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
2Ten Harley Street, London W1G 9PF, 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.
Tel: +44-1227-823022, Fax: +44-1227-763-912
E-mail: d.k.griffin@kent.ac.uk
Received 2005-02-24 Accepted 2005-07-06
DOI: 10.1111/j.1745-7262.2005.00068.x
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.
|