Jean-Pierre Milazzo

Rapid Screening of Cryopreservation Protocols for Murine Prepubertal Testicular Tissue by Histology and PCNA Immunostaining

Numerous parameters have to be tested to identify optimal conditions for prepubertal testicular tissue banking. Our study evaluated 19 different cryopreservation conditions for immature testicular tissue using a rapid screening method. Immature mice testes were cryopreserved using either 1,2-propanediol (PROH) or dimethyl sulfoxide (DMSO) at a concentration of 0.75 or 1.5 M using a controlled slow-cooling rate protocol with (S+) or without seeding (S+). Equilibration was performed either at room temperature or at 4°C for 15 or 30 minutes. Seminiferous cord cryodamage was determined by scoring morphologic alterations. Cell proliferation ability was evaluated using a proliferating cell nuclear antigen (PCNA) antibody. Testes cryopreserved with optimal conditions were grafted into immunodeficient mice. The highest proportions of PCNA-positive nuclei and lowest morphologic alterations were observed with 1.5 M DMSO. Tissues were more altered with 0.75 M DMSO or PROH. Complete germ cell maturation was observed after allografting of testicular pieces previously frozen with 1.5 M DMSO, S+, 30 minutes. The 1.5 M DMSO, S+ or S+ protocol preserved prepubertal mice testicular tissue architecture and germ cell and Sertoli cell proliferation potential. Allografting of thawed testis fragments into immunodeficient mice confirmed that the 1.5 M DMSO, S+, 30 minutes protocol maintained testicular somatic and germ cell functions. Postthaw histologic evaluation and PCNA immunostaining are useful to rapidly test numerous freeze-thaw parameters. They may also be efficient tools to control human prepubertal frozen testis quality, within the context of a clinical application.

Retinol Improves In Vitro Differentiation of Pre-Pubertal Mouse Spermatogonial Stem Cells into Sperm during the First Wave of Spermatogenesis

Testicular tissue freezing has been proposed for fertility preservation in pre-pubertal boys. Thawed frozen testicular tissue must undergo a maturation process to restore sperm production. The purpose of the current study was to evaluate the ability of retinol to improve the in vitro differentiation of pre-pubertal mouse spermatogonial stem cells into sperm. Testes from pre-pubertal mice, aged 2.5 and 6.5 days post-partum, were cultured on agarose gel at a gas-liquid interphase for 34, 38 and 60 days (D) and for 16, 30 and 36 D respectively. Assessment of basal medium (BM) supplemented with retinol (RE) alone, FSH/LH alone or a combination of both, was performed. Stereological analyses and tissue lesion scoring were performed at the culture time points indicated above. Sperm production was quantified at D30 and D34 after mechanical dissection of the testicular tissues. FSH/LH significantly increased the percentage of round spermatids at D30 and D38, when compared to BM alone. However, RE significantly increased the percentages of round but also elongated spermatids at D30 and D34. Moreover, RE significantly increased the number of spermatozoa per milligram of tissue at D30 and D34 when compared to BM. Therefore, RE improved the in vitro production of spermatids and spermatozoa from pre-pubertal SSCs during the first wave of spermatogenesis. The use of RE could be a useful tool for in vitro spermatogenesis from pre-pubertal human testicular tissue.

Assessment of freezing procedures for rat immature testicular tissue

Fertility preservation has been included in the management of childhood cancer treatment. Cryopreservation of immature testicular tissue is the only available solution for pre-pubertal boys. Different freezing protocols have been developed in several species but without a clearly identified procedure. We tried to evaluate several protocols for cryopreservation of rat immature testicular tissue. Twelve different freezing protocols using different (i) cryoprotectant (dimethylsulphoxide [DMSO] or 1,2-propanediol [PROH]), (ii) cryoprotectant concentration (1.5M or 3M), (iii) equilibration time (30 or 60 min), (iv) equilibration temperature (4 °C or room temperature), (v) size of testicular fragment (7.5mg or 15 mg), (vi) package (straws or cryovials), were compared using cord morphological damage evaluation. A testicular tissue piece of 7.5mg cryopreserved in cryovial using 1.5M DMSO, an equilibration time of 30 min at 4 °C showed fewer morphological alterations than the other protocols tested. The selected freezing protocol was able to maintain rat immature testicular tissue architecture, functionality after testicular pieces organotypic culture, and could be proposed in a human application.

Assessment of the optimal vitrification protocol for pre-pubertal mice testes leading to successful in vitro production of flagellated spermatozoa.

Testicular tissue cryopreservation offers the hope of preserved future fertility to pre‐pubertal boys with cancer before exposition to gonadotoxic treatments. The objective of this study was to compare controlled slow freezing (CSF) with five vitrification techniques for cryopreservation of murine pre‐pubertal testicular tissue and to evaluate the best protocol that could provide a successful completion of spermatogenesis after in vitro maturation. Testicular tissue from 24 mice at 6.5 days post‐partum (dpp) was used to compare several vitrification protocols with one another, as well as with a CSF protocol. Toxicity test using additional 12 mice was performed for all cryopreservation solutions. Fresh tissue (FT) from six mice was used as a control. Once the optimal vitrification protocol was selected [the modified solid surface vitrification No. 1 (mSSV1)], testes from 18 mice were cultured in vitro for 30 days with (i) fresh, (ii) slow‐frozen/thawed and (iii) vitrified/warmed tissues. Testes from six mice at 36.5 dpp were used as controls. At day 30 of in vitro culture, germ cells of the seminiferous tubules showed a high ability to proliferate and elongated spermatids were observed after both freezing techniques, confirming the successful completion of in vitro spermatogenesis. However, after mSSV1, the morphological alterations and the percentage of pyknotic seminiferous tubules were lower than CSF (4.67 ± 0.53 vs. 10.1 ± 1.12 and 22.7 ± 2.83% vs. 37.3 ± 4.24% respectively). Moreover, the number of flagellated spermatozoa produced per mg of tissue was higher for mSSV1 than for CSF (35 ± 3 vs. 9 ± 4 cells), with amounts of secreted testosterone during the culture close to those of FT. The mSSV1 protocol resulted in success rates better than CSF in maintaining testicular tissue structure, tubular morphology and tissue functions not solely for immediate frozen/thawed tissues but also after a long‐term in vitro culture.

Effects of Vitamin A on In Vitro Maturation of Pre-Pubertal Mouse Spermatogonial Stem Cells

Testicular tissue cryopreservation is the only potential option for fertility preservation in pre-pubertal boys exposed to gonadotoxic treatment. Completion of spermatogenesis after in vitro maturation is one of the future uses of harvested testicular tissue. The purpose of the current study was to evaluate the effects of vitamin A on in vitro maturation of fresh and frozen-thawed mouse pre-pubertal spermatogonial stem cells in an organ culture system. Pre-pubertal CD1 mouse fresh testes were cultured for 7 (D7), 9 (D9) and 11 (D11) days using an organ culture system. Basal medium was supplemented with different concentrations of retinol (Re) or retinoic acid (RA) alone or in combination. Seminiferous tubule morphology (tubule diameter, intra-tubular cell type), intra-tubular cell death and proliferation (PCNA antibody) and testosterone level were assessed at D7, D9 and D11. Pre-pubertal mouse testicular tissue were frozen after a soaking temperature performed at -7°C, -8°C or -9°C and after thawing, were cultured for 9 days, using the culture medium preserving the best fresh tissue functionality. Retinoic acid at 10-6M and retinol at 3.3.10-7M, as well as retinol 10-6M are favourable for seminiferous tubule growth, maintenance of intra-tubular cell proliferation and germ cell differentiation of fresh pre-pubertal mouse spermatogonia. Structural and functional integrity of frozen-thawed testicular tissue appeared to be well-preserved after soaking temperature at -8°C, after 9 days of organotypic culture using 10-6M retinol. RA and Re can control in vitro germ cell proliferation and differentiation. Re at a concentration of 10-6M maintains intra-tubular cell proliferation and the ability of spermatogonia to initiate spermatogenesis in fresh and frozen pre-pubertal mouse testicular tissue using a soaking temperature at -8°C. Our data suggested a possible human application for in vitro maturation of cryopreserved pre-pubertal testicular tissue.

Ovarian tissue thawing: A comparison of two conditions

Two different protocols to thaw cryopreserved human ovarian cortex have been evaluated using a histological analysis. The slower one, based on a progressive dilution of cryoprotectants, seems to maintain an optimal follicle morphology.

Concomitant unilateral and synchronous bilateral testis cancer in azoospermic dizygotic twins: differential management of fertility preservation.

OBJECTIVE To optimize fertility preservation management in unilateral or bilateral testicular cancer. DESIGN Case series. SETTING Urology department and reproductive biology laboratory. PATIENT(S) Dizygotic azoospermic twins presenting unilateral and bilateral synchronous testicular tumors. INTERVENTION(S) Testicular sperm extraction (TESE) and orchiectomy. MAIN OUTCOME MEASURE(S) Semen analysis, histologic diagnosis. RESULT(S) No spermatozoa were cryopreserved for the first case, because fertility preservation was proposed after orchiectomy. Spermatozoa were retrieved after TESE for his brother with bilateral tumor. CONCLUSION(S) Clinicians should be aware of the need to recommend sperm banking before treatments may alter spermatogenesis. TESE may be the sole option for fertility preservation in bilateral testicular cancer.

Éclosion 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.

Precocious initiation of spermatogenesis in a 19-month-old boy with Hurler syndrome

Mucopolysaccharidosis type IH (MPS IH) is a rare autosomal recessive lysosomal storage disorder. Haematopoietic stem cell transplantation (HSCT) has been proposed for the treatment of MPS IH patients and offers the possibility to grow into their adulthood. Precocious puberty has been described in few MPS patients. We report, to the best of our knowledge and for the first time, the initiation of the first waves of spermatogenesis fortuitously observed in seminiferous tubules of a pre-pubertal 19-month-old boy, affected by MPS IH and who did not present any clinical signs of precocious puberty. This patient benefited from testicular tissue cryopreservation before HSCT. Seminiferous tubule size, germ cell differentiation and Sertoli cell expression of androgen receptor and anti-müllerian hormone corresponded to the pattern observed in a pubertal boy. The Hurler syndrome may be responsible for the precocious initiation of spermatogenesis. A specific follow-up during childhood may be useful to confirm if such abnormal testis development is common in young boys with MPS IH and if it may lead to precocious onset of puberty in survivors despite HSCT. Furthermore, we have observed that Sertoli cell maturation (up-regulation of AR expression, down-regulation of AMH expression) occurred before the clinical signs of puberty and before the increase of testosterone plasmatic level.RésuméLa Mucopolysaccharidose de type IH (MPS IH) est une maladie rare lysosomale de transmission récessive autosomique. L’allogreffe de cellules souches hématopoïétiques (CSH) a été proposée pour le traitement des patients atteints de MPS IH et offre la possibilité d’une survie de ces patients jusqu’à l’âge adulte. La puberté précoce a été décrite chez quelques patients MPS. Nous rapportons, à notre connaissance et pour la première fois, l’initiation de la première vague de spermatogenèse observée fortuitement dans les tubules séminifères d’un garçon de 19 mois, atteint de MPS IH et sans signes cliniques de puberté précoce. Ce patient a bénéficié d’une cryoconservation de tissu testiculaire avant la greffe de CHS. Le diamètre des tubes séminifères, la différenciation des cellules germinales et l’expression du récepteur aux androgènes et de l’hormone antimüllérienne dans les cellules de Sertoli présentent les caractéristiques retrouvées dans le testicule d’un garçon pubère. Le syndrome de Hurler peut être responsable de l’initiation précoce de la spermatogenèse. Un suivi spécifique pendant l’enfance peut être utile pour confirmer si cette maturation prématurée du testicule est fréquente chez les jeunes garçons atteints de MPS IH et si elle peut conduire à une puberté précoce chez les patients malgré la greffe de CSH. En outre, notre observation démontre que la maturation des cellules de Sertoli se produit avant les signes cliniques de puberté et avant l’augmentation de la concentration plasmatique de testostérone.

Spermatogenèse in vitro… nouvel horizon pour restaurer la fertilité ?

The survival of the young boy after cancer has considerably progressed in recent years due to the efficiency of chemo/radiotherapy against the tumor cells. However, this treatment causes adverse effects on healthy tissues, including fertility. Freezing testicular tissue before highly gonadotoxic treatment is a prerequisite for preserving fertility in prepubertal boys that do not produce sperm yet. But which strategy proposes to restore fertility from frozen-thawed testicular tissue? One potential solution would be to consider an in vitro maturation of spermatogonial stem cells. In this article we trace the chronological development of in vitro spermatogenesis that resulted in mouse sperm production in vitro and give an overview of new challenges for the future.