1 University Department of Growth and Reproduction, Rigshospitalet, The Juliane Marie Centre and 2 Department of Biostatistics, University of Copenhagen, Copenhagen, Denmark
3 To whom correspondence should be addressed at: University Department of Growth and Reproduction, Blegdamsvej 9, Section GR-5064, DK-2100 Copenhagen, Denmark. E-mail: niels.joergensen{at}rh.hosp.dk
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Abstract |
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Key words: finger ratio/HOX genes/semen quality/sex hormones/testicular dysgenesis syndrome
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Introduction |
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Materials and methods |
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Physical examination and finger ratio measurement
At the day of attendance, a physical examination was performed by one of four physicians. They examined 38.9, 11.9, 47.8 and 1.4% of men respectively. Information on weight and height was obtained, the genital development and the possible presence of any genital malformation was recorded. Assessment of testicular volume was done by orchidometer and ultrasound. Additionally, information on date of birth of the men was obtained.
As a part of the physical examination, the men had an outline of the ventral surface of their right hand made by the examining physician. These drawings were used for the measurement of the 2nd and 4th finger length, from the basal crease of the finger to the tip. The assessments of all the drawings were done by one investigator only. Before starting the assessment, the quality of the evaluation of the drawings had been assured by a blinded control assessment of the finger length of 10 participants in a random selection. The investigator measured the same drawings twice randomly on two different days.
Blood sample
A blood sample was drawn from each participant and serum was kept frozen at 20°C until the analysis. The hormone assessments were done simultaneously to reduce intra-laboratory variations. Serum levels of FSH, LH and sex hormone-binding globulin (SHGB) were determined, using a time-resolved immunofluorometric assay. Testosterone levels were determined using a time-resolved fluoroimmunoassay. Free androgen index was calculated as total testosteronex100/SHBG. Inhibin B levels were determined by a specific two-sided enzyme immunometric assay. Intra- and inter-assay coefficients of variation (CV) for measurements of both FSH and LH were 3 and 4.5%. CV for both testosterone and SHBG were <8 and <5%, and CV for inhibin B were 15 and 18% respectively.
Semen sample
Semen samples were obtained by masturbation. The participants had been advised to keep an ejaculatory abstinence period of 48 h prior to sampling. The actual abstinence period was assessed as the time between current and previous ejaculation. The semen samples were produced in the privacy of a room near the laboratory and kept in the laboratory at 37°C until analysis. Semen volume was estimated by weighing the collection tube with the semen sample and subtracting the predetermined weight of the empty tube, assuming that 1 ml = 1 g. For sperm motility assessment, 10 µl of well-mixed semen was placed on a clean glass slide (which had been kept at 37°C) and covered with a 22x22 mm coverslip. The preparation was placed on the heating stage of a microscope (37°C) and immediately examined at x400 magnification. The spermatozoas were classified as either motile [World Health Organization (WHO 1999
) motility class A + B + C] or immotile (WHO motility class D), in order to report the percentage of motile sperm. For the assessment of the sperm concentration the samples were diluted in a solution of 0.6 mol/l NaHCO3 and 0.4% (v/v) formaldehyde in distilled water. The sperm concentration was subsequently assessed using a BürkerTürk haemocytometer. Only spermatozoas with tails were counted.
Statistics
For the descriptive statistics, mean and median values, SD and 595th percentiles were calculated for the different variables.
Between-group differences of the different parameters in the Copenhagen group and the Aalborg group were tested by MannWhitney test. From this, a significant difference between the variables of the men from Copenhagen and the men from Aalborg in relation to height, BMI and mean testis size (orchidometer) became evident (P < 0.05) (see also Table I). Further analysis of these variables were therefore done separately for men from each city as well as combined. For the remaining variables, no significant differences between the two groups of men were detected and all further analyses were therefore combined in one group.
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For the blinded control assessments of the drawings, Wilcoxon signed ranks test was used. The result was a non-significant difference between the 2nd finger measurement (P = 0.32) and the 4th finger measurement (P = 0.10), the difference between the ratio also being non-significant (P = 0.46).
SPSS version 11.0 (2001) for Windows was used to perform all statistical analyses.
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Results |
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The MannWhitney test only showed a statistically significant difference between the results from Copenhagen and Aalborg in relation to height (P < 0.0005), BMI (P = 0.002) and mean testis size assessed by orchidometry (P = 0.002). Although a statistical difference was detected, the actual differences are regarded as minor and without biological relevance. Therefore, the young men from Copenhagen and Aalborg were combined into one group for further analysis.
Statistically, only the Spearman correlations between 2D:4D and the variables FSH (P = 0.03) and inhibin B/FSH (P = 0.04) reached statistical significance (Table I).
For the regression analyses the ejaculatory abstinence period was considered a possible confounder for the semen parameters. A duration of abstinence of ejaculation up to 96 h increased significantly the semen volume (P = 0.002), sperm concentration (P = 0.020), total sperm count (P = 0.001) and motility (P = 0.006). Duration of abstinence >96 h could not be shown to possess a confounding influence on the semen parameters. Only the duration of abstinence up to 96 h was therefore included in the further analysis of these parameters.
Assessment delay in minutes (time from ejaculation to start of analysis) was also evaluated as a possible confounder of the motility. The assessment delay ranged from 5 minutes to 2 h 30 min, mean 31 min, median 30 min and SD 10 min. Assessment delay did not have a significant confounding influence on the sperm motility (P = 0.23) and was hence not included in the analysis as a confounder.
The hour of the day of blood sampling was evaluated as a possible confounder for the hormone values. The blood sampling was performed in Copenhagen between 08:25 and 13:35 and in Aalborg between 07:50 and 19:40 respectively. In Copenhagen the majority of blood samples (73.4%) were drawn between 08:25 and 10:00. In Aalborg the majority were drawn between 16:00 and 19:40 (64.8%). The hour of the day of the blood sampling had a statistically significant impact in relation to the inhibin B/FSH level in the Copenhagen subgroup (P = 0.05) with higher values in the early morning hours. Consequently, the hour of the day of blood sampling was included in the further analysis of this parameter.
Additionally, the confounding effect of the examining physician was non-significant (P = 0.11) and therefore not included in the final model for the regression analysis.
Table II shows the results of the linear regression analyses. A statistically significant association with the 2D:4D finger ratio was found only in relation to ln FSH in group B (men having 2D:4D >1, P = 0.036, B/10 = 0.28), and in group A (men having 2D:4D 1) in relation to the total sperm count (P = 0.045, B/10 = 0.81). In group A, a statistical significance was found also in relation to the height (P = 0.002, B/10 = 344). However, the (2D:4D ratio)2 for height also showed a statistical significance, thereby indicating a non-linear association. For other tested parameters this quadratic term was non-significant.
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These results of the linear regression analyses indicate that an alteration of the 2D:4D finger ratio with, for example, 0.1 has an increasing effect on the FSH level of e(0.28) = 1.324 in group B and on the total sperm count of e(0.81) = 2.255 in group A. Consequently, this means that if the 2D:4D ratio is increased by 0.1 (e.g from 1.0 to 1.1), the FSH level should accordingly increase with 32.4% (95% CI 272), but only in the group of young men with 2D:4D ratio >1. In the group of young men with 2D:4D finger ratio 1, an increase of the finger ratio of 0.1 should consequently result in an increase in the total sperm count of 125.5% (95% CI 2400).
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Discussion |
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The increased FSH level was not found to coincide with altered semen parameters, hence indicating a possible compensated reduced spermatogenic capacity. From a biological point of view, this finding supports the Manning theory that the 2D:4D finger ratio >1 correlates with reduced male testis function. Contrary to this, increasing 2D:4D finger ratio was associated with increasing total sperm count in the group of young men with 2D:4D finger ratios 1. This finding contradicts the Manning theory, because an increased total sperm count should accordingly not coincide with an increased finger ratio. The mean 2D:4D ratio of 1.02 detected in our study is slightly larger than the original mean ratio of 0.99 observed by Manning et al. (1998)
. However, this is not likely to influence the interpretation of our results. The effects of increasing or decreasing ratios will be the same.
The validity of the present study is good. The examined young men are representative of the general population of young men in Denmark and the semen and sex hormone parameters used in this study are relevant for the evaluation of the testicular function. In addition, the sampling procedures and the methodology of the semen analysis and the hormone analysis were standardized. The findings of only two direct associations could not be explained, neither by the differences in length of abstinence or the hour of blood sampling, nor by the differences between the two cities nor differences between the doctors who performed the examinations, as these confounders already have been accounted for in the different analyses. The measurement of the fingers was done from a drawing of the right hand, using the ventral surface and the finger length from the crease of the finger to the top according to specifications by Manning et al. (1998). The drawing of the right hand was done by the same four physicians and the measurement of the drawing was done by one investigator only under controlled circumstances.
Manning et al. (1998) have found a direct significant association between the finger ratios of 2D:4D of the right hand and LH, prolactin, and estrogen level respectively, and an inverse significant association between the finger ratio and the testosterone levels. Unlike our study, Manning et al. (1998)
also found a statistically significant inverse association with sperm count but no significant association between the 2D:4D finger ratio and the FSH level, which was the only parameter that showed significance in both correlation and linear regression in our study. Manning et al. (2004)
investigated the relationship between 2D:4D finger ratio (both right and left hand) and testicular function in men attending an infertility clinic and in men included from the UK general population. They found a direct association between the 2D:4D finger ratio and the FSH and LH respectively, and an inverse association with testosterone level and testis volume in men with compromised testicular function, but not in men with normal testicular function. The only common denominator between the results of that particular study and our study is the direct association of FSH and the 2D:4D finger ratio.
Firman et al. (2003) found no significant association between 2D:4D ratios and measures of semen quality, neither for right nor left hands. However, they did find a significant inverse association between body asymmetry (i.e. subtracting the left from the right side values of the ear, wrist, elbow, ankle and foot) and sperm count as well as sperm motility.
TDS is probably caused by an abnormal fetal development, and the aetiology may in some cases be due to an increased fetal exposure to estrogenic or anti-androgenic substances (Skakkebæk et al., 2001; Sharpe, 2003
). The HOXA and HOXD genes are essential for both limb and genital development (Kondo et al., 1997
), and studies have shown that HOX gene expression can be reduced by estrogen exposure (Taylor et al., 1999
; Block et al., 2000
). However, the specific association between TDS and the deregulation of HOX genes remains uncertain.
In conclusion, we have shown direct associations between the ratio of the 2nd to 4th finger length and the FSH level and the total sperm count in a subset of young Danish men, thereby indicating the presence of TDS symptoms. Nevertheless, these results are mutually contradictory and one must question whether our statistically significant result is actually a false positive (statistical type I error) rather than a true association. We cannot exclude a true biological relationship between finger length and testicular function, but overall one could now interpret the data as indicating that Mannings original hypothesis-generating study was on a small scale, suggesting an association between semen quality and 2D:4D in 67 men (Manning et al., 1998) which he was unable to replicate in a second study (Manning et al., 2004
). Furthermore, the study by Firman et al. (2003)
, although also on a small scale (n = 50), does not support Mannings hypothesis. Our relatively large study of young, normal Danish men shows no reliable association between 2D:4D finger ratio and testicular function and therefore does not support Mannings hypothesis. Thus, taken together, drawings of finger lengths do not have the power to predict the testicular function of adult men.
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Acknowledgements |
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References |
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Firman RC, Simmons LW, Cummins JM and Matson PL (2003) Are body fluctuating asymmetry and the ratio of 2nd to 4th digit length reliable predictors of semen quality? Hum Reprod 18,808812.
Kondo T, Zákány J, Innis JW and Duboule D (1997) Of fingers, toes and penises. Nature 390,29.[CrossRef][ISI][Medline]
Manning JT, Scutt D, Wilson J and Lewis-Jones DI (1998) The ratio of 2nd to 4th digit length: a predictor of sperm numbers and concentrations of testosterone, luteinizing hormone and oestrogen. Hum Reprod 13,30003004.[Abstract]
Manning JT, Wood S, Vang E, Walton J, Bundred PE, van Heyningen C and Lewis-Jones DI (2004) Second to fourth digit ratio (2D:4D) and testosterone in men. Asian J Androl 6,211215.[ISI][Medline]
Sharpe RM (2003) The oestrogen hypothesis where do we stand now? Int J Androl 26,215.[CrossRef][ISI][Medline]
Skakkebæk NE, Rajpert-De Meyts E and Main KM (2001) Testicular dysgenesis syndrome: an increasingly common development disorder with environmental aspects. Hum prod 16,972978.
Taylor HS, Igarashi P, Olive DL and Arici A (1999) Sex steroids mediate HOXA11 expression in the human peri-implantation endometrium. J Clin Endocrinol Metab 84,11291135.
World Health Organization (1999) WHO laboratory manual for the examination of human semen and sperm-cervical mucus interaction, fourth edition. Cambridge University Press.
Submitted on April 15, 2005; resubmitted on May 25, 2005; accepted on May 31, 2005.