Editor-in-Chief
Prof. Yi-Fei WANG,
Asian J Androl 2005; 7 (4): 419-425 |
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This web only provides the extract of this article. If you want to read the figures and tables, please reference the PDF full text on Blackwell Synergy. Thank you. - Complementary Medicine - Significant reduction of sperm disomy in six men: effect of traditional Chinese medicine? Helen G. Tempest1, Sheryl T. Homa2, Xiao-Ping Zhai2, Darren K. Griffin1 1Department of Biosciences, University of Kent, Canterbury CT2 7NJ, UK Abstract Aim: To test the hypothesis that levels of sperm disomy fell significantly in six men treated by traditional Chinese medicine (TCM). Methods: Fluorescence in situ hybridization (FISH) was done on the sperm heads of six men before and during treatment by TCM. Results: There was a significant reduction in sperm disomy in all six men. This coincided with TCM treatment. Conclusion: This is the first study reporting a significant reduction in sperm disomy in men over a given time course. The fact that this coincided with TCM treatment is intriguing but no conclusions can be drawn from this until placebo-controlled clinical trials are implemented. (Asian J Androl 2005 Dec; 7: 419-425) Keywords: traditional Chinese medicine; sperm disomy; chromosome; male infertility Corresponence to: Dr Darren K. Griffin, Department of Biosciences,
University of Kent, Canterbury CT2 7NJ, UK.
1 Introduction Sperm aneuploidy is the presence of one or more extra or missing chromosomes in the sperm head. Because of the difficulties of probe hybridization with distinguishing nullisomy (absence of a chromosome), disomy (an extra chromosome) is usually only noted [1]. The level of sperm disomy therefore can be defined as the proportion of sperm with an extra given chromosome. A number of factors have been associated with increased levels of sperm disomy including age [2], smoking [3], chemotherapy [4] and, more significantly, severely compromised semen parameters [1]. Men with abnormal levels of sperm concentration, motility and/or morphology (e.g. severe oligoasthenoteratozoospermia [OAT]), have been shown to have up to a 30-fold increase in levels of sperm disomy compared with normal controls [1]. This is by far the most significant correlate of increased sperm disomy; for example, by comparison, an increase in age from 25 to 55 correlates to only a two-fold increase in sperm disomy for the sex chromosomes [2]. Men with severely compromised semen parameters, e.g. severe OAT (and thus often a high incidence of sperm disomy), are frequently treated by intracytoplasmic sperm injection (ICSI). The subsequent potential for non-Mendelian transmission of diseases such as Klinefelter Syndrome and Down Syndrome, as well as the increased propensity for ICSI failure through injection of a disomic sperm, has raised considerable debate in both the scientific and popular press [1, 5]. To date, however, clinical intervention has been limited to screening for defects with a view to genetic counseling of individuals with abnormally high levels about 1) the risks of affected children and 2) the likely success of the ICSI procedure itself [5]. To our knowledge, there have been no studies reporting that these high levels in infertile men can, potentially, be reduced. In this paper, we assayed sperm disomy levels of six men at various time points. By coincidence, all were under treatment for infertility by traditional Chinese medicine (TCM). TCM originated in China over 5 000 years ago and is still used as a primary therapy in a number of Asian countries, often in parallel with Western medicine [6]. The diagnostic techniques and therapeutic philosophy in TCM differ from Western medicine in that, rather than target specific biochemical pathways, they define health in terms of balance. Simplistically, TCM is based around the doctrines of Yin-Yang that are defined as two fundamental principles that oppose and complement each other and are present in all things. Yin-Yang is a balance between the anabolic and catabolic processes, and healthy physiology and metabolism depend on the "vital energy", or Qi that flows throughout the body [6]. Thus both the body in isolation and that in relationship to the environment should be balanced, and any alteration in this balance (between Yin and Yang) results in disease. The application of TCM either in the form of acupuncture or herbal formulations has been used to treat a range of infertility phenotypes, primarily those affecting female infertility and male infertile factors, including varicocoele, seminal plasma abnormalities and semen parameters. Studies carried out using TCM have been shown to significantly improve the quality and quantity of sperm (for a review of the TCM treatment of female and male infertility see Xu et al. [6]). To the best of our knowledge, this is the first study performed to assess sperm aneuploidy levels as a result of, or coincident with, any medical intervention.
2 Materials and methods
Six men undergoing TCM treatment for infertility at the Zhai Clinic, Harley Street, gave informed consent for the levels of sperm chromosome abnormalities to be assayed during the time that they attended the clinic. Controls were normal and OAT donors who did not receive TCM treatment. The age of patients and controls ranged from 33 to 39 years with a mean of 35.5 years; none of the patients were smokers nor drug-users, and none were receiving chemotherapy treatment. Individuals were given a physical examination, and their semen quality was assessed according to the WHO guidelines and Kruger strict criteria for assessment of morphology. The TCM treatment that they received consisted of a cocktail of 10-20 herbs (taken as an infusion). A list of the herbs is given in Table 1, (for more information contact the Zhai clinic, 112 Harley Street, London, W1N 1AF, UK; Tel:+44-207-9083-866; E-mail: zhaiclinic@btopenworld.com). In line with TCM practice the composition of the treatments and the doses varied depending on the results of the physical examination, which included examinations of the patients¡¯ pulse, diet, appearance of the tongue, lifestyle and semen assessment. That is, patients took 5 g and 15 g of each herb, mixed together in a 1-L infusion. Sperm disomy was assessed according to Griffin et al. [2, 7] for chromosomes X, Y and 21 (trisomies of which can lead to Klinefelter or Down Syndrome). All sperm samples were taken on the day of donation, resuspended three times in buffer (10 mmol/L Tris. HCl, 10 mmol/L NaCl) then spread on to a glass slide. Fluorescence in situ hybridization (FISH) followed standard protocols, (i.e. swelling using 10 mmol/L Dithiothreitol [DTT]), simultaneous denaturation of probe and target at 72 ºC followed by formamide stringency washes and 4,6 Diamidinophenylindole (DAPI) counterstain. Probes used were supplied by Vysis (Downers Grove, IL, USA) and labeled with spectrum orange (Chromosomes 21 and X) and spectrum green (chromosomes X and Y). Thus, chromosome 21 appeared in red, X chromosome in yellow and Y chromosome in green. The total number of 5 000 sperm was chosen in line with the majority of studies that have investigated the association between sperm disomy and compromised semen parameters [1]. The number of sperm with an extra one of these chromosomes (out of 5 000) was noted for each sample using a Leica epifluoresence microscope equipped with Photometric CCD camera and Smart Capture software (Digital Scientific, Cambridge, UK). Samples were taken at entry into the clinic then at subsequent intervals between 6 weeks and 6 months. The results were controlled in three ways. First, disomy levels were compared to samples from each of the four control males who were of proven fertility (a total of nine samples); this was designated control group 1. Second, patient disomy levels were compared to one sample from each of the four males with severe OAT (control group 2). Finally, all disomy levels for each patient were compared with the first sample and the sample immediately preceding it to address the question of whether the disomy levels had fallen. All preparations were scored "blind" by at least two independent observers (i.e. without prior knowledge of the origin of the sample); strict scoring criteria [2, 7] were applied.
3 Results
In all six patients, initial samples were significantly greater than the mean of control group 1 (normal) and comparable to the mean of control group 2 (severe OAT). Sperm disomy subsequently fell to levels not significantly different to that of the mean of control group 1. That is, levels in the final sample were all significantly lower than the initial sample when all chromosomes were taken into account. Table 2 showed the levels for each chromosome pair; Table 3 showed the levels collectively for all chromosomes assayed as well as information about the semen assessments. Patients had varying degrees of aberrant semen parameters; however, we found no associations between semen quality and improved sperm disomy.
4 Discussion
As far as we are aware, this is the first study to report a significant improvement in the levels of sperm disomy in men with compromised semen parameters. The effects of smoking [3] and chemotherapy [4] on sperm disomy have been well reported and thus the inference was that individuals who either quit smoking or complete their course of chemotherapy treatment might return to normal levels. Indeed, Robbins et al. [4] reported a decline back to pre-treatment levels around 100 days after the cessation of chemotherapy treatment. In this study, however, males who began with very high levels of sperm disomy showed a significant improvement. Given that the issue of the possible transmission of chromosome abnormalities via ICSI raises significant concerns [2]; any approach that was effective in reducing sperm disomy could ultimately be used prior to ICSI to improve success rates (i.e. to increase the chances of injecting a chromosomally normal sperm). While we do not claim that we have yet discovered such a protocol that we have at least demonstrated that high chromosome abnormalities levels can be reduced. This leaves us to speculate on the reasons why the levels fell significantly in each patient. The association with the TCM treatment and the significant reduction coincident with this treatment is intriguing. It would be unwise at this stage to suggest that the TCM treatment itself was the reason for the improvement. Nevertheless it is not unrea-sonable, given these results, to propose that double-blind placebo-controlled clinical trials should ensue to test this hypothesis. Of course, good, well-designed studies should require no other justification; however, it is our opinion and experience that such a trial would be unlikely to be supported either financially or by ethical committees until a degree of evidence was found that suggested that the trial might be effective and thus of patient benefit. Indeed Yuan and Lin [8] emphasized the importance of open tube studies as forerunners for double- blind, placebo-controlled clinical trials of new pharmaceutical regimes; this was an example of such a study. In order to address the contentious issue of changing prescriptions and potential variability in herb potency, we suggested that a future double-blind placebo-controlled clinical trial should mirror that of Bensoussan et al. [9]. That is, the TCM practitioner would examine every patient in the traditional way and make the prescription. The first group would receive the herbs as prescribed. The second group would, unbeknown to the practitioner, receive, not the herbs as prescribed, but a predetermined combination standardized for potency that would not change. The last group would (again unbeknown to the practitioner) receive a placebo and neither patient nor practitioner would be aware of which patients were in which group. There are few reports in the literature of either natural remedies or traditional treatments having an effect on fertility [6]; however, Gurfinkel et al. [10] reported a positive effect of acupuncture and moxa treatment on the semen parameters of 19 men in a controlled, blind study. Moreover, St. John¡¯s Wort has been shown to have a negative effect on sperm motility if the sperm are exposed to it directly [11]. One possible explanation for our observations might be that the mechanism causing increased abnormalities involves an excess of estrogens and/or reactive oxidant species to which these males are particularly susceptible. Indeed smoking, in part through causing the formation of excessive reactive oxidative species, is believed to reduce male fertility and increase levels of sperm chromosome abnormalities, perhaps through impeding DNA repair or spindle formation mechanisms in meiosis [3]. The role of estrogen in testicular function is evident from reports of sexually mature, male, estrogen receptor knockout mice that are infertile due to seminiferous tubule dysmorphogenesis and impaired spermatogenesis [12]. Male, androgen receptor knockout mice show a steady, age-related fertility decline with disrupted spermatogenesis at early spermatid stage and Leydig cell hyperplasia; low levels of dietary phytoestrogens partially prevent these disruptions [13]. Furthermore, Feng et al. [14] reported a positive effect of ligustrum fruit extract on the reproductive potential of diabetic rats. While we are not aware of studies correlating endocrine disruption and sperm disomy. The action of these chemicals have been associated, sometimes controversially, with an increase in testicular dysgenesis syndrome over the last 70 years [14]. Moreover, one recent study correlated the increase in aneuploidy in endocrine disrupted mice [16]. If small quantities of endocrine disruptors or reactive oxidative chemicals were contributing to increased levels of sperm disomy in susceptible men, this might provide the basis for a mechanistic explanation as to why these levels subsequently improved in our patients. The presence of antiestrogens or antioxidants in the form of Chinese herbs might be one possibility; however, in the absence of properly controlled trials, this remains to be established. Indeed Rolf et al. [17] reported no significant changes in semen parameters and no pregnancies in asthenozoospermic and oligoasthenozoospermic patients exposed to anti-oxidant treatment. It might be noted, however that, in a related study (Tempest et al. unpublished data), we have found evidence of antiestrogenic and antioxidant activity in the herbs used to treat these men. Further studies involving controlled clinical trials and careful chemical analyses of the herbs used to treat them should make some significant steps towards establishing whether TCM does indeed have a positive effect in male fertility and sperm disomy. References 1 Tempest HG, Griffin DK. The relationship between male infertility and increased levels of sperm disomy. Cytogenet Gen Res 2004; 107: 83-94. 2 Griffin DK, Abruzzo MA, Millie EA, Sheean LA, Feingold E, Sherman SL, et al. Non-disjunction in human sperm: evidence for an effect of increasing paternal age. Hum Mol Genet 1995; 4: 2227-32. 3 Rubes J, Lowe X, Moore D 2nd, Perreault S, Slott V, Evenson D, et al. Smoking cigarettes is associated with increased sperm disomy in teenage men. Fertil Steril 1998; 70: 715-23. 4 Robbins WA, Meistrich ML, Moore D, Hagemeister FB, Weier HU, Cassel MJ, et al. Chemotherapy induces transient sex chromosomal and autosomal aneuploidy in human sperm. Nat Genet 1997; 16: 74-8. 5 Griffin DK, Hyland P, Tempest H, Homa ST. Safety issues in assisted reproduction technology: Shouldmen undergoing ICSI be screened for chromosome abnormalities in their sperm? Hum Reprod 2003; 18: 229-35. 6 Xu X, Yin H, Tang D, Zhang L, Gosden RG. Application of traditional Chinese medicine in the treatment of infertility. Hum Fertil (Camb) 2003; 6: 161-8. 7 Griffin DK, Abruzzo MA, Millie E, Hassold TJ. Sex ratio in normal and disomic sperm: evidence that the extra chromosome 21 preferentially segregates with the Y chromosome. Am J Hum Genet 1996; 59: 1108-13. 8 Yuan R, Lin Y. Traditional Chinese medicine: an approach to scientific proof and clinical validation. Pharmacol Ther 2000; 86: 191-8. 9 Bensoussan A, Talley NJ, Hing M, Menzies R, Guo A, Ngu M. Treatment of irritable bowel syndrome with Chinese herbal medicine: a randomized controlled trial. JAMA 1998; 280: 1585-9. 10 Gurfinkel E, Cedenho AP, Yamamura Y, Srougi M. Effects of acupuncture and moxa treatment in patients with semen abnormalities. Asian J Androl 2003; 5: 345-8. 11 Ondrizek RR, Chan PJ, Patton WC, King A. Inhibition of human sperm motility by specific herbs used in alternative medicine. J Assist Reprod Genet 1999; 16: 87-91. 12 Eddy EM, Washburn TF, Bunch DO, Goulding EH, Gladen BC, Lubahn DB, et al. Targeted disruption of the estrogen receptor gene in male mice causes alteration of spermatogenesis and infertility. Endocrinology 1996; 137: 4796-805. 13 Robertson KM, O'Donnell L, Simpson ER, Jones ME. The phenotype of the aromatase knockout mouse reveals dietary phytoestrogens impact significantly on testis function. Endocrinology 2002; 143: 2913-21. 14 Feng SL, Li SH, Wang Y, Chen CC, Gao B. Effect of ligustrum fruit extract on reproduction in experimental diabetic rats. Asian J Androl 2001; 3: 71-3. 15 Sharpe RM, Irvine DS. How strong is the evidence of a link between environmental chemicals and adverse effects on human reproductive health? BMJ 2004; 328: 447-51. 16 Hunt PA, Koehler KE, Susiarjo M, Hodges CA, Ilagan A, Voigt RC, et al. Bisphenol A exposure causes meiotic aneuploidy in the female mouse. Curr Biol 2003; 13: 546-53. 17 Rolf C, Cooper TG, Yeung CH, Nieschlag E. Antioxidant treatment of patients with asthenozoospermia or moderate oligoasthenozoospermia with high dose vitamin C and vitamin E: a randomised, placebo-controlled, double-blind study. Hum Reprod 1999; 14: 1028-33. |
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