradiol concentration on the day on which ovulation was triggered of 1640 ± 1028 pg/ml. Three cycles were cancelled due to ovarian hyperstimulation syndrome (n = 1), poor response (n = 1) and treatment error (n = 1). A mean of 8.2 ± 4.8 oocytes were retrieved, with 6.1 ± 4.2 mature oocytes and 4.4 ± 3.3 pronuclear-stage embryos per cycle. The mean number of embryos frozen per cycle was 4.2 ± 3.1. During follow-up, three patients died from the consequences of their disease. For the 49 surviving patients, 22.5% of the couples concerned (n = 11) requested embryo replacement. A total of 33 embryos were thawed with a post-thawing survival rate of 76%. Embryo replacement was finally performed for 10 couples with a total of 25 embryos transferred, leading to one biochemical pregnancy, one miscarriage and three live births. Clinical pregnancy rate and live birth per couple who wanted a pregnancy after cancer were, respectively, 36% (95% CI = 10.9-69.2%) and 27% (95% CI = 6.0-61%). LIMITATIONS, REASONS FOR CAUTION: The overall response rate for clinics was 53.6%. Therefore, it is not only that patients may not have been included, but also that those that were included were biased towards the University sector with a response rate of 83% (25/30) for a small number of patients. WIDER IMPLICATIONS OF THE FINDINGS: According to literature, malignant disease is a risk factor for a poor response to COS. However, patients having emergency IVF before gonadotoxic treatment have a reasonable chance of pregnancy after embryo replacement. Embryo freezing is a valuable approach that should be included among the strategies used to preserve fertility. STUDY FUNDING/COMPETING INTEREST(S): No external funding was sought for this study. None of the authors has any conflict of interest to declare.

Relationship between conventional sperm parameters and motile sperm organelle morphology examination (MSOME).

With the motile sperm organelle morphology examination (MSOME), spermatozoa morphology may be assessed directly on motile spermatozoa at high magnification (up to 6600×). This procedure describes more precisely spermatozoa abnormalities, especially head vacuoles. However, no consensus has been established concerning normal or abnormal MSOME criteria. The aim of our study was to define MSOME vacuole criteria assessed objectively with a digital imaging system software to establish a potential relationship between conventional semen parameters. A total of 440 semen samples were obtained from males consulting in Rouen University Hospital Reproductive Biology Laboratory. Conventional semen analysis (volume, sperm concentration, progressive motility, vitality and morphology) and MSOME assessment {sperm head length, width and area as well as vacuole number, vacuole area and relative vacuole area to sperm head [RVA (%) = [vacuole area (µm(2))/head area (µm(2))] × 100)]} were performed for each semen sample. Among our 440 males, 109 presented normal conventional semen parameters and 331 abnormal ones. Sperm head vacuoles were significantly larger in abnormal semen samples (p < 0.0001). RVA was the most discriminative MSOME criterion between normal and abnormal semen samples according to ROC curves analysis, and was negatively correlated with poor sperm morphology (r = -0.53, p < 0.0001). We concluded to (i) the normal occurrence of vacuoles in sperm head whatever the normality or abnormality of semen parameters, (ii) the discriminative function of the RVA to distinguish semen samples with normal and abnormal parameters, and (iii) the strong correlation between high RVA and poor sperm morphology.

Management of male infertility due to congenital bilateral absence of vas deferens should not ignore the diagnosis of cystic fibrosis.

Microsurgical or percutaneous epididymal sperm aspiration and intracytoplasmic sperm injection (ICSI) are proposed to overcome male infertility due to congenital bilateral absence of vas deferens (CBAVD). CBAVD has been associated with mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene and consequently, genetic counselling has to be addressed before beginning ICSI procedure. However, management of male infertility due to CBAVD should not ignore a mild form of cystic fibrosis. We describe the case of cystic fibrosis late diagnosis performed in a 49-year-old infertile men with CBAVD. CFTR molecular testing detected two mutations F508del and A455E corresponding to a cystic fibrosis genotype. Pneumological evaluation revealed a severe obstructive respiratory disease, bronchiectasis and high sweat chloride levels. Symptoms consistent with a cystic fibrosis have to be identified in infertile men with CBAVD before beginning assisted reproductive procedures.

[Assisted reproductive technologies with sperm donation]. / Assistance médicale à la procréation avec don de spermatozoïdes.

INTRODUCTION: The purpose of this review is to relate to the operating rules of CECOS in France and the legal, medical and ethical issues raised by sperm donation. MATERIAL AND METHODS: Review of articles and consensus conferences on this subject published in Medline (PubMed) selected from 1973 and 2011 according to their relevance and Acts recorded on official legislative French websites. RESULTS: The operating rules of CECOS were established by the Act of July 29, 2004, revised 6 August 2004 and July 7, 2011. Of the 21,759 children born of ART in France in 2009, 5.1% are from a sperm donation. From 1973 to 2006, 44,045 children are born after a sperm donation. Between 1973 and 2006, 16,971 donors are presented in the CECOS and only 10,347 donors have completely made their donation process. The main indication for use of donor sperm (75% of applications) is represented by men of infertile couples with nonobstructive azoospermia, the second indication is infertile men with oligospermia. In azoospermia, the application is usually performed after failure of testicular or epididymal surgical specimen. In oligozoospermia, claims made most often after several failures of intraconjugal ART. CONCLUSION: Many questions are still present around the conception of children by sperm donation. The legitimacy of maintaining anonymity in the gift remains widely debated.

Motivations and personality characteristics of candidate sperm and oocyte donors according to parenthood status: a national study from the French CECOS network.

STUDY QUESTION: In a non-commercial national gamete donation programme, do the motivations and personality characteristics of candidate sperm and oocyte donors differ according to their parenthood status? SUMMARY ANSWER: Moderate differences exist between non-parent and parent candidate donors in motivations for gamete donation and representations as well as in personality characteristics. WHAT IS KNOWN ALREADY: Several studies have analysed the motivations and experiences of oocyte or sperm donors, but mainly in countries where gamete donation is a commercial transaction, and very few studies have reported results of personality traits using personality inventory tests. No study has specifically investigated the motivations and personality characteristics of candidate gamete donors according to parenthood status. STUDY DESIGN SIZE DURATION: A prospective study was carried out including 1021 candidate donors from 21 centres (in university hospitals) of the national sperm and egg banking network in France between November 2016 and December 2018. PARTICIPANTS/MATERIALS SETTING METHODS: In total, 1021 candidate gamete donors were included in the study. During their first visit, male (n = 488) and female candidate donors (n = 533) completed a questionnaire on sociodemographic characteristics, their motivations for donation and their representations of donation, infertility and family. Secondly, a NEO Personality Inventory (NEO-PI-R) exploring the Big Five personality traits was completed online. Results were compared between parent and non-parent candidate donors. MAIN RESULTS AND THE ROLE OF CHANCE: Altruistic values were the principal motive for donation irrespective of parenthood status. Reassurance about their fertility or preservation of sperm for future use was more often reported in non-parent than in parent candidate donors. With regard to representation of gamete donation or of the family, independently of their parenthood status, candidate donors more frequently selected social rather than biological representations. Mean personality characteristics were in the normal range. Non-parent candidate donors had higher scores on openness and depression than parents, while parent candidate donors appeared more social than non-parents. LIMITATIONS REASONS FOR CAUTION: The personality characteristics inventory was not completed by all candidate donors included in the study. However, family status did not differ between the two groups (NEO-PI-R completed (n = 525) or not), while the group who completed the NEO-PI-R had a higher educational level. This national study was performed in a country where gamete donation is subject to strict legislation. WIDER IMPLICATIONS OF THE FINDINGS: In a global context where reproductive medicine is commercialized and gamete donor resources are limited, this study found that altruism and social representations of gamete donation and family are the main motivations for gamete donation in a country which prohibits financial incentive. These findings are relevant for health policy and for gamete donation information campaigns. STUDY FUNDING/COMPETING INTERESTS: Grant from the Agence de la Biomédecine, France. The authors have nothing to disclose related to this study. TRIAL REGISTRATION NUMBER: N/A.

Assessment of acrosome and nuclear abnormalities in human spermatozoa with large vacuoles.

BACKGROUND: Spermatozoa with large vacuoles (SLV) may have a negative impact on embryo development. The origin of these vacuoles is unknown. We evaluated acrosome and nucleus alterations in isolated SLV, versus unselected spermatozoa. METHODS: We studied 20 patients with teratozoospermia. Spermatozoa from the native semen sample and spermatozoa presenting a vacuole occupying >13.0% total head area, isolated under high magnification (×6600), were assessed. Confocal and transmission electron microscope evaluations were performed on SLV and native sperm, respectively. Acrosome morphology and DNA fragmentation were analysed using proacrosin immunolabelling (monoclonal antibody 4D4) and terminal deoxynucleotidyl transferase-mediated dUTP nick end labelling assay. Chromatin condensation was evaluated with aniline blue staining. Sperm aneuploidy was assessed using fluorescence in situ hybridization. RESULTS: SLV represented 38.0 ± 5.10% of motile spermatozoa obtained after gradient density centrifugation. Vacuoles were mainly in the anterior and median sperm head (45.7 ± 2.90 and 46.1 ± 3.00%, respectively). Abnormal acrosomes were increased in SLV compared with unselected spermatozoa (77.8 ± 2.49 versus 70.6 ± 2.62%; P = 0.014). Microscopic observations showed an exclusively nuclear localization of large vacuoles. Complete DNA fragmentation was higher in native spermatozoa (P < 0.0001) than SLV, while chromatin condensation was altered in SLV (P < 0.0001). Aneuploidy and diploidy rates were increased in SLV (P < 0.0001). CONCLUSIONS: Sperm vacuoles were exclusively nuclear. In our selected teratozoospermic population, aneuploidy and chromatin condensation defects were the main alterations observed in SLV. Based on results from this small sample of spermatozoa, we propose a global impairment of the spermatogenesis process as a common origin of the morphological alterations.

Fertility preservation in boys: recent developments and new insights †.

BACKGROUND: Infertility is an important side effect of treatments used for cancer and other non-malignant conditions in males. This may be due to the loss of spermatogonial stem cells (SSCs) and/or altered functionality of testicular somatic cells (e.g. Sertoli cells, Leydig cells). Whereas sperm cryopreservation is the first-line procedure to preserve fertility in post-pubertal males, this option does not exist for prepubertal boys. For patients unable to produce sperm and at high risk of losing their fertility, testicular tissue freezing is now proposed as an alternative experimental option to safeguard their fertility. OBJECTIVE AND RATIONALE: With this review, we aim to provide an update on clinical practices and experimental methods, as well as to describe patient management inclusion strategies used to preserve and restore the fertility of prepubertal boys at high risk of fertility loss. SEARCH METHODS: Based on the expertise of the participating centres and a literature search of the progress in clinical practices, patient management strategies and experimental methods used to preserve and restore the fertility of prepubertal boys at high risk of fertility loss were identified. In addition, a survey was conducted amongst European and North American centres/networks that have published papers on their testicular tissue banking activity. OUTCOMES: Since the first publication on murine SSC transplantation in 1994, remarkable progress has been made towards clinical application: cryopreservation protocols for testicular tissue have been developed in animal models and are now offered to patients in clinics as a still experimental procedure. Transplantation methods have been adapted for human testis, and the efficiency and safety of the technique are being evaluated in mouse and primate models. However, important practical, medical and ethical issues must be resolved before fertility restoration can be applied in the clinic.Since the previous survey conducted in 2012, the implementation of testicular tissue cryopreservation as a means to preserve the fertility of prepubertal boys has increased. Data have been collected from 24 co-ordinating centres worldwide, which are actively offering testis tissue cryobanking to safeguard the future fertility of boys. More than 1033 young patients (age range 3 months to 18 years) have already undergone testicular tissue retrieval and storage for fertility preservation. LIMITATIONS REASONS FOR CAUTION: The review does not include the data of all reproductive centres worldwide. Other centres might be offering testicular tissue cryopreservation. Therefore, the numbers might be not representative for the entire field in reproductive medicine and biology worldwide. The key ethical issue regarding fertility preservation in prepubertal boys remains the experimental nature of the intervention. WIDER IMPLICATIONS: The revised procedures can be implemented by the multi-disciplinary teams offering and/or developing treatment strategies to preserve the fertility of prepubertal boys who have a high risk of fertility loss. STUDY FUNDING/COMPETING INTERESTS: The work was funded by ESHRE. None of the authors has a conflict of interest.

Fertility preservation in adolescent males: experience over 22 years at Rouen University Hospital.

BACKGROUND: Sperm banking is a suitable procedure to prevent infertility after cancer therapy in male adolescents. We evaluated the feasibility of semen preservation in 156 adolescents aged between 13 and 20 years and then we assessed fertility outcome after treatment. METHODS: Age, urogenital history, indications for cryopreservation, histological diagnosis and semen parameters were recorded. Fertility status after treatment was assessed by a questionnaire addressed to those patients who had utilized sperm storage. Post-treatment semen analysis was performed for 22 patients. RESULTS: Cryopreservation was possible in 88.5% of cases. Azoospermia was detected in 2.6% of the patients at the time of diagnosis. Malignant disease accounted for 84% of our male adolescents. In this type of disease, semen parameters were significantly altered only among patients with metastatic malignant bone tumour. After treatment, nine patients presented azoospermia, five patients achieved pregnancy spontaneously, two achieved it after assisted reproductive technique using fresh ejaculated spermatozoa and one following sperm donation. Three failed with cryopreserved sperm. CONCLUSIONS: Semen cryopreservation is possible for most adolescents and, regardless of disease type, may be a means of preserving fertility prior to gonadotoxic treatment that might impair the spermatogenesis process.

[Laser assisted hatching: Rouen University Hospital outcomes]. / Eclosion embryonnaire assistée: évaluation des indications au CHU de Rouen.

OBJECTIVE: Despite technical progress in In Vitro Fertilisation (IVF) procedure, embryo implantation rate remains low. Assisted hatching has been proposed to facilitate natural embryo hatching and implantation. PATIENTS AND METHODS: Our study has evaluated whether laser assisted hatching improves implantation, pregnancy and live birth rates in different cases. We studied retrospectively 143 IVF cycles concerning more than 38 years old women, 166 IVF cycles after two previous implantation failures and 180 frozen-thawed embryo transfers. RESULTS: Population characteristics were comparable in hatched and control groups. Implantation, pregnancy and live birth rates in women more than 38 years old were comparable with or without assisted hatching. Concerning repeated implantation failures, even if implantation, pregnancy and live birth rates were higher in assisted hatching group (FIV or ICSI), the differences were not significant. After frozen-thawed embryo transfers, implantation rate was significantly better with assisted hatching (19.14% vs 8.84% [p=0.02]). DISCUSSION AND CONCLUSION: Assisted hatching improves embryo implantation rate after frozen-thawed embryo transfer.

Comparison of conditions for cryopreservation of testicular tissue from immature mice.

BACKGROUND: Cryopreservation of immature testicular tissue could be considered as a major step in fertility preservation for young boys with cancer. In the present study, eight different freezing protocols were evaluated in immature mice testis. METHODS: Testis from six-day-old mice were frozen using either 1,2-propanediol (PROH) or dimethylsulphoxide (DMSO: D) at 1.5 M. Different cooling rate curves were tested: (i) controlled slow protocol with seeding (CS+) or (ii) without seeding (CS-), (iii) controlled rapid protocol and (iv) non-controlled protocol. Cryodamage of seminiferous cords was semi-quantitatively determined, establishing a scoring of alterations. Cell viability and apoptosis induction were assessed on testicular cell suspensions immediately after digestion (D0) and after a 20-h culture period (D1). Cells recovered after digestion of 100 mg tissue and the rate of living and non-apoptotic cells were quantified at D0 and D1. A long-term culture (9 days) of testis pieces was carried out for the protocol offering the best survival. Testosterone production, intratubular cell proliferation and tubule growth were assessed. RESULTS: DMSO produced optimal results in the different cooling rate curves tested when compared with PROH. Optimal results were obtained for the DCS- procedure (P < 0.05). Testosterone production, tubule growth and cell proliferation of post-thaw pieces were similar to fresh samples. CONCLUSIONS: Testis freezing with 1.5 M DMSO in a CS- procedure was found to maintain not only immature testicular tissue architecture, but also viability of testicular cells, endocrine and partial exocrine functions of the testis. Semi-quantitative evaluation of seminiferous cord cryodamage can be effectively used to rapidly screen optimal freezing conditions and as a possible quality control in a human application.

[Comparison of oestradiol and testosterone levels in peripheral blood and spermatic cord blood in patients with secretory azoospermia]. / Comparaison des taux d'estradiol et de testostérone dans le sang périphérique et dans le sang spermatique chez les patients avec azoospermie sécrétoire.

OBJECTIVE: Comparison of testosterone and oestradiol levels in spermatic cord blood and peripheral blood as a function of the results of testicular sperm extraction by surgical biopsy in a population of patients with nonobstructive azoospermia (NOA). MATERIAL AND METHODS: Prospective, comparative, teaching hospital-sponsored, ethics committee-approved study of 30 patients with NOA. Plasma testosterone and oestradiol assays in peripheral blood and blood taken from the spermatic vein during testicular biopsy were performed simultaneously for each patient. Statistical analyses were performed with Mann-Whitney test and Spearman's correlation coefficient by univariate and multivariate analysis of variance (p<0.05). RESULTS: No significant correlation was demonstrated between spermatic and peripheral blood levels of testosterone and oestradiol, regardless of the biopsy results. The spermatic oestradiol/testosterone ratio was significantly increased when testicular sperm extraction was negative (p=0.018). CONCLUSION: The increase of the oestradiol/testosterone ratio in spermatic cord blood in the case of negative testicular sperm extraction suggests the hypothesis of greater conversion of testosterone to oestradiol in the testes. This could reflect increased aromatase activity in the absence of germ cells. Further studies using tissue markers of spermatogenesis should provide a better understanding of the physiological role of oestrogens in spermatogenesis and to refine the indications for testicular biopsy in patients with NOA.

[Laparoscopic ovarian transposition and cryopreservation of ovarian tissue before chemo-radiotherapy for rectal cancer]. / Transposition ovarienne par coelioscopie et cryopréservation de tissu ovarien avant radiochimiothérapie d'un cancer du rectum.

We report a case of laparoscopic ovarian transposition and ovarian cryopreservation in a nulliparous 25-year-old woman with rectal cancer. The authors focus on the surgical technique and the importance of preserving ovarian function.