Hugh C. Hensleigh

Testicular Trauma: Potential Impact on Reproductive Function

PURPOSE The long-term effects of testicular trauma on reproductive function are unknown. In an effort to define the relationship between testicular injury and fertility in humans, we identified patients with a history of testicular trauma and assessed parameters commonly associated with fertility. MATERIALS AND METHODS We reviewed 15 patients 23 to 59 years old who underwent immediate exploration after testicular trauma between 1972 and 1991. Of the patients 11 were contacted and 8 returned for prospective followup. Reproductive and sexual histories, physical examination, measurements of serum hormones and antisperm antibodies, semen analysis and scrotal ultrasound were done. RESULTS Of the 8 patients 1 (13%) achieved and 7 (87%) did not attempt conception. Hormonal status was normal in all 8 patients. Six men had objective evidence of subfertility by semen analysis only, although none had severe oligospermia or asthenospermia and only 1 had severe teratospermia. Five of 9 traumatized testes were atrophic. Interestingly, only 1 patient had antisperm antibodies, the levels of which were probably low enough to be clinically insignificant. CONCLUSIONS There was definite evidence of subfertility as assessed by abnormal semen analyses and atrophic testes following testicular trauma. However, the subfertility did not appear to be immune mediated nor did the patients present with infertility. Since only 1 patient had severely compromised fertility according to semen analysis we conclude that early repair can help preserve hormonal function as well as fertility.

Epidermal growth factor in human follicular fluid stimulates mouse oocyte maturation in vitro

OBJECTIVE To study the effect of human follicular fluid (FF) and the specific contribution of its epidermal growth factor (EGF) component on the in vitro maturation of cumulus-enclosed mouse oocytes. DESIGN A previously described mouse oocyte model system was used to study the effect of FF on oocyte maturation before and after extraction of EGF by immunoprecipitation. Follicular fluid specimens enclosing both mature and immature human oocytes were tested. MAIN OUTCOME MEASURES The endpoints assessed were the percentage of oocytes undergoing germinal vesicle breakdown (GVBD) and polar body one formation at different intervals over a 24-hour period and the final degree of cumulus expansion achieved. RESULTS A concentration-related stimulatory effect of mature FF was noted when compared with the spontaneous increase of GVBD and polar body one formation observed for the EGF-free control medium. Overall, the effect of immature FF was inhibitory. After extraction of EGF from FF by immunoprecipitation from both immature and mature FF, the rates of GVBD and polar body one formation were decreased in both groups. The addition of 5 ng/mL of EGF to the extracted groups reversed this effect on polar body one formation. Cumulus expansion was maximal for oocytes incubated with mature FF and minimal for those incubated with EGF-free media. CONCLUSIONS The positive effect of mature human FF on mouse oocyte maturation and cumulus expansion is to a large extent because of the presence of EGF.

Prostate specific origin of dipeptidylpeptidase IV (CD-26) in human seminal plasma.

PURPOSE A number of peptidases which can metabolize certain bioactive peptides and growth factors have been identified in seminal plasma. Our goal in this study was to determine molecular properties and the tissue source(s) for one of these peptidases, dipeptidylpeptidase IV (DPP IV), in human seminal plasma. MATERIALS AND METHODS We measured the activities of DPP IV with the dipeptide glycylprolyl-p-nitroanalide and its molecular forms using immunoblotting of seminal plasmas of men who were vasectomized or with different sperm concentrations, and in prostatic and seminal vesicle secretions of men undergoing prostatic surgery. RESULTS DPP IV in seminal plasma of vasectomized men was a membrane associated dimer comprised of subunits of approximately 110 kDa. Its activity did not differ in seminal plasmas of vasectomized, azoospermic, oligozoospermic and normozoospermic men indicating no correlation with the concentration of sperm originally present in the semen. The DPP IV antigen (CD -26) and enzymic activity were present in prostatic secretion, but absent from that of the seminal vesicles. These data indicate that the prostate gland is the primary source of DPP IV activity in seminal plasma. There was little variation in its activities in repeat seminal plasma samples from the same individual, and there was no change in its activity with age to 50 years. CONCLUSIONS DPP IV in seminal plasma was derived from the prostate gland and it may be useful as a bioindicator of prostate function and/or disease with age in men.

A comparison of 17β-hydroxysteroid oxidoreductase type 1 and type 2 activity of cytosol and microsomes from human term placenta, ovarian stroma and granulosa-luteal cells

A large body of evidence suggests multiple forms of 17 beta-hydroxysteroid oxidoreductase (17-HOR) regulate estrogen and androgen levels within gonadal and peripheral tissues. Two kinetically-differing 17-HOR activities have been detected in placental homogenates. 17-HOR type 1, found mainly in the cytosol, is highly reactive with estradiol-17 beta (E2) and estrone (E1) but not testosterone (T) (high E2/T activity ratio). Microsomal 17-HOR type 2 is reactive with both E2 and T (low E2/T activity ratio). In this study, 17-HOR activity of cytosol and microsomes from term placenta, ovarian stroma and granulosa-luteal cells was assayed under conditions which specifically differentiate between the two forms of the enzyme. Placenta had the highest activity with either E2 or T in both cytosol and microsomes and stroma the lowest. The highest specific activity with E2 and E1 was cytosolic in all samples. The highest specific activity with T was microsomal in placenta and ovarian stroma. E2/E1 activity ratios were comparable for cytosol and microsomes while E2/T activity ratios were comparable for placenta and stroma, but markedly elevated in granulosa-luteal (G-L) cell cytosol and microsomes. The results indicate trophoblast and ovarian stroma have more 17-HOR type 2 relative to type 1. G-L cells, in contrast, are relatively enriched in 17-HOR type 1 and thus have a greater capacity for net conversion of E1 to E2 under physiologic conditions. These differences may contribute to increasing serum and follicular fluid E2/E1 ratios during development of the dominant follicle.

Vibratory stimulation for treatment of anejaculation in quadriplegic men

Sexual dysfunction and infertility are common problems following spinal cord injury. Most men with complete spinal cord lesions do not ejaculate during coitus. Vibratory stimulation applied to the frenulum of the penis in six quadriplegic male subjects produced ejaculates for intrauterine inseminations. Pregnancies occurred in five of the six partners. Vibratory stimulation is a relatively safe and effective means to produce an ejaculation in men with quadriplegia.

Analysis of Fibronectin on Human Sperm

PURPOSE The purpose of this study was to localize fibronectin on human sperm and correlate its distribution with the morphological and functional integrity of sperm. MATERIALS AND METHODS Semen samples were collected and sperm fractionated by swim-up. Subsets of the swim-up sperm were capacitated and acrosome reacted. Damage to swim-up sperm was induced by freezing and thawing. The presence of fibronectin on the surface of sperm was determined by immunocytochemistry. RESULTS FN immunoreactivity was variable but staining on the sperm tail was consistently highest, whereas FN immunoreactivity over the acrosome and equatorial band was consistently lowest. Capacitation and acrosome reaction did not substantially change the distribution of FN staining. However, swim-up sperm had significantly less FN immunoreactivity (4%) than sperm that were unable to swim-up (12%; p < 0.01). Sperm that were deliberately damaged by freeze/thaw showed significantly increased FN binding (p < 0.01). FN immunoreactivity was inversely correlated with sperm viability (r = -0.68), motility (r = -0.70), and morphology (r = -0.63). CONCLUSIONS This study demonstrates that only a minority of the sperm in an ejaculate stain positive for FN and the localization of FN in positive sperm is primarily to the tail. Inferior sperm stain more frequently for FN leading to an inverse correlation between FN staining and sperm quality. Taken together, these results do not support a role for FN in sperm-egg binding. However, FN staining may provide a method for selecting the highest quality sperm for use in assisted reproduction techniques.

Immunocytochemical localization of μ-opioid receptors in follicular cells and preimplantation mouse embryos

Abstract It has been demonstrated that opioid peptides are involved in the regulation of mammalian reproduction. In our previous studies we demonstrated direct effects of opioids on preimplantation mouse embryos, and hypothesized the existence in preimplantation embryos of receptors similar to opioid receptors in the central neuronal system of adult animals. In the present study we addressed this issue by employing immunocytochemical staining for μ-opioid receptors using antisera raised against the C-terminal portion of the cloned μ-opioid receptors (MOR1, NHQLENLEAETAPLP, 384–398) predicted from the cloned receptor. Diffuse MOR1 immunoreactivity of moderate intensity has been revealed in one-cell embryos, while in follicular cells MOR1 staining was of high intensity and appeared to be associated with plasma membrane. No MOR1 immunoreactivity has been observed in two-cell to morula stages of development. However, blastocysts displayed intense MOR1-labeling that was particularly prominent in cells within the inner cell mass. MOR1-staining was most likely specific because preincubation of MOR1 antisera with cognate peptide completely abolished the staining. Our findings suggest the presence of opioid receptors during preimplantation development, long before the formation of the nervous system. Embryonic opioid receptors may play a role in the regulation of preimplantation development and implantation.

Effect of nicotine on in vitro human sperm penetrability through cervical mucus and motility parameters

Nicotine at concentrations found in the cervical mucus of female smokers appeared to enhance in vitro human sperm penetrability through ovulatory bovine cervical mucus. Sperm motility parameters were not affected by the addition of nicotine to semen samples incubated with BWW medium. Overall, these results suggest that a direct inhibitory effect of nicotine on sperm penetrability through cervical mucus is not responsible for the apparent increase in cervical factor infertility present in smoking women.

Evaluation for antisperm antibodies after storage of sperm in TEST-yolk buffer**Supported by the Minnesota Medical Foundation, Minneapolis, Minnesota.

OBJECTIVE To determine if TEST-yolk buffer, consisting of TES (N-tris [hydroxymethyl]-methyl-2-aminoethanesufonic acid), Tris (Tris[hydroxymethyl]aninomethane), and chicken egg yolk, affects the presence of antisperm antibodies on the sperm surface as detected by the immunobead test. DESIGN A prospective study of antisperm antibodies on sperm surface before and after incubation in TEST-yolk buffer. Direct immunobead test and indirect immunobead test were done the day of collection of the semen sample to detect the presence of human immunoglobulin class G (IgG) and immunoglobulin class A (IgA); immunobead tests were repeated on the same sperm samples after 24 hours of storage in TEST buffer. SETTING Academic tertiary institution. PARTICIPANTS Patients undergoing evaluation for infertility. RESULTS There was no significant difference in the outcome of the direct immunobead test after extending semen samples with TEST-yolk buffer for 24 hours at 4 degrees C. Eleven samples that were initially negative for IgG and 13 samples that were negative for IgA remained negative after 24-hour storage in TEST-yolk buffer. Eleven samples that were positive for IgG and nine samples that were positive for IgA by the direct immunobead test the first day remained positive the next day. Five extended sperm samples used in the indirect immunobead test with IgG positive serum gave positive results and four of five used with IgA positive serum gave positive results. CONCLUSIONS These findings suggest that TEST-yolk buffer can be used to extend semen without affecting the presence of antibodies on the sperm surface as indicated by the direct immunobead test. The higher variability of the indirect immunobead tests indicates there may be some alteration of sperm antigens after storing in TEST-yolk buffer. These findings indicate that TEST-yolk buffer can be used to store semen for batched processing of samples or as a transport medium for delivery to a central laboratory for antibody testing.

Direct positive effect of epidermal growth factor on the cytoplasmic maturation of mouse and human oocytes**Supported in part by the research grant RO1 AA07733 (awarded to B.S.L.) from the National Institute on Alcohol Abuse and Alcoholism, Rockville, Maryland.

OBJECTIVE Immature mammalian oocytes cultured in vitro undergo inadequate cytoplasmic maturation and hence have a limited potential for fertilization. Our primary objective was to determine if the addition of epidermal growth factor (EGF) to the in vitro culture system would have a positive effect on oocyte cytoplasmic maturation. DESIGN We studied the effect of different EGF concentrations on both denuded and cumulus-enclosed mouse oocytes cultured in vitro. MAIN OUTCOME MEASURES The percentage of oocytes undergoing germinal vesicle breakdown (GVBD) and polar body one formation over time as a function of EGF concentration was determined. RESULTS A dose-related positive effect of EGF on both GVBD and polar body one formation over time was observed for mouse oocytes. As well, a similar effect of EGF was seen on immature human oocytes that had not been stimulated with exogenous gonadotropins. CONCLUSIONS The use of EGF may allow for the performance of successful in vitro fertilization procedures using immature human oocytes retrieved during unstimulated cycles.