Participant selection: Study survey was conducted for two years from 2009 to 2011. To maintain a homogenous population only those participants were included in the analysis whose both parents originally belonged to central Indian population. This reduces the population size from 1500 to 964 including both female (n = 464) and male (n = 500) subjects [Table/Fig-1]. The mean age for males was 24±6.44 and for females the mean age was 22.6±7.23. Volunteer subjects were recruited from students and staff of Chirayu Medical College Hospital, Bhopal, Peoples College of Medical Sciences and Research Centre, DB City Mall Bhopal, New Market, Bhopal. Subject’s willingness to participate in continuing study was asked and contact details were recorded for future correspondence. Out of 964 subjects only 135 subject showed interest in the future study.

Participation in the study was purely on voluntary basis. Subjects were asked about their parent’s ethnicity, age, birth order history (number of elder siblings and their sex and order). History regarding any miscarriage in the birth order was also asked. Finger lengths 2D (index finger) and 4D (ring finger) of both the hands (Palmer surface) were measured by direct measurement using a calibrated digital vernier caliper from the most proximal crease at the base of the digit to the tip of the finger [15].

The study was designed to assess the finger length ratio of the mentioned population and to find out fraternal birth order effect on the finger length ratio between the first born and second born child.

Exclusion Criteria: Subjects with any kind of arthropathy, history of cardiac disease, hyper/hypothyroidism, diabetes, hypertension, tuberculosis, any kind of restrictive or obstructive disease of lung, present history of liver disease, viral myositis, and other muscular diseases and if not 1^st or 2^nd child of the mother were excluded. Female subjects with hormonal disorders including polycystic ovarian disease and adrenal hyperplasia were excluded. If the subject was smoker or using tobacco in any form were excluded from the study.

Study population was categorized in several groups depending upon the sex and birth order.

Depending upon the birth order history three groups was made:

Group A: First born child,

Group B: With elder brother,

Group C: With elder sister

For both male and female subject groups.

Data was expressed as mean±Standard Deviation. The p<0.05 was considered as statistically significant. T-test was performed for comparison of the variables between the groups. Analysis of Variance (ANOVA) was applied to examine the fraternal birth order effect on finger length ratio.

Finger length ratio (2D:4D) for the males in the above mentioned population found to be 0.967 (SD±0.033) for right hand and 0.963 (SD±0.037) for left hand. For females the digit ratio found to be 0.982 (SD±0.027) for right hand and 0.974 (SD±0.034) for the left hand [Table/Fig-2,3]. Total study population mean 2D:4D ratio for right hand appears 0.976 (SD±0.031) and 0.969 (SD±0.035) for the left hand. The finger length ratio found to be smaller in male in comparison to females in both hands [Table/Fig-2]. The difference of finger length ratio was significant (p=0.03) for right hand [Table/Fig-3]. Birth order effect (Fraternal Birth order) did not show any significant variation in finger length ratio for both hands in case of males and females [Table/Fig-4,5].

Difference of length of Index (2D) and ring finger (4D) was measured for both the hands in both the sexes. Difference of lengths for right hand (R_(2D-4D)) was significantly more in males (-2.43±2.50, p = 0.024) [Table/Fig-3].

The research related to finger length ratio in Indian context came in to limelight post 2001 era [1] and slowly increasing linearly with time. Scientists are trying to explore the relationship between finger length ratio and different physiological factors in Indian population [16-18]. The present study is the first to examine the 2D:4D ratio in a large sample from Central India regional population. We took up a particular view point to examine that whether the fraternal birth order has any relationship with finger length ratio.

Developmental biology research shows that the prenatal androgen exposure that is the intrauterine sex steroid levels are responsible for this sexually dimorphic structural change which takes place around thirteenth week of gestational age [3]. High level of sex steroid during intra uterine life is supposed to be the reason for the lower 2D:4D ratio which is termed as more masculine pattern [3]. Studies have found that there is a significant sex difference in 2D:4D ratio of right hand between males and females with men having lower 2D:4D ratio than female [4]. The 2D:4D ratio has prominent overlapping and varies appreciably by ethnicity [5]. But interestingly the sex difference in 2D:4D ratio exists across the various ethnic groups. We find from the earlier studies that, the finger length ratio (2D:4D) is around 0.99 to 1.00 for Polish, Spanish and English people, 0.93 to 0.95 among Zulus, Finns, Jamaicans and 0.96 to 0.97 among Indian, German and Hungarian people [2]. India is a vast country with people from various ethnic backgrounds. We find a wide range values regarding 2D:4D ratio among different geographical locations and population of India [Table/Fig-6] [16-23]. Central India region consists of three states of Madhya Pradesh, Chhattisgarh and Maharashtra [24]. Due to its immense geopolitical significance and since the region was considered as the passage way between indo-gangetic plains and deccan, amalgamation of different ethnic population is observed [25]. Prior available data regarding Central India population for the same was not found on extensive search.

Digit ratio research use four different ways: left 2D:4D, right 2D:4D, mean 2D:4D, difference between right and left 2D:4D (called Dr-l) and rarely used 4D/height [11]. In our present study we found that the difference of index to ring finger length is significantly more (p = 0.024) for right hand in males than females. The significance was not found for the left hand (p=0.24).

Breedlove informed about his findings that 2D:4D ratio is influenced and significantly varies with the number of older brothers [26].

Sexual orientation, finger length ratio and birth order are studied frequently. It was indicated that later birth order is related to homosexuality in males. That is chances of homosexuality increase with having older brother. This relation between high fraternal order and homosexuality is termed as the fraternal birth order effect [26-28].

Blanchard and Bogaert derived a hypothesis known as maternal immune hypothesis which can be summarized as H-Y antigen (Y linked minor histocompatibility antigens) which is present in the cell surfaces of male fetus may cross the placenta and produce an immune reaction to some mothers [27,28]. In response mother produce the antibody which cross the placenta and alter the fetal brain development. It has also been reported that H-Y specific CD8 T cells were functional in mothers with previous male pregnancies for after eight years of their last male pregnancy [29]. The response to the antigen increase with each male fetus and thus the probability of homosexuality increases with each elder male sibling. Four male specific proteins encoded by SMCY, PCDH11Y, NLGN4Y, TBL1Y genes were considered as a possible antigenic substance underlying the FBO which were found to support the maternal immune hypothesis. Among these only the product of PCDH11Y isoform-a, was found to be exclusively expressed in the brain [28]. The other physiological aspects are also been correlated with this fraternal birth order. Birth weight and placental weight has negative and positive correlation with fraternal birth order. Animal studies where maternal immunization was done with paternal antigens have also shown that it affect fetal birth weight, placental weight or both. Zuckermann, Blanchard and several one indicate that prior male pregnancies decrease fetal weight for subsequent males [27].

These studies point towards the assertion that this fraternal birth order effect certainly causes alteration in growth of the male fetus. Williams found that men with more older brothers tend to have shorter index fingers relative to their ring fingers (i.e., low 2D:4D ratios). A replicated study done by Manning failed to confirm the finding of Williams study [26]. The study of Nicola S reported that males with more elder brother show more masculine 2D:4D ratios, and with more number of elder sisters the ratio appeared to be more feminine [30]. It was suggested that as the Y linked proteins those are targeted by maternal immune response are not exclusively limited to brain and may be found in other sites of the body [29]. The same effect in females was absent. In our present study the group consisting elder sister was made to make sure to avoid the effect of any maternal immune response. It was observed in our study that though mean value shows some differences similar to the previous studies but the difference between the groups is not significant. In our study we did not observe any birth order effect on finger length ratio. Our study observes that the fraternal birth order did not affect finger length ratio between the second born male or female and first born (elder) male. We observed that the applicability of the fraternal birth order theory is limited to males and this theory is not applicable to females because of lacking of Y linked proteins in them. Further researches show that different set of genes Wnt5a, Ihh, Bmp6, Fgfr2, lgfbp2/5, Sox9 and Runx2 play an important role in digit development and are regulated by the steroid [31].

Findings suggested that Androgen Receptor (AR) is a negative modulator of Wnt 5a expression in digits. 4D [31,32] has higher levels of AR and Estrogen Receptor (ER) than 2D. AR and ER activities influence the 2D:4D ratio by modulating levels of skeletogenic gene expression and cell proliferation in a digit-specific manner [31]. Another interesting fact was reported that the intrauterine timing of phalangeal growth regulation by AR and ER correlates with the timing of masculinization or feminization of brain by the steroids. It was also observed that several genes have role in the development of brain and different sexually dimorphic structures [33,34]. So, finger length ratio or 2D:4D ratio which was proposed as a reflection of prenatal androgen exposure is found to be caused by androgen and estrogen signaling [35].

Most of the research what we found regarding prenatal androgen exposure is related to sexual orientation and behavioural studies [1]. As earlier mentioned several studies show that there is considerable effect of prenatal androgen exposure (2D:4D ratio) on other physiological functions. More studies are required to establish the exact mechanism leading to adult diseases associated with finger length ratio.

Fraternal birth order effect on finger length ratio was not established in the study. Authors realize that more detailed study in the field to reach the depth of the finger length ratio and its correlation with different physiological functions in Indian population is required.

Our study is in accordance with the previous studies and results show that there is considerable sexual dimorphism in central India population. Along with the other existing traditional calculation method used in the finger length study we used difference of 2D and 4D (2D-4D). We find this difference is significantly more in male right hand than in female.