Pasquale Patrizio

Long-term survival of human spermatogonial stem cells in mouse testes

OBJECTIVE To evaluate colonizing ability of human spermatogonial stem cells in mouse testes. DESIGN Transplantation of human testis cells into the seminiferous tubules of immunodeficient mice. SETTING University hospital and academic laboratory. PATIENT(S) Men with obstructive azoospermia or maturation arrest of spermatogenesis. Analyzed up to 6 months after transplantation. Also analyzed: cryopreservation of donor cells, donor cell concentrations, and leuprolide treatment of recipients. MAIN OUTCOME MEASURE(S) Detection of human donor cells in recipient testes using whole-mount immunohistochemistry with antibodies that react with human germ cells. RESULT(S) Mouse testes were colonized by human testis cells obtained from each of 6 patients; overall, human spermatogonia were found in 16 of 22 (73%) recipient testes. Human spermatogonial stem cells survived in mouse testes for at least 6 months and proliferated during the first month after transplantation. No human-differentiating spermatogonia were identified, and meiotic differentiation did not occur in mouse testes. In this initial study, human stem cell colonization was not influenced by cryopreservation of donor cells, donor cell concentration, or leuprolide treatment of recipient mice. CONCLUSION(S) Xenogeneic transplantation of human germ cells using mice as recipients is feasible and could be used as a biological assay system to further characterize human spermatogonial stem cells. This study might provide a mechanism to evaluate the status of the stem cell population in selected infertile male patients.

Mutations of the Cystic Fibrosis Gene and Congenital Absence of the Vas Deferens

It is estimated that about 30 to 40% of couples seeking fertility treatments are diagnosed with male factor infertility. These males have a range of gonadal dysfunctions which include azoospermia (i.e., no sperm in the ejaculate); oligozoospermia (i.e. sperm count less than 20 million/ml), asthenozoospermia (i.e.sperm motility less than 50%) and teratozoospermia (i.e. sperm with normal morphology less than 30%). The group of patients with azoospermia represent about 25% of the total and, of these, about 30% have an obstructive process (obstructive azoospermia) while the remaining have a primary testicular failure (non-obstructive azoospermia). In the obstructive azoospermia group, 25% of males have congenital bilateral absence of the vas deferens (CBAVD), while the incidence among all infertile males is about 2%. In the USA, it is estimated that approximately 16000 males are affected by CBAVD. Anatomically, CBAVD is a disorder characterized by regression bilaterally of variable portions of the epididymis, vas deferens, and, in about 80% of cases, absence of the seminal vesicles. In about 10 to 20% of the patients, a renal anomaly is also present. These anatomical hallmarks are so strikingly similar to those observed in men with cystic fibrosis (CF) that, as early as 1971 (Holsclaw et al. 1971), these two apparently unrelated disorders were hypothesized to have the same genetic origin. The hypothesis was proven when mutations in the Cystic Fibrosis Transmembrane conductance Regulator (CFTR) gene (Kerem et al. 1989; Riordan et al. 1989;Rommens et al. 1989) were found in patients with CF as well patients with isolated CBAVD (Dumur et al. 1990; Anguiano et al. 1992; Patrizio et al. 1993).

Molecular evaluation of two major human sperm fibrous sheath proteins, pro-hAKAP82 and hAKAP82, in stump tail sperm

OBJECTIVE To determine whether mutations in the pro-hAKAP82 gene and the resulting pro-hAKAP82 and hAKAP82 proteins were associated with the infertility seen in a patient with stump tail sperm. DESIGN Case report. SETTING Academic research and teaching environment, tertiary care hospital. PATIENT(S) A single, infertile Caucasian male diagnosed with essentially 100% stump tail sperm. INTERVENTION(S) None. MAIN OUTCOME MEASURE(S) Electrophoresis, silver staining, and immunoblotting of patient and control sperm proteins; RII (type II regulatory subunit of protein kinase-A) overlay assay of patient and control sperm proteins, partial DNA sequence analysis of patients pro-hAKAP82 gene; indirect immunofluorescence and immunogold electron microscopy of patient and control sperm. RESULTS(S) No significant abnormalities in the size or amount of pro-hAKAP82 and hAKAP82 or in the ability of these proteins to bind the regulatory subunit of protein kinase-A were identified in the patients sperm. Partial sequence analysis of the patients pro-hAKAP82 gene was identical to the published normal sequence. Indirect immunofluorescence and immunoelectron microscopy of sperm localized pro-hAKAP82/hAKAP82 to the sperm flagellum and demonstrated that the proteins were present in a disorganized, amorphous region, which apparently represented the fibrous sheath. CONCLUSION(S) These results suggest that, although pro-hAKAP82 and hAKAP82 localize to the correct structural component of the flagellum and are not directly responsible for the stump tail phenotype, they are unable to assemble normally into the fibrous sheath. Although this study did not identify abnormalities in the pro-hAKAP82 gene or its resulting proteins in a patient with stump tail sperm, several regions of the gene and protein remain to be examined.

Identification of meiotic and postmeiotic gene expression in testicular tissue of patients histologically classified as Sertoli cell only

OBJECTIVE To determine whether meiotic and postmeiotic germ cell gene products could be detected in biopsy specimen from patients with Sertoli cell only (SCO) and maturation arrest. DESIGN Prospective clinical study. SETTING University-based departments and laboratories. PATIENT(S) Nine patients, seven with nonobstructive azoospermia (12 biopsies) and two with obstructive azoospermia (controls) (2 biopsies). INTERVENTION(S) Specimens were divided into three parts: IVF laboratory, histology, and molecular analysis. Germ cell-specific messenger RNAs (mRNAs) were detected by extracting total RNA for Northern blotting or reverse transcription-polymerase chain reaction. MAIN OUTCOME MEASURE(S) Detection of meiotic (lactate dehydrogenase C4) and postmeiotic (transition protein 1 and protamine 1 and 2) gene expression and correlation with histologic and IVF laboratory findings. RESULT(S) The IVF laboratory identified spermatozoa in 3 of 14 biopsies (controls and severe hypospermatogenesis). Histologically, 6 of 14 biopsies (43%) were diagnosed as SCO, 4 (29%) maturation arrest, 2 (14%) severe hypospermatogenesis, and 2 normal. Molecular analysis showed mRNA for meiotic and postmeiotic genes in 12 of 14 biopsies (86%) (P =. 006), of which 4 (67%) in SCO and 3 (75%) in maturation arrest. CONCLUSION(S) Differentiated germ cells are present in biopsies of men histologically diagnosed as SCO. Absence of these molecular markers strengthens the histologic diagnosis and helps the physician in counseling the infertile couple.

Sperm cryopreservation for male patients with cancer: an epidemiological analysis

Many cancers strike young males who have not yet started or completed families. Since cancer treatments such as chemotherapy and radiation can irreversibly affect spermatogenesis, sperm cryopreservation is an important option for storing male reproductive potential. In this report, we review our database of 10 years of experience with cryostorage for male cancer patients. We assess types of cancer, timing of collection, sperm quality, and utilization for reproductive purposes. We also report specimen disposal and rates of patient death. There were a total of 164 oncology patients electing to freeze sperm at our institution during the study period. Types of cancer were varied, with testicular cancer, Hodgkins lymphoma, leukemia, and gastrointestinal cancers comprising the largest groups. Evaluation of semen parameters for these groups revealed that oligospermia, even prior to initiation of cancer therapy, was common. Sperm counts, motility, and morphology did not differ by type of cancer. Interestingly, less than 5% of patients utilized their specimens for reproductive purposes. Seven insemination cycles yielded no pregnancies, while one of two IVF attempts and the single ICSI case were successful. In conclusion, the epidemiological review of our database suggests that sperm cryostorage for fertility preservation in male cancer patients is under-utilized. Additionally, there is minimal use of cryopreserved specimens for reproductive purposes. We speculate that this under-utilization may be due to the paucity of reports regarding reproductive outcome after freezing. It is our objective to provide a compilation of data that will prove useful to both physicians and patients who are considering sperm cryopreservation.