Gail S. Prins

Testosterone protects cerebellar granule cells from oxidative stress-induced cell death through a receptor mediated mechanism

It is known that steroid hormones can affect neuronal susceptibility to different types of insults, including oxidative stress. Using an in vitro/ex vivo model, we have previously shown that cerebellar granule cells prepared from neonatal rats treated with a single dose of testosterone are less vulnerable to oxidative stress-induced cell death, via a mechanism involving an upregulation of the cellular antioxidant defenses. Whether the testosterone protective action on cerebellar granule cells was direct or indirect remained to be clarified. Therefore, in this study we have investigated the effects of in vitro testosterone treatment, to see whether it also protects cerebellar granule cells from oxidative stress-induced damage. Cerebellar granule cells treated with 10(-6) M testosterone for 48 h were found less susceptible to damage induced by 50 microM hydrogen peroxide, as shown by a 30% decrease in the number of cells with apoptotic morphology. The addition of the androgen receptor antagonist flutamide abolished the protective effect of testosterone, suggesting an androgen receptor-mediated mechanism. This hypothesis was further supported by the presence of the androgen receptor in cultured cerebellar granule cells. The activity of the antioxidant enzyme catalase was also measured, and a 2-fold increase was detected in the testosterone treated cells, but not in the cells co-treated with flutamide. The present results demonstrate that cerebellar granule cells treated in vitro with testosterone are protected from oxidative stress via a mechanism mediated by the androgen receptor. Similarly to what we observed after in vivo administration of testosterone, the potentiation of the antioxidant defences seems to play a major role in the protection afforded by testosterone.

In vitro fertilization outcomes after intracytoplasmic sperm injection with fresh or frozen-thawed testicular spermatozoa

OBJECTIVEnTo compare the outcomes of intracytoplasmic sperm injection (ICSI) and in vitro fertilization (IVF) with fresh and cryopreserved testicular spermatozoa in patients with obstructive and nonobstructive azoospermia.nnnDESIGNnRetrospective analysis of consecutive ICSI cycles.nnnSETTINGnLarge urban reproductive medicine program.nnnPATIENT(S)nTwenty-nine patients with obstructive and nonobstructive azoospermia undergoing testicular sperm extraction for a total of 46 IVF-ICSI cycles (12 fresh, 34 frozen).nnnINTERVENTION(S)nTesticular sperm extraction, cryopreservation, and IVF-ICSI with fresh or frozen-thawed spermatozoa.nnnMAIN OUTCOME MEASURE(S)nFertilization rates, embryo cleavage rates, embryo implantation rates, clinical pregnancy rates per cycle and per embryo transfer, and delivery and spontaneous abortion rates.nnnRESULT(S)nNo statistically significant differences were noted in any of the parameters examined between IVF-ICSI cycles from fresh or frozen-thawed testicular spermatozoa. Fertilization rates were 56% with fresh vs. 61% with frozen-thawed testicular sperm, cleavage rates 92% vs. 95%, implantation rates 26% vs. 17%, clinical pregnancy rates per cycle 33% vs. 41%, and pregnancy rates per embryo transfer 33% vs. 45%, respectively. Delivery rates were 75% with fresh vs. 69.2% with frozen-thawed testicular sperm, and spontaneous abortion rates 25% and 30.8%, respectively.nnnCONCLUSION(S)nNo differences were found in IVF-ICSI outcomes between cryopreserved and fresh testicular sperm. In addition, cryopreservation provides several advantages for the patients and reproductive team.

Bisphenol A Promotes Human Prostate Stem-Progenitor Cell Self-Renewal and Increases In Vivo Carcinogenesis in Human Prostate Epithelium

Previous studies in rodent models have shown that early-life exposure to bisphenol A (BPA) reprograms the prostate and enhances its susceptibility to hormonal carcinogenesis with aging. To determine whether the human prostate is similarly sensitive to BPA, the current study used human prostate epithelial stem-like cells cultured from prostates of young, disease-free donors. Similar to estradiol-17β (E2), BPA increased stem-progenitor cell self-renewal and expression of stem-related genes in a dose-dependent manner. Further, 10 nM BPA and E2 possessed equimolar membrane-initiated signaling with robust induction of p-Akt and p-Erk at 15 minutes. To assess in vivo carcinogenicity, human prostate stem-progenitor cells combined with rat mesenchyme were grown as renal grafts in nude mice, forming normal human prostate epithelium at 1 month. Developmental BPA exposure was achieved through oral administration of 100 or 250 μg BPA/kg body weight to hosts for 2 weeks after grafting, producing free BPA levels of 0.39 and 1.35 ng/mL serum, respectively. Carcinogenesis was driven by testosterone plus E2 treatment for 2 to 4 months to model rising E2 levels in aging men. The incidence of high-grade prostate intraepithelial neoplasia and adenocarcinoma markedly increased from 13% in oil-fed controls to 33% to 36% in grafts exposed in vivo to BPA (P < .05). Continuous developmental BPA exposure through in vitro (200 nM) plus in vivo (250 μg/kg body weight) treatments increased high-grade prostate intraepithelial neoplasia/cancer incidence to 45% (P < .01). Together, the present findings demonstrate that human prostate stem-progenitor cells are direct BPA targets and that developmental exposure to BPA at low doses increases hormone-dependent cancer risk in the human prostate epithelium.

Differential regulation of androgen receptors in the separate rat prostate lobes: androgen independent expression in the lateral lobe

In order to understand the hormonal regulation of androgen receptors (AR) in the separate lobes of the rat prostate gland, the present study examined AR levels in the ventral, dorsal and lateral prostate lobes as a function of androgen withdrawal to complete prostatic regression and subsequent testosterone replacement. In the intact rat, the 3 prostate lobes contained significantly different amounts of androgen binding sites. Mean number of total cellular AR in the ventral, dorsal and lateral lobes was 7370, 1690, and 1015 fm/mg DNA, respectively. These receptors were primarily localized within the nuclear fraction of homogenized tissue: ventral, 86%; dorsal, 83%; and lateral, 100% nuclear localization. Androgen withdrawal was initiated via castration and rats were sacrificed 1, 2, 3, 5, 7, 10 and 14 days thereafter. Nuclear AR levels fell rapidly to 5, 24 and 30% of intact values by 48 h in the ventral, dorsal and lateral lobes, respectively. Levels of nuclear AR continued to decline in the ventral and dorsal lobes to undetectable levels by Day 10. In marked contrast, lateral lobe nuclear AR began to increase on Day 3 postcastration, reaching intact values by Day 7 and 133% intact levels by Day 14. Cytosolic AR in the ventral and dorsal lobes initially increased following castration, but subsequently declined to low levels by Day 14. Cytosolic AR were not detectable in the lateral prostate at any time point following castration. To determine the nuclear AR response to testosterone at this time, 14 day castrate rats were given 2 cm testosterone implants and sacrificed 1, 3, 5, 7, 10 and 14 days thereafter. As expected, nuclear AR rapidly returned in the ventral and dorsal lobes by Day 1 and reached a plateau by Day 5. A short term response to androgen exposure occurred in the lateral lobe where an immediate 9-fold increase in nuclear AR quantity was observed; however, these levels rapidly declined to pre-implant values by Day 5 and remained at that level despite continued exposure to testosterone. These f findings indicate that while nuclear AR levels in the ventral and dorsal prostate are primarily regulated by androgens, a testosterone-independent component exists within the lateral lobe.

A comparative study of buffer systems as cryoprotectants for human spermatozoa

Several cryoprotective media currently in use for human sperm cryopreservation have been directly compared with each other by use of a static vapor freezing technique. The TEST-citrate-yolk buffers with glycerol resulted in the highest motility and longevity after thawing; thus, their use is recommended.

Gonadotropins augment maturation and fertilization of human immature oocytes cultured in vitro

The inclusion of hMG in the culture medium for immature human oocytes results in improved maturation and fertilization rates. The resulting increased number of conceptuses available for transfer may improve the incidence of IVF pregnancy.

Prevalence of Sperm Bound Antibodies in Infertile Men with Varicocele: The Effect of Varicocele Ligation on Antibody Levels and Semen Response

An increased level of antisperm antibodies has been demonstrated in infertile men with varicocele compared with normal fertile men, suggesting a possible cause and effect relationship. To evaluate the possible etiological role of antisperm antibodies in varicocele patients, we performed a prospective study of 32 infertile men undergoing varicocele ligation. Semen analyses and antisperm antibodies as measured by the immunobead test were performed preoperatively and postoperatively at 3 and 6 months. Of the infertile men with varicocele 28% had a positive immunobead test compared with 0% of normal fertile men. The average total motile sperm count was significantly different (p < 0.05, 2-tailed t test) for 9 varicocele patients with sperm-bound antibody (3.2 x 10(6)) compared with 23 without antibody (8.4 x 10(6)). Postoperatively, 68% of all patients exhibited improved semen parameters, with no change in antibody status in either group. Among the antibody positive group 71% showed an increase in motile sperm per ml. of 2.8 x 10(6) to 17.2 x 10(6) (525% increase, p < 0.05), while in the antibody negative group 67% showed an increase of 3.8 x 10(6) to 24.9 x 10(6) (553% increase, p < 0.05). Our study suggests that there is an increased incidence of sperm-bound immunoglobulin in infertile varicocele patients and an apparent adverse effect on semen parameters in these patients. However, the presence of sperm-bound immunoglobulin did not affect the percentage response to surgical correction, nor can we postulate an immunological mechanism as a major etiological factor in varicocele induced infertility.

Life table analysis of intrauterine insemination pregnancy rates

One hundred twenty-eight couples undergoing intrauterine inseminations were retrospectively reviewed. Life table methodology was used to analyze cumulative pregnancy rates and monthly fecundability. Respective 6- and 12-month cumulative pregnancy rates for each diagnostic group receiving intrauterine insemination were: cervical factor, 28.6% and 42.8%; male factor, 16.7% and 16.7%; female immune factor, 66.7% and 100.0%; male immune factor, 37.5% and 68.8%; and empiric treatment, 60.0% and 60.0%. There was no difference in pregnancy rates between sperm processed with a swim-up in Hams F-10 or a two-gradient Percoll system. Abnormal sperm penetration assay results in patients with male factor did significantly (p = 0.05) lower the pregnancy rate. It is concluded that if no pregnancy has occurred after six cycles of inseminations, further workup or other treatment may be initiated, but additional pregnancies can be achieved from the seventh through the twelfth cycles of intrauterine insemination.

Comparison of urinary human follicle-stimulating hormone and human menopausal gonadotropins for ovarian stimulation in an in vitro fertilization program*†

This report compares the effects of human menopausal gonadotropin (hMG) and purified urinary human follicle-stimulating hormone (hFSH) protocols in patients with irreparable tubal disease as the sole indication for in vitro fertilization-embryo transfer (IVF-ET). The hFSH protocol was associated with significantly more uniform folliculogenesis and more effective steroidogenesis than the one using hMG. In addition, the hFSH protocol showed a trend toward more oocytes per laparoscopy and more embryos per transfer than the hMG group, although the difference was not statistically significant. More oocytes in the hMG group were classified as immature when compared with the hFSH group (P less than 0.05). Pregnancy rates in both groups were not significantly different. An allergic drug reaction that occurred in one patient on the hFSH protocol is the first such reaction reported with hFSH in the literature. The hFSH protocol is associated with a trend toward parameters that correlate with improved success rates in the IVF-ET program.

Antibody binding patterns in infertile males and females as detected by immunobead test, gel-agglutination test, and sperm immobilization test

Sera from 214 infertile patients were assayed for antisperm antibodies using the IBT, GAT, and SIT. The new IBT methodology was compared with the more classical tests. Although SIT and GAT did not correlate to any particular antibody class, both were negative if IgA was present alone. The immunoglobulin class presented a preferential sperm region binding site: IgG to the head and tail, IgM to tail tip only, IgA to head and tail. Furthermore, these immunoglobulins from the serum of male patients bound differently to sperm than immunoglobulins from female serum. Finally, we were able to calculate the relative sensitivity, specificity, and predictive values for these tests.