Panayiotis M. Zavos

Effects of Smoking on Testicular Function, Semen Quality and Sperm Fertilizing Capacity

PURPOSE The effects of smoking on testicular function and sperm physiology were studied. MATERIALS AND METHODS Left testicular biopsy was performed in 49 smokers and 28 nonsmokers. Seminal specimens from these men were analyzed. RESULTS Testosterone levels in the left testicular vein, left testicular androgen-binding protein secretion rate (in vitro), sperm motility, percentage of morphologically normal spermatozoa, sperm morphometric parameters and outcome of sperm function tests were significantly lower (p < 0.05) in smokers than in nonsmokers. CONCLUSIONS Morphological sperm abnormalities due to secretory dysfunction of the Leydig and Sertoli cells may be the cause of impaired sperm fertilizing capacity in smokers.

An Electron Microscope Study of the Axonemal Ultrastructure in Human Spermatozoa From Male Smokers and Nonsmokers

OBJECTIVE To investigate possible abnormalities or deterioration of the sperm axonemal ultrastructure in men who have smoked a large quantity of cigarettes (> 20 per day) for a prolonged period. DESIGN Semen specimens were collected by patients via masturbation; qualitative characteristics of the sperm were assessed and ultrastructural analysis of the sperm axoneme was performed using standard operating procedures for electron transmission microscopy. SETTING The Andrology Institute of Lexington, Lexington, Kentucky, and the Department of Histology and Embryology, University of Salonika, Greece (collaborative effort). PATIENT(S) Twenty-nine men (mean age +/- SD, 30.7 +/- 2.1 years) who smoked a mean (+/- SD) of 30.7 +/- 2.1 cigarettes per day for 10.7 +/- 0.7 years and 15 men who never smoked (mean age +/- SD, 30.4 +/- 2.2 years) participated in this study. MAIN OUTCOME MEASURE(S) Ultrastructural organization of the sperm axoneme in male smokers and nonsmokers. RESULT(S) Changes in the number and the arrangement of axonemal microtubules were noted in the smoker group when compared to the nonsmoker group. The incidence of axonemal abnormalities was higher in spermatozoa from smokers compared with that in spermatozoa from nonsmokers. CONCLUSION(S) Smoking a large quantity of cigarettes per day, under the conditions of the current study, severely affected the ultrastructure of the flagellum and, more specifically, it affected the axoneme of the human spermatozoon.

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.

Antisperm Antibody Treatment Mode: Levels of Antisperm Antibodies After Incubation with TEST-Yolk Buffer and Filtration Using the SpermPrep™II Method

OBJECTIVE To assess whether incubation in TEST-yolk buffer (TYB) or human tubal fluid (HTF) could alter the sperm membrane characteristics and its relationship to antisperm antibodies (ASA) and/or antigen detachment from the sperm membrane and to evaluate the filtration of those specimens and possible recovery of ASA-free spermatozoa. DESIGN A prospective clinical study. SETTING Andrology Institute of Lexington, Lexington, Kentucky. PATIENT(S) Twenty patients undergoing infertility treatment. MAIN OUTCOME MEASURE(S) Recovery of spermatozoa with reduced levels or antisperm antibody-free sperm after treatment with TYB or HTF, followed by filtration using the SpermPrepII method (Sephadex based). RESULT(S) Assessment of ASA using the direct immunobead test showed no significant differences between specimens incubated for 2 hours in seminal plasma (fresh) or HTF with regard to levels of IgA and IgG. The percentage binding of anti-IgA and anti-IgG immunobeads was significantly reduced in specimens incubated for 2 hours in TYB compared with specimens incubated in seminal plasma or HTF. Furthermore, selection of spermatozoa using the SpermPrepII filtration method significantly reduced the percentage binding of anti-IgA and anti-IgG immunobeads compared with specimens incubated in HTF. CONCLUSION(S) The results suggest that TYB either altered the sperm membrane properties so that there was a decreased affinity at the antibody and/or antigen sites or that the egg yolk proteins were absorbing the antibodies and/or antigens complexes from the sperm membrane surface. Incubation of spermatozoa in TYB followed by filtration with the SpermPrepII method improved the recovery of ASA-free spermatozoa by selectively entrapping spermatozoa with ASA bound to its surface.

Viability of human semen specimens cryostored and transported at 5 C using the Bio-Tranz shipping system.

Objective: Transport of unprocessed human semen specimens from the production site to distant laboratories for andrological evaluation and clinical use requires the development of proper protocols and devices for the shipment and maintenance of sperm viability during transport. Factors such as maintenance of proper temperature and the specific diluent used are considered to affect the viability of semen specimens during transport. The Bio-Tranz shipper (ZDL, Inc., Lexington, KY, USA), which was designed to cool specimens (5 C) during transport, consists of a properly refrigerated Styrofoam box, TEST-yolk Buffer (TYB), a nonspermicidal condom-shaped semen collection kit (Hygene Kit; ZDL, Inc.) and test tubes (15.0 mL). The viability of semen specimens stored from the time of collection to the time at which the specimens were to be processed and used (24 hours post-collection) was evaluated using the Bio-Tranz shipping technology.Methods: Semen specimens (n = 30) were assessed for percentage and grade of motility (0-4), and for the sperm membrane functional integrity as measured by the hypoosmotic swelling (HOS) test at collection time and 24 hours after storage in the Bio-Tranz shipper. The specimens were produced at intercourse via the use of the Hygene collection kit, assessed for sperm characteristics, split into two aliquots and then transferred to 15.0 mL test tubes. Aliquot 1 was mixed 1:1 (v/v) with TYB media and aliquot 2 was used as raw (unprocessed). The specimens were then placed and secured in the Bio-Tranz shipper and assessed for sperm qualitative characteristics following 24 hours of storage.Results: The results of the sperm parameters assessed among the various seminal treatments are shown below:Sperm characteristics were significantly improved when preparing the specimens using TYB (0 h; P <.05). Significant differences in all sperm qualitative characteristics assessed were noticed between raw specimens and those prepared via TYB after 24 hours of storage (P <.05). Most interestingly, sperm characteristics between raw specimens (0 h) and specimens prepared using TYB and stored via the use of the Bio-Tranz shipper for 24 hours were not different (P <.05).Conclusion: The results obtained show that collection and shipment of semen specimens via the Bio-Tranz shipper system is possible. The Bio-Tranz shipper maintains adequate sperm viability after 24 hours of cryostorage. The use of the Bio-Tranz shipper is extremely convenient for patients that request semen processing services such as semen cryostorage, semen evaluation, semen preparation for IUI purposes, or other assisted reproductive technologies. The technique could be of significant clinical and economic importance to the patient and to the treating physician at locations across the United States.

Outcome of in vitro fertilization (IVF) and intracytoplasmic sperm injection (ICSI) using human sperm prepared via a new standardized swim-up technique fit for office use

Objective: Recent developments in the assisted reproductive technology (ART) areas necessitate the use of new and more efficient and acceptable modes of gamete in vitro manipulation techniques. As established via the use of various forms of ART, the natural process of fertilization has been largely bypassed and the gametes are obtained and manipulated outside of the normal means of conception. For the male, improvements in sperm quality have involved the use of various techniques that aim to recover a small percentage of healthy spermatozoa that can be further used for the different ART procedures. Previous IVF results showed no differences in fertilization rates between ZSC and Percoll recovered sperm (Zarmakoupis-Zavos et al. ACOG, 1997). The current study was designed to study the fertilizing capacity of ZSC recovered spermatozoa and subsequent embryonic development and pregnancy rates established via IVF and ICSI procedures.Methods: Sperm specimens (n = 30) were prepared via the use of the standardized ZSC swim-up technique, which was designed to harvest almost all overlayered media at the end of the procedure to maximize the sperm recovery. The ZSC device consists of a column with a conical cavity where the semen is placed, and from where the sperm swim-up into the overlayered media and are subsequently recovered at the end of the procedure. Semen specimens were initially assessed and then prepared (0.5 mL each) via the ZSC swim-up technique. The enhancement of the routine semen parameters was assessed and the recovered specimens were used for either conventional IVF or ICSI.Results: The results of the fertilization and pregnancy via IVF and ICSI are shown below:The generated results point out that ZSC recovered specimens yielded acceptable levels of fertilization and pregnancy rates in our IVF and ICSI program. Furthermore, the IVF inseminations yielded higher fertilization rates with a slight increase in polyspermic oocytes when compared to the ICSI inseminations.Conclusion: The data generated in this study tend to point out that the ZSC, besides being a simple one-step standardized system, can also be effectively employed in an ART program and can generate adequate fertilization and pregnancy rates. We have previously shown that the quality and fertilizing capacity of ZSC recovered sperm is comparable to that recovered via Percoll gradients. However, the ZSC when compared to the Percoll method was less time consuming (5 minutes of actual labor) and less tedious by eliminating the dilution and centrifugation steps of Percoll preparation, since it washes the sperm while simultaneously selecting it. The ZSC is a standardized semen preparation method and could be the method of choice in an ART program.

Selection of single-stranded deoxyribonucleic acid spermatozoa via the SpermPrep**SpermPrep, ZBL, Inc., Lexington, Kentucky. filtration column

Eighteen semen samples were collected from 18 normospermic men. Two aliquots (1 mL) were prepared from each ejaculate, washed with Hams F-10, and each washed sperm pellet was reconstituted in 2 mL volume of Hams F-10 medium. Each aliquot one was stained using the AO-staining method. Each aliquot two was filtered via the SpermPrep II method, and the recovered spermatozoa were stained similarly. The proportion of single-stranded DNA (red) spermatozoa to double-stranded (green) spermatozoa was significantly higher in aliquot one than in the postfiltered sample (aliquot two), suggesting that the SpermPrep filtration procedure selectively entrapped the spermatozoa with abnormal DNA.