title: Developmental potential of human spermatogenic cells co-cultured with Sertoli cells.
abstract: BACKGROUND
  Development of an in-vitro culture system capable of supporting human early germ cell differentiation would be important for treatment of azoospermic patients.
  
  
  METHODS
  Sertoli cells, spermatogonia and spermatocytes were isolated from testicular biopsies of 61 non-obstructive azoospermic patients, and co-cultured using Vero cell conditioned medium only or supplemented with recombinant (r)FSH or rFSH plus testosterone. Germ cell purity was checked by fluorescent in-situ hybridization (FISH) analysis.
  
  
  RESULTS
  Best results were achieved with both hormones, which elicited 6.9% of meiosis index and 22.7% of differentiation into normal late spermatids after 2-3 weeks of culture. In-vitro matured spermatids were microinjected into oocytes to study their developmental potential. Round spermatids elicited 37.5% of fertilization and 28.6% blastocyst rates. Abnormal elongating and elongated spermatids enabled 8.3 and 27.3% fertilization rates respectively, but none achieved the blastocyst stage. Normal elongating and elongated spermatids elicited 30.5% fertilization and 42.9% of blastocyst rates. FISH analysis showed sex chromosome anomalies in all embryos, except in the case of morulae from normal late spermatids.
  
  
  CONCLUSIONS
  Results suggest that meiosis and spermiogenesis can be resumed in vitro, with normal differentiated spermatids showing a low fertilization potential but regular rates of blastocyst formation. However, most of the embryos did not reach the morula stage and showed major sex chromosome abnormalities.
authors.affiliation: Department of Medical Genetics, Faculty of Medicine, State University of Porto, Porto, Portugal. msousa@icbas.up.pt
authors.forename: Mário
authors.initials: M
authors.lastname: Sousa
authors.forename: Nieves
authors.initials: N
authors.lastname: Cremades
authors.forename: Cláudia
authors.initials: C
authors.lastname: Alves
authors.forename: Joaquina
authors.initials: J
authors.lastname: Silva
authors.forename: Alberto
authors.initials: A
authors.lastname: Barros
mesh_terms: D000818:Animals; D001706:Biopsy; D002454:Cell Differentiation; D002478:Cells, Cultured; D002522:Chlorocebus aethiops; D018920:Coculture Techniques; D017077:Culture Media, Conditioned; D004622:Embryo, Mammalian / Q000648:ultrastructure; D005260:Female; D005307:Fertilization in Vitro; D005640:Follicle Stimulating Hormone / Q000494:pharmacology; D006801:Humans; D017404:In Situ Hybridization, Fluorescence; D008297:Male; D008540:Meiosis; D009845:Oligospermia / Q000473:pathology; D011994:Recombinant Proteins / Q000494:pharmacology; D012708:Sertoli Cells / Q000502:physiology*; D020554:Sperm Injections, Intracytoplasmic; D013087:Spermatids / Q000502:physiology; D013091:Spermatogenesis; D013093:Spermatogonia / Q000502:physiology*; D013094:Spermatozoa / Q000502:physiology*; D013737:Testis / Q000473:pathology; D013739:Testosterone / Q000494:pharmacology; D020858:Tissue and Organ Harvesting; D014709:Vero Cells; D014960:X Chromosome
chemical_list: D017077:Culture Media, Conditioned; D011994:Recombinant Proteins; D013739:Testosterone; D005640:Follicle Stimulating Hormone