M. Feliciano

Xenogeneic transplantation of human spermatogonia

In the last 3 years, several studies have shown that xenogeneic transplantation of rodent spermatogonia is feasible. The treatment of infertile patients with spermatogenic arrest using the injection of immature germ cells has yielded only poor results. We attempted to establish a complete spermatogenetic line in the testes of mutant aspermatogenic (W/Wv) and severe combined immunodeficient mice (SCID) transplanted with germ cells from azoospermic men. Spermatogenic cells were obtained from testicular biopsy specimens of men (average age of 34.3 +/- 9 years) undergoing infertility treatment because of obstructive and non-obstructive azoospermia. Testicular tissue was digested with collagenase to promote separation of individual spermatogenic cells. The germ cells were injected into mouse testicular seminiferous tubules using a microneedle (40 microm inner diameter) on a 10 ml syringe. To assess the penetration of the cell suspension into the tubules, trypan blue was used as an indicator. Mice were maintained for 50 to 150 days to allow time for germ cell colonisation and development prior to them being killed. Testes were then fixed for histological examination and approximately 100 cross-sectioned tubules were examined for human spermatogenic cells. A total of 26 testicular cell samples, 16 frozen and 10 fresh, were obtained from 24 men. The origin of the azoospermia was obstructive (OA) in 16 patients and non-obstructive (NOA) in 8 patients. The concentration of spermatogenic cells in the OA group was 6.6 x 10(6) cells/ml, and 1.3 x 10(6) cells/ml in the NOA group (p < 0.01). The different spermatogenic cell types were distributed equally in the OA samples, ranging from spermatogenia to fully developed spermatozoa, but in the NOA group the majority of cells were spermatogonia and spermatocytes. A total of 23 testes from 14 W/Wv mice and 24 testes from 12 SCID mice were injected successfully, as judged by the presence of spermatogenic cells in histological sections of testes removed immediately after the injection. However, sections from the remaining testes examined up to 150 days after injection showed tubules lined with Sertoli cells and xenogeneic germ cells were not found. The reason why the two strains of mouse used as recipients did not allow the implantation of human germ cells is probably due to interspecies specificity involving non-compatible cell adhesion molecules and/or immunological rejection.

Localization and identification of sumoylated proteins in human sperm: excessive sumoylation is a marker of defective spermatozoa

BACKGROUND Sumoylation is a type of post-translational modification that is implicated in the regulation of numerous cellular events. However, its role in the function of human sperm has not yet been characterized. METHODS AND RESULTS In this study, both immunofluorescence and electron microscopy revealed that small ubiquitin-like modifiers (SUMO) SUMO1 and SUMO2/3 were highly enriched in the neck area of human sperm that is associated with the redundant nuclear envelope and were also detectable in the flagella and some head regions. Similar localization patterns of SUMO were also observed in mouse and fly sperm. Nonmotile, two-tailed, curled tailed, misshapen, microcephalic (small head) and aciphalic (no head) sperm exhibited abnormally high levels of sumoylation in their neck and tail regions relative to normal sperm. Numerous sumoylated proteins, ranging from 20 to 260 kDa, were detected via western blotting and identified by mass spectrometry, and 55 SUMO targets that were present specifically in human sperm, and not in the control fraction, corresponded to flagella proteins, proteins involved in the maturation and differentiation of sperm, heat shock proteins and important glycolytic and mitochondrial enzymes. The targets that were identified included proteins with specific functions in germ cells and sperm, such as heat shock-related 70-kDa protein 2, outer dense fiber protein 3, A-kinase anchor proteins 3 and 4, L-lactate dehydrogenase C, sperm protein associated with the nucleus on the X chromosome B/F, valosin-containing protein, seminogelins, histone H4 and ubiquitin. Coimmunoprecipitation experiments confirmed the sumoylation of semenogelin and indicated that some sperm proteins are modified by sumoylation and ubiquitination simultaneously. CONCLUSIONS Numerous proteins are modified by sumoylation in human sperm; excessive sumoylation is a marker of defective spermatozoa.

Cigarette smoke affects posttranslational modifications and inhibits capacitation-induced changes in human sperm proteins

Sperm are highly dependent on posttranslational modifications of proteins. Massive phosphorylation on tyrosine residue is required for sperm capacitation. Sumoylation has also been recently implicated in spermatogenesis and sperm functions. Cigarette smoke is known to cause oxidative stress in different tissues, and several studies suggest that it causes oxidative stress in sperm. Whether tobacco affects posttranslational modifications in human sperm is currently unknown. In this study, we show that a short exposure of human sperm to physiological concentrations of cigarette smoke extract (CSE) causes the partial de-sumoylation of many sperm proteins. Furthermore, the presence of a low concentration of CSE in the human tubal fluid during an induction of in vitro capacitation inhibits the capacitation-associated increase in protein phosphorylation. Collectively, changes in posttranslational modifications may be one of the mechanisms through which exposure to tobacco can negatively affect sperm functions and cause fertility problems.

PD68-04 IMPACT OF CFTR MUTATIONS ON PHENOTYPE AND REPRODUCTIVE OUTCOMES IN MEN WITH CONGENITAL ABSENCE OF THE VAS DEFERENS

RESULTS: A total of 198 men (mean age 35 8 years) were included. Fertilization and pregnancy rates were 44% and 38%, respectively. On univariable analysis, sperm motility and the lack of acrosome defects were associated with higher fertilization and pregnancy rates while an increasing number of total sperm abnormalities was negatively associated with fertilization, but not pregnancy, rates (Table). On MVA, only sperm motility was associated with fertilization rates (p<0.001) while both sperm motility (p1⁄40.004) and the lack of acrosome abnormalities (p1⁄40.018) were associated with pregnancy rates. Female age (mean age 31 5 years) was not associated with fertilization or pregnancy rates on MVA. CONCLUSIONS: This study is the first to assess the relationship between injected testicular sperm characteristics and reproductive outcomes in ICSI. All men with NOA have abnormal sperm morphology and our experience suggests that no specific sperm abnormality, including injection of non-motile sperm, precludes the chance of pregnancy. Further study of sperm characteristics may influence how aggressively surgeons should seek better sperm quality during microTESE procedures. Use of elongating spermatids (blunt-tailed sperm) did not adversely affect the chance of pregnancy despite concerns for lower fertilization with severely blunt-tailed sperm.

V9-08 MALE INFERTILITY MICROSURGERY TRAINING – TRICKS OF THE TRADE

INTRODUCTION AND OBJECTIVES: Gender confirmation surgery is an essential component in the management of gender identity disorder. However, short vaginal length, vaginal stenosis, or complications in the perineal dissection are significant limitations of current techniques in male to female surgery. Here we describe our technique for the robot assisted penile inversion vaginoplasty that addresses these needs. METHODS: The patient is prepped and draped in low lithotomy position. The penis is degloved through a circumcision incision. The neurovascular bundle, urethra and corpora cavernosa are dissected out. A six cm bulbar perineal incision is then made, and the dissection is carried to the bulbar urethra. The dissected urethra, neurovascular bundle, glans and corpora are delivered through this incision. The bilateral corpora are transected at their most proximal limit and overswen. The penile skin is inverted and gently retracted to allow a two cm incision above the neovagina for the neoclitoris. Immediately below this, an incision for the neomeatus is made. The urethra is brought through this incision and sutured to the skin. The remaining urethral tissue is used as an inlay onto the incised dorsal epithelial surface of the penile skin. The robotic portion of the surgery uses 4 port incisions: periumbiical Gelport with two pre-placed robotic trocars, right and left lateral ports, and an assistant port in the upper right abdomen. The dissection is from the posterior prostate, staying above Denonviller’s fascia to reach the endopelvic fascia. Under direct vision, the endopelvics are opened sharply from below and opened to a width of two fingerbreadths. The neovagina is passed into robotic field and pexed to the anterior reflection of the posterior peritoneum. The peritoneal reflection is then closed. The neoclitoris is fashioned from the glans penis and approximated. Labia majora and minora are fashioned with local skin flaps. A foley catheter is left indwelling as well as a vaginal stent. RESULTS: The index case required 7 hours of surgical time with an estimated blood loss of 100 mL. The vaginal length was greater than 15 cm. The patient was discharged home on post-operative day three, with no complications. The patient endorses sensation at the neoclitoris and anterior neovagina, and finds the vaginal depth satisfactory CONCLUSIONS: Our novel method for robot assisted penile inversion vaginoplasty is an important step in optimizing outcomes for our patients. This technique achieves maximal vaginal length in a safe and reproducible manner.