A study of finger lengths, semen quality and sex hormones in 360 young men from the general Danish population

A.K. Bang1, E. Carlsen1, M. Holm1, J.H. Petersen1,2, N.E. Skakkebæk1 and N. Jørgensen1,3

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


    Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
BACKGROUND: It has been suggested that finger length may correlate with function or disorders of the male reproductive system. This is based on the HOXA and HOXD genes’ common embryological control of finger development and differentiation of the genital bud. The objective of this study was to explore the association between the ratio of 2nd to 4th finger length (2D:4D ratio) and testis function in a sample of young Danish men from the general population. METHODS: Semen samples and finger measurements were obtained from a total of 360 young Danish men in addition to blood samples for sex hormone analysis to describe the possible association between 2D:4D and semen and sex-hormone parameters. RESULTS: A statistically significant inverse association with the 2D:4D was found only in relation to hormone levels of FSH in the group of young men with a 2D:4D >1 (P = 0.036) and a direct association with the total sperm count in the group of young men with a 2D:4D #1 (P = 0.045). CONCLUSION: The statistically significant results may be ‘false positives’ (type I error) rather than representing true associations. This relatively large study of young, normal Danish men shows no reliable association between 2D:4D finger ratio and testicular function. Measurements of finger lengths do not have the power to predict the testicular function of adult men.

Key words: finger ratio/HOX genes/semen quality/sex hormones/testicular dysgenesis syndrome


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
During recent years, there has been a decline in human semen quality and at the same time an increase in the occurrence of testicular germ cell cancer, cryptorchidism and hypospadias (Skakkebæk et al., 2001Go). Experimental and epidemiological studies suggest that testicular cancer, poor semen quality, hypospadias and undescended testis may all be symptoms of a main syndrome—the testicular dysgenesis syndrome (TDS) (Skakkebæk et al., 2001Go). The cause of the syndrome is believed to be mainly environmental, including lifestyle factors disrupting embryonic development and thereby gonadal development during fetal life, possibly against a background of genetic susceptibility. The influencing environmental factors are suggested to act estrogen-like or anti-androgen-like (Skakkebæk et al., 2001Go; Sharpe, 2003Go). Manning et al. (1998)Go reported an association between the development of the fingers and the prenatal level of testosterone and estrogen and suggested that the ratio of 2nd finger to 4th finger length (2D:4D ratio) is inversely associated with prenatal testosterone and directly with prenatal estrogen. Thus, the adult finger ratio may be indicative of sex steroid exposure in utero because the two groups of Homeobox genes known as HOXA and HOXD are required for the differentiation of the genital bud, as well as for the growth of fingers (Kondo et al., 1997Go). The common control of finger development and differentiation of the genital bud has raised the possibility that dysregulation of these HOX genes may at the same time alter the relative lengths of fingers and affect the development and the function of the male reproductive system. The objective of this study was to explore a possible association between the 2D:4D ratio and testis function in a group of young Danish men from the general population. A possible association could be used to indicate presence of TDS in larger scale population studies.


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
Study population
In Denmark, all young men at 18–25 years of age (with the exception of those suffering from chronic diseases, which is <15%) are required to attend a compulsory medical examination before they are considered for military service. Men appearing before the draft board are therefore representative of the general population of young, healthy men in Denmark. Since autumn 1996, young men from the Copenhagen area in Denmark have been invited for a study of reproductive function. They were invited for the study on the day they attended the compulsory medical examination and received written information and were offered further verbal information when the military health authorities had completed their examination, independent of whether they were accepted for military service. For men agreeing to participate, an appointment for examination at University Department of Growth and Reproduction, Rigshospitalet was made. A similar study using the same invitation procedure and inclusion criteria was undertaken—also by staff from Rigshospitalet—in the city of Aalborg in the northern part of Denmark in November–December 2001. The study in Aalborg was undertaken at the Department of Urology, Aalborg Sygehus Nord. The men investigated in Copenhagen from September 1, 2001 to January 21, 2002 and the men examined in Aalborg had a drawing of their right hand made in addition to the investigations included in the semen study itself. A total of 370 men out of the 1198 invited to participate actually did so (participation rate 31%). Ten men did not have a drawing of their right hand made for unknown reasons and were therefore excluded from the statistical analysis. Of the 360 men that had a drawing of their right hand made, 255 were from Copenhagen and 105 were from Aalborg. Examination of the right hand was chosen because Manning et al. (1998)Go found a particularly strong relationship between 2D:4D of the right hand and testicular function and only a weak relationship to the left hand.

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 1999Go) 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 Bürker–Türk haemocytometer. Only spermatozoas with tails were counted.

Statistics
For the descriptive statistics, mean and median values, SD and 5–95th 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 Mann–Whitney 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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Table I. Basic descriptions including 2D:4D finger ratio, sex hormones and semen parameters of young Danish men.

 
For a visual inspection of the relationship between the 2D:4D ratio and the different variables, scatter plots with fitted lines were performed. In some of the scatter plots, the fitted line (lowess) had a tendency towards bending in the region of the 2D:4D finger ratio = 1. This tendency was seen for weight, height, mean testis size (orchidometer and ultrasound), free androgen index, sperm concentration, total sperm count, and motility. As a consequence of the apparent effect of 2D:4D >1.0 or <1.0, the men were therefore divided into two new groups: group A consisting of the men with a 2D:4D finger ratio ≤1.0, and group B consisting of the men with a 2D:4D ratio >1.0. Finally, linear regression analysis was performed separately on the two groups (A and B). In order to obtain a normal distribution of the residuals, the data of semen parameters and sex hormones were natural log-transformed before analysis. To test linearity between the tested parameter and the 2D:4D ratio, the (2D:4D ratio)2 was included in the model. Significance of this indicated a non-linear association. Potentially relevant confounders, including duration of ejaculatory abstinence, physician and others were evaluated and included in the final analysis if significant. Duration of ejaculation abstinence entered the model as piecewise linear functions (linear splines); e.g. one straight line for abstinence <48 h, another straight line for abstinence periods 48–96 h etc. The final linear regression models were subjected to standard check of the residuals.

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.


    Results
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
The basic descriptions of the study population are summarized in Table I, which also shows 2D:4D ratio, sex hormones and semen parameters of the young men from the general Danish population. The Mann–Whitney test showed non-significant differences between Copenhagen and Aalborg for the finger lengths and ratios (2nd finger length: P = 0.21; 4th finger length: P = 0.60; 2D:4D: P = 0.16).

The Mann–Whitney 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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Table II. Analyses of linearity between semen parameters, sex hormones, physical appearance and 2D:4D ratio

 

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 2–72), 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 2–400).


    Discussion
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
An association between the ratio of 2nd to 4th (2D:4D) finger length and testis function was found in relation to the FSH level among the young Danish men having 2D:4D >1, and in relation to the total sperm count among the young men having a ratio ≤1.

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)Go. 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)Go. 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)Go 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)Go 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)Go 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)Go 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., 2001Go; Sharpe, 2003Go). The HOXA and HOXD genes are essential for both limb and genital development (Kondo et al., 1997Go), and studies have shown that HOX gene expression can be reduced by estrogen exposure (Taylor et al., 1999Go; Block et al., 2000Go). 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 Manning’s original hypothesis-generating study was on a small scale, suggesting an association between semen quality and 2D:4D in 67 men (Manning et al., 1998Go) which he was unable to replicate in a second study (Manning et al., 2004Go). Furthermore, the study by Firman et al. (2003)Go, although also on a small scale (n = 50), does not support Manning’s 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 Manning’s hypothesis. Thus, taken together, drawings of finger lengths do not have the power to predict the testicular function of adult men.


    Acknowledgements
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
The volunteers who participated in the study are thanked. Dr Richard Sharpe, MRC Centre for Reproductive Biology, Edinburgh, UK is thanked for inspiring this study, and Ms Julie Køning is thanked for assessment of the finger lengths. The study was supported by the European Union (QLK4-CT-1999-01422) and the ‘Svend Andersen’s Foundation’.


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
Block K, Kardana A, Igarashi P and Taylor HS (2000) In utero diethylstilbestrol (DES) exposure alters Hox gene expression in the developing Müllerian system. FASEB J 14,1101–1108.[Abstract/Free Full Text]

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 18,808–812.[Abstract/Free Full Text]

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 13,3000–3004.[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 6,211–215.[ISI][Medline]

Sharpe RM (2003) The ‘oestrogen hypothesis’ – where do we stand now? Int J Androl 26,2–15.[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 16,972–978.

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 84,1129–1135.[Abstract/Free Full Text]

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.