Relationship between semen quality and tobacco chewing in infertile men

Relationship between semen quality and tobacco chewing in infertile men

approximately 6.4% have already had or are currently having difficulty conceiving with their partner. Considering the significant abnormalities on sem...

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approximately 6.4% have already had or are currently having difficulty conceiving with their partner. Considering the significant abnormalities on semen analysis and that 68% of these patients were either unmarried or had never attempted conception, it is extremely likely that this percentage is considerably underestimated. Our data supports prior recommendations for a comprehensive evaluation of all men presenting with subfertility. [1] Honig SC, Lipshultz LI, Jarow J. Significant medical pathology uncovered by a comprehensive male infertility evaluation. Fertil Steril. 1994 Nov; 62(5):1028 –34. Supported by: None.

MALE REPRODUCTION AND UROLOGY: RESEARCH P-124 Withdrawn P-125 Caⴙ2 and cholesterol and mitochondrial activity in sperm from fertile and infertile men. M. Meseguer Sr., N. Garrido Sr., J. A. MartinezConejero Sr., C. Simon Sr., A. Pellicer Sr., J. Remohi Sr. IVI, Valencia, Spain; Department of Paediatrics, Obstetrics and Gynaecology, School of Medicine, Valencia, Spain; Hospital Universitario Dr. Peset, Valencia, Spain.

P-123 Continued evaluation of a new semen collection technique/container in subfertile and infertile individuals using a cross-species model. D. Johnson, C. Dehn, S. Prien. Texas Tech University, Lubbock, TX; YL Ranch, Albany, TX; Texas Tech University Health Sciences Center, Lubbock, TX. OBJECTIVE: To date, the common thread in the use of semen collection/ extension techniques, whether being used for intrauterine insemination, cryopreserved semen, or advanced reproductive techniques such as in vitro fertilization, is that the samples are collected into a dry, unprotected specimen container. This container, by its nature, allows for drastic shifts in temperature and pH resulting in damage to the spermatozoa. Preliminary studies from this laboratory using a new collection container/technique to collect semen from canines and humans demonstrated an improvement in most semen parameters. The modified collection device optimizes semen parameters by controlling temperature, pH, and osmotic stress. The objective of the current study was to further investigate the usefulness of the modified device in improving semen parameters using a cross-species model focusing on sub- and infertile individuals. DESIGN: A cross-species evaluation of a new semen collection technology. MATERIALS AND METHODS: Semen samples were collected from canine (n⫽10), equine (n⫽8) and human (n⫽12) donors using standard techniques. In the case of the equine and canine donors, a splitter was placed in the artificial vagina to produce a true split sample into a standard collection vessel and the modified collection unit. Human donors were collected into both devices in sequential samples at three day intervals. Standard semen parameters (viability, motility, morphology, concentration) and biochemical markers were evaluated for all donors at 0, 1, 3, 6, 12, 18, and 24 hr post-processing. Canine and Equine samples were further evaluated at 24 hr intervals until the samples had reached 0% motility. All donors were then classified as either fertile or sub-fertile based upon species standard for count and/or motility. Data analysis within species were performed with SPSS using the general linear model and appropriate t-tests. RESULTS: As in previous studies, semen quality at 24 hr (as reflected by motility) was significantly higher in samples collected in the new collection container as compared to the control regardless of species (human 20% vs 14%, P ⬍ .001; canine 38% vs 8%, P ⬍ .001; equine 55% vs 37%, P⬍ .001; treatment vs control respectively). Further, collection of the samples into the new device extended the time to last full insemination dose (dose is species specific) an average of 7 fold (7 hrs vs 50 hrs) P⬍.03 in the canine and 2 fold (148 hrs vs 228 hrs) P⬍.001in the equine over their respective control. This improvement was even more dramatic in animals classified as subfertile, where collection into the new device extended the time to last full insemination dose an average of 15 fold (6 hrs vs 91 hrs) in the canine and 3.5 fold (120 hrs vs 312 hrs) in the equine over their respective controls. Improvement was also seen in viability and biochemical parameters which will be discussed in detail at presentation. CONCLUSION: Modification of the semen collection/extension procedure resulted in improved semen parameters for extended time-periods post-collection. The data suggests the described technique can yield significantly more motile sperm by placing the sample into a physiologically favorable environment (eliminating pH and cold shock), thus making more sperm available for use in a variety of infertility treatments. Further studies, evaluating pregnancy rates, will be needed to confirm these observations. Supported by: None


OBJECTIVE: A significant proportion of the males suffer from unknown origin infertility, although semen parameters, evaluated under WHO criteria are normal. This suggests that male infertility could be caused by different deficiencies not described yet. To date, data regarding molecular aspects of male infertility, useful to be employed as a diagnostic test are lacking. The aim of the current study was to correlate the levels of concrete sperm function markers as cholesterol (CH) and calcium (Ca⫹2), together with mitochondrial activity (MA) with the standard semen parameters and to compare these between fertile and infertile men. MATERIALS AND METHODS: Semen samples of proven fertility donors (n⫽91), and from infertile males undergoing ART (n⫽ 60), were obtained after 3–5 days of sexual abstinence together with previous signed consent. All samples were studied according with WHO statements for volume, concentration, motility and morphology in the routine semen analysis by two different observers. We determined Ca⫹2 and CH concentrations on seminal plasma by enzymoimmunoanalysis techniques with an Alcyon analyzer (Abbot), and intracellular Ca⫹2 by incubation with Fluo Ca⫹2 indicator Fluo-4 AM (Molecular Probes) (30⬘ 37°C), CH concentrations in the sperm plasma membrane dissolved in chorophorm-methanol (2:1) solution by using Amplex Red Cholesterol Assay kit (Molecular Probes) together with MA by 100nM MitoTracker Green in PBS (Molecular Probes)(30⬘37°C) all of them by fluorometry (Fluoroskan, Labsystems)..T test was employed to detect differences between groups of fertile and infertile regarding the molecular markers studied. RESULTS: There were not significant differences between fertile and infertile males in any of the sperm parameters studied. Subsequently, their cause of infertility is not related with any macro or microscopical sperm alteration. There was a significant positive correlation between sperm membrane CH content and sperm morphology (r⫽ 0.395, p⫽0.011). Intracellular Ca⫹2 was significantly lower in infertile patients compared to sperm donors (0.53 ⫾ 0.05 fluorescence arbitrary units (fau)/50 million sperm vs 0.81 ⫾ 0.13 fau/50, p⫽0.020). No differences were found regarding Ca⫹2 and CH concentrations in seminal plasma, and MA. As expected by previous observations MA is directly related with sperm motility (r⫽0.785, p⫽0.001). CONCLUSION: Intracellular concentrations of Ca⫹2 and the proportion of CH in the sperm membrane are two important markers of the sperm quality due to its direct relationship with sperm morphology and fertility potential. We have also corroborated the effectiveness of the measurement of MA in the sperm motility quantification. To this end, we should study the regulation mechanism of the Ca⫹2 flux into the sperm cells and second, we could revise which external factors are affecting CH concentrations in the sperm membrane. Knowing the ability of sperm cells to exchange lipids with external medium an exogenous addition of CH to culture media could improve the sperm quality.

P-126 Relationship between semen quality and tobacco chewing in infertile men. G. Ranga, T. M. Said, A. Agarwal. Karthekeya Medical Research and Diagnostic Center, Santa Cruz, India; Cleveland Clinic Foundation, Cleveland, OH. OBJECTIVE: Male fertility is affected by a variety of occupational and environmental factors, as well as life style habits that include tobacco consumption. A large population of Indian men is addicted to tobacco chewing. The objective of our study was to assess the relationship between plain tobacco chewing and semen characteristics of the male partners of infertile couples. DESIGN: Retrospective study.

Vol. 82, Suppl. 2, September 2004

MATERIALS AND METHODS: Data was collected from 640 male patients undergoing infertility evaluation from November 1998 to December 2003. All subjects included in our study were in the age range of 18 ⫺ 40 years, had a history of tobacco chewing for 4 ⫺ 10 years, and no history of other relevant social habits. Patients were grouped according to the frequency of their habit of tobacco chewing as: mild (⬍ 3 times/day, n ⫽ 180), moderate (⬎ 6 times/day, n ⫽ 263), and severe (⬎ 18 times/ day, n ⫽ 197). Semen parameters were assessed according to WHO (1999) criteria. RESULTS: Sperm count, percentage motility, morphology and percentage vitality were significantly higher in mild group compared to moderate, and in the moderate compared to the severely addicted group (P ⬍ 0.0001 in all comparisons). The percentage of azoospermia observed in the 3 groups was, 1%, 3%, and 14% respectively indicating a highly significant increase in prevalence in the severely addicted group (P ⬍ 0.001). Similarly, the incidence of oligoasthenoteratozoospermia (OAT) showed an increasing trend from mild (2%) to moderate (8%) to severe addiction (25%). The incidence of OAT was highly significant in the severely addicted group as compared to mild and moderate users (P ⬍ 0.001). No significant changes in semen parameters were observed in men with mild habit compared to normal standard values of WHO. Median, and interquartile range (25th, 75th percentiles) of sperm count, percentage motility, morphology and percentage vitality in all 3 groups are illustrated in Table 1. CONCLUSION: The decrease in semen quality (sperm count, motility, morphology and vitality) in infertile male patients is strongly associated with tobacco chewing habit. Oligoasthenozoospermia or azoospermia was more prevalent in men severely addicted to tobacco chewing. Infertile men should be counseled about the adverse effects of tobacco chewing on their semen quality. The decrease in semen quality may be due to effects of tobacco on spermatogenesis, however, further studies are needed to substantiate its exact mode of action. Supported by: None

P-127 Characteristics of human sperm DNA and its relationship with male infertility. S. Acharyya, A. K. Duttagupta, A. Agarwal, A. K. Bhattacharyya. University of Calcutta, Calcutta, India; Cleveland Clinic Foundation, Cleveland, OH. OBJECTIVE: To determine variation in sperm chromatin among different categories of human semen samples. DESIGN: 34 human semen samples including 16 normozoospermic, 15 asthenozoospermic and 3 oligoasthenozoospermic. MATERIALS AND METHODS: Assessment of the purity of the DNA samples was performed by spectral characteristic analysis (within a wavelength of 230 –290nm) as well as calculation of A260/A280 ratio. Estimation of the DNA content, RNA and protein as contaminants were done by diphenylamine method, orcinol method and Lowry’s method respectively. RAPD analyses of sperm DNA samples (categorized on the basis of motility difference) were done using random decamer primer (5’AATCGGGCTG 3’) solely to detect polymorphism within different categories of semen samples. A dissimilarity matrix was prepared for identifying the extent of variation. Band frequencies were also calculated. RESULTS: Spectral analysis revealed no observable differences for respective DNA samples. The A260/A280 ratios (1.98⫾ 0.2) indicated pure DNA samples. Results of DNA content estimation showed a lack of correlation with the nature of the semen samples. RNA was absent in all cases and some of the DNA samples had protein contamination. Identification of the major bands for each category of DNA samples was performed only on higher values of the band frequencies (0.75–1.0). Four major bands were recognized in sperm DNA from samples with normal motility (70 – 80%),


with higher dissimilarity indices of 1.0 and 0.8. Five major bands were identified in sperm DNA with 60 –70% motility with low values of dissimilarity indices (0.07– 0.53). RAPD of sperm DNA with 50 – 60% motility also showed 5 major bands with frequency of 1.0. The number of major bands recognized in sperm DNA with a motility percentage of (40 –50), (30 – 40) and (0 –30) were 1, 5 and 10, respectively. Sperm DNA in two of the oligoasthenozoospermic samples revealed 100% similarity. RAPD analysis of sperm DNA in different semen categories revealed the lack of existence of even a single band with a band frequency of 1.0. CONCLUSION: The present investigation provides a clear view of the genetic variation among different categories of human semen specimens at the level of sperm chromatin. Supported by: None

P-128 Trinucleotide (CAG) repeat length in exon 1 of the androgen receptor gene of Turkish males with idiopathic infertility. A. C. Tufan, N. L. Satiroglu-Tufan, B. Aydinuraz, M. H. Satiroglu. Pamukkale University School of Medicine, Denizli, Turkey; Gen-Art Women Health, IVF and Reproductive Biotechnology, Ankara, Turkey; Ankara University School of Medicine, Ankara, Turkey. OBJECTIVE: Androgens acting via the androgen receptor are required for normal spermatogenesis. The androgen receptor gene (AR) has a repetitive DNA sequence in exon 1 that encodes a polymorphic polyglutamine tract. A link between idiopathic male infertility and expansion of this polymorphic (CAG) tract in the AR has been suggested by several recent studies, whereas several others were unable to demonstrate an association. Thus, ethnic background of the population studied may play an important role in this association. The objective of this study was to determine whether changes in (CAG) repeat length of the AR are associated with spermatogenic defects in Turkish patients with idiopathic male infertility. DESIGN: A prospective case-control study. MATERIALS AND METHODS: Forty-seven infertile patients and 32 fertility proven controls were included in this study. Clinical analyses included cause of infertility, semen analysis, and reproductive hormone (LH, FSH, and testosterone) concentrations. Analysis of trinucleotide (CAG) repeat length was performed by polymerase chain reaction (PCR) and sequencing targeting the AR (CAG) tract using blood DNA of each infertile patient and control. Results were statistically evaluated using the Mann-Whitney U-test and the test for difference between two population proportions. P⬍0.05 was taken as statistically significant for both tests. RESULTS: The mean serum LH level in the infertile group was significantly higher than that of the control group [6.99 ⫾ 0.74 IU/L (range 2.2–13.8) vs. 3.35 ⫾ 0.43 IU/L (range 1.9 –5.9), respectively; P⬍0.0001]. The mean serum FSH level in the infertile group was also significantly higher than that in the control group [13.53 ⫾ 1.87 IU/L (range 3.44 –36) vs. 3.54 ⫾ 0.51 IU/L (range 1.1–5.43), respectively; P⬍0.0001]. In contrast, the mean serum testosterone level in the infertile group did not differ significantly from that in the control group [18.45 ⫾ 1.41 nmol/L (range 3.8 –27.8) vs. 21.79 ⫾ 1.92 nmol/L (range 10.3–27.5), respectively; P⫽0.16]. The mean length of the AR (CAG) tract in the infertile group did also not differ significantly from that in the control group [22.28 ⫾ 0.37 (range 18 –29) vs. 22.41 ⫾ 0.54 (range 16 –29), respectively; P⫽0.84]. In addition, the frequency of having a ⱖ24 AR (CAG) repeat number was also comparable in between the infertile patients and fertile controls [31.9 % vs. 40.6 %, respectively; P⫽0.21]. CONCLUSION: The results from analyses of reproductive hormones with elevated LH and FSH, and normal or low testosterone levels are suggestive of partial impairment of testicular function and are in agreement with results from previously published studies. On the other hand, analysis of the AR (CAG) tract length showed no statistically significant relationship between the size of the (CAG) repeat of the polyglutamine tract and idiopathic impaired sperm production in the population studied. The variability of the results by other researchers may be explained by variations of the ethnic backgrounds of the populations studied. In addition, it is highly probable that the infertile males included in previously published reports may represent a heterogeneous group with respect to the cause of defective spermatogenesis. Supported by: Pamukkale University Scientific Research Grants Fund.