N. Sofikitis

Effects of paternal cigarette smoking on testicular function, sperm fertilizing capacity, embryonic development, and blastocyst capacity for implantation in rats

Summary. We evaluated the effects of paternal smoking on testicular function, sperm fertilizing capacity, embryonic development, and blastocyst capacity for implantation. Rats of group A were exposed to cigarette smoke for 10 weeks. Rats of group B were exposed to the smoke of incense sticks for 10 weeks. Rats of group C served as a control group. Rats of group D were exposed to cigarette smoke for 7 weeks only. Experimental period was 10 weeks in all groups. At the end of the experimental period serum testosterone responses to human chorionic gonadotropin stimulation, andro‐gen‐binding protein activity in testicular cytosols, epididymal sperm motility, and oocyte fertilization rate, oocyte cleavage rate, and blastocyst development rate after in vitro fertilization (IVF) trials were significantly smaller in group A compared with groups B and C. In contrast, fertilization rate, cleavage rate, and blastocyst development rate after intracytoplasmic sperm injection (ICSI) procedures were not significantly different among groups A, B, C, and D. Both after IVF trials and ICSI techniques, the proportion of the alive offspring to the number of transferred oocytes was significantly smaller in group A than in groups B and C. Cigarette smoke‐exposure results in a secretory deficiency of Leydig and Sertoli cells leading to an impaired epididymal sperm maturation process and diminished capacity of spermatozoa to penetrate oocytes. In addition paternal cigarette smoke exposure affects the embryonic ability for implantation.

Differences in seminal parameters in specimens collected via intercourse and incomplete intercourse (coitus interruptus)

Whatever the mechanism of such improvements may be, the results in this study point out that coitus interruptus in the human may not be the method of choice for collection of semen specimens, especially in patients with spermatogenic dysfunctions such as hypospermia, oligospermia, and asthenospermia. It should also be noted at this point that, for whatever purpose (semen analysis or artificial insemination by husband), the collected specimen should as closely as possible resemble the ejaculate delivered during intercourse. The complete coitus method, as applied in this study, showed that completion of the ejaculatory process during intercourse as compared with the coitus interruptus method, may assist in the improvement of the collected specimen and should closely resemble the ejaculate obtained during intercourse without the use of Silastic condoms. Furthermore, on the basis of the results generated in this study, the complete coitus method should always be the method of choice for male infertility patients with ejaculatory and spermatogenic dysfunctions as well as for scientists and clinicians who deal in the field of infertility diagnosis and treatment.

Influence of sexual stimulation on sperm parameters in semen samples collected via masturbation from normozoospermic men or cryptozoospermic men participating in an assisted reproduction programme.

To evaluate the influence of sexual stimulation via sexually stimulating videotaped visual images (VIM) on sperm function, two semen samples were collected from each of 19 normozoospermic men via masturbation with VIM. Two additional samples were collected from each man via masturbation without VIM. The volume of seminal plasma, total sperm count, sperm motility, percentage of morphologically normal spermatozoa, outcome of hypo‐osmotic swelling test and zona‐free hamster oocyte sperm penetration assay, and markers of the secretory function of prostate were significantly larger in semen samples collected via masturbation with VIM than masturbation without VIM. The improved sperm parameters in the samples collected via masturbation with VIM may reflect an enhanced prostatic secretory function and increased loading of the vas deferens at that time. In a similar protocol, two semen samples were collected via masturbation with VIM from each of 22 non‐obstructed azoospermic men. Semen samples from these men had been occasionally positive in the past for a very small number of spermatozoa (cryptozoospermic men). Two additional samples were collected from each cryptozoospermic man via masturbation without VIM. The volume of seminal plasma, total sperm count, sperm motility, and a marker of the secretory function of prostate were significantly larger in semen samples collected via masturbation with VIM. Fourteen out of the 22 men were negative for spermatozoa in both samples collected via masturbation without VIM. These men demonstrated spermatozoa in both samples collected via masturbation with VIM. Six men with immotile spermatozoa in both samples collected via masturbation without VIM exposed motile spermatozoa in both samples collected via masturbation with VIM. High sexual stimulation during masturbation with VIM results in recovery of spermatozoa of greater fertilizing potential both in normozoospermic and cryptozoospermic men. The appearance of spermatozoa after masturbation with VIM in the vast majority of cryptozoospermic men is of clinical significance in programmes applying intracytoplasmic sperm injections for the management of severe male infertility and obviates the need for testicular biopsy.

Effects of erythropoietin, bromocryptine and hydralazine on testicular function in rats with chronic renal failure

Summary. We evaluated the effects of chronic renal failure (CRF) on testicular function and semen physiology. A CRF model was created in 48 male rats by performance of five‐sixths nephrec‐tomies in two‐stage procedures, and a control (group A) by two‐stage sham operation on six male rats. Seven weeks later, serum urea and creatinine concentrations were assessed, and the nephrectomized rats were then equally divided into four groups, B, C, D and E, and treated with saline, erythropoietin, bromocryptine and hydralazine, respectively. Seventeen weeks after the first surgical procedure, the number of fertile rats, the mean values of epididymal sperm content and motility, the outcome of in vitro fertilization, and peripheral serum testosterone concentrations and responses to human chorionic gonadotropin were significantly higher (P<0.05) in groups A, G and D than in groups B and E. Serum prolactin concentration was significantly higher (P<0.05) in all groups of nephrectomized rats than in group A. Our results indicate that bromocryptine and erythropoetin improve Leydig cell function, sper‐matogenesis, epididymal sperm maturation, and sperm fertilizing capacity in rats with CRF.

Comparisons of sperm quality, morphometry and function among human sperm populations recovered via SpermPrep@II filtration, swim-up and Percoll density gradient methods

Summary. The purpose of this study was to compare the morphology/morphometry and fertilizing capacity of human spermatozoa recovered via swim‐up method, Percoll density gradient method, and SpermPrep™ II filtration method. Thirty‐three ejaculates were equally divided into 2 aliquots. Aliquot 1 was processed via the direct swim‐up method, whereas aliquot 2 was filtered via a SpermPrep™ II column. The Percoll density gradient method was compared with the SpermPrep™ II method in a similar protocol using 43 ejaculates. Sperm populations recovered via the SpermPrep™ II filtration method showed significantly higher hypoosmotic swelling test results, acrosin profiles, and percentage of hyperactivated spermatozoa than sperm fractions recovered by the swim‐up method. Furthermore, significant differences were found in most of sperm morpho‐metric parameters between the above sperm populations. However, sperm fractions recovered via the SpermPrep™ II method did not show significantly different values for these same tests and for most of sperm morphometric parameters compared to the Percoll density gradient method. These results suggest that the SpermPrep™ II filtration and Percoll density gradient method are equally efficient in isolating sperm subpopulations with better functional parameters than the swim‐up method.

Role of testicular tissue telomerase assay for the prediction of the presence of testicular spermatozoa in azoospermic men with varicoceles, pre- and post-varicocelectomy.

Summary.u2003 We evaluated the reproductive potential of frozen/thawed testicular spermatozoa of azoospermic men with left varicocele. The role of testicular tissue telomerase assay (TTA) in the prediction of the presence of testicular spermatozoa pre‐ and post‐varicocelectomy was investigated, as well. Therapeutic testicular biopsy and TTA were performed in 82 nonobstructed azoospermic (NOA) men with varicoceles. Testicular spermatozoa were found in 33 men and processed for cryopreservation. Oocytes were later recovered from the spouses of the latter azoospermic men with varicoceles and injected with frozen/thawed testicular spermatozoa. Among the 49 men who were negative for testicular spermatozoa, 22 men underwent subsequently subinguinal microsurgical varicocelectomy. A total of 198 mature oocytes were successfully injected and 101 were normally fertilized and subsequently cleaved. Transfer of these 101 embryos in 26 women resulted in nine full‐term pregnancies. Thirteen healthy babies were delivered. A cut‐off value of TTA of 39u2003TPGu2003Uu2003μg−1 protein had an overall diagnostic accuracy equal to 90.2% to predict the presence of testicular spermatozoa pre‐varicocelectomy. Within the group of men who were negative for testicular spermatozoa a cut‐off value of TTA equal to 28u2003TPGu2003Uu2003μg−1 protein (pre‐varicocelectomy) had a 84.2 % diagnostic accuracy to recognize the men who would become positive for either ejaculated or testicular spermatozoa post‐varicocelectomy. Testicular spermatozoa can be found in 40% of NOA men with left varicocele. Ooplasmic injections with frozen/thawed testicular spermatozoa have a role in the therapeutic management of non‐obstructive azoospermia associated with varicocele. Pre‐varicocelectomy, a TTA cut‐off value equal to 39u2003TPGu2003Uu2003μg−1 protein has a 90.2% diagnostic accuracy to indicate the men positive/negative for testicular spermatozoa. In addition, pre‐varicocelectomy, a cut‐off value equal to 28u2003TPGu2003Uu2003μg−1 protein has a 84.2% diagnostic accuracy to identify those men with varicoceles without testicular spermatozoa, who will become positive/negative for spermatozoa (either ejaculated or testicular) post‐varicocelectomy.

Cryptorchidism: seasonal variations in Greece do not support the theory of light

To examine seasonal trends of cryptorchidism in Greece, 583 males with true isolated cryptorchidism were analyzed. All 208u2003912 live‐born boys born during the same period were used as a comparison group. Seasonality by month of birth was evaluated using both Edwards model with adjusted frequencies and exact θi, and Walter‐Elwood method with exact θi. Both tests resulted in consistent findings. The incidence of cryptorchid births in Greece follows a documented cyclic pattern of simple harmonic type with spring being the season of statistical predominance (peak in March with a second, almost equivalent, peak in May). In contrast, in autumn the incidence of cryptorchid births was considerably lower (trough in September). Given the fact that no significant differences in daylight length are found among seasons in Greece, the detection of a significant seasonal variation suggests that factors other than light are involved in the pathogenesis of cryptorchidism. Low environmental temperature is proposed as a causative factor negatively influencing the maternal hCG profiles and the inguinoscrotal phase of testicular descent. This is further supported by: (i) the similarity of our results to those reported by other European countries of different longitude and geographical width and (ii) our data showing significantly smaller maternal hCG profiles at the 26th week of gestation during winter compared with summer.

The role of ultrasonographically guided puncture of the human rete testis in the therapeutic management of nonobstructive azoospermia.

We attempted to characterize the cells collected from the rete testis via ultrasonographically guided puncture. Unilateral puncture of the rete testis was performed in nine men with obstructive azoospermia and 51 men with nonobstructive azoospermia. All the aspirated samples from the rete testis were observed via confocal scanning laser microscope and some of them after fluorescent in situ hybridization techniques. Then therapeutic testicular biopsy was performed in the punctured testis of each man. Spermatozoa were found in all rete testis samples and all biopsy samples from obstructed men. Twenty‐two nonobstructed men demonstrated absence of spermatozoa in biopsy samples. Twenty‐nine nonobstructed men showed spermatozoa in biopsy material and 24 of these men (82%) had demonstrated spermatozoa in rete testis samples. There were no significant differences in fertilization and cleavage rate between intracytoplasmic sperm injection trials using biopsy spermatozoa and rete testis spermatozoa both in obstructed and nonobstructed men. Considering that puncture of the rete testis does not reduce the volume of testicular parenchyma, is less invasive and apparently causes less detrimental effect on testicular vasculature than biopsy, puncture of rete testis is recommended as first line approach for the treatment of azoospermic men. If puncture is negative for spermatozoa in nonobstructed men, biopsy is indicated.

P-065 Postmeiotic modifications of spermatogenic cells are accompanied by inhibition of telomerase activity (TA)

We investigated whether testicular telomerase activity is due to telomerase expression in all cells or expression in a limited number of cells. Telomerase activity was assayed in highly purified fractions of spermatogonia cells plus primary spermatocytes, secondary spermatocytes plus round spermatids, secondary spermatocytes plus spermatids plus spermatozoa, round spermatids, or spermatozoa prepared from healthy or cryptorchid animals. Telomerase activity was additionally assayed in testicular tissue of prepubertal animals and animals with Sertoli cell only pathophysiology. Telomerase activity was detected in fractions containing primary spermatocytes and/or secondary spermatocytes and/or spermatids. Fractions enriched in round spermatids were positive for telomerase activity. In contrast, spermatozoa or Sertoli cell fractions were negative for telomerase activity. Using the relative telomerase activity assay and the sensitive quantitative telomerase assay to quantify telomerase activity, we showed that induction of cryptorchidism does not result in quantitative alterations in testicular tissue telomerase activity. In addition, elimination of round spermatids does not lead to significant alterations in testicular tissue telomerase activity. The present results suggest that the male gamete telomerase activity is inhibited during spermiogenesis. Furthermore, it appears that spermatogonia/primary spermatocytes are the main sources of telomerase activity in the testis.