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PLOS One logoLink to PLOS One
. 2020 Jul 20;15(7):e0236134. doi: 10.1371/journal.pone.0236134

Rhesus-minus phenotype as a predictor of sexual desire and behavior, wellbeing, mental health, and fecundity

Jaroslav Flegr 1,2,*, Radim Kuba 2,3, Robin Kopecký 2
Editor: Calogero Caruso4
PMCID: PMC7371180  PMID: 32687529

Abstract

Background

Since its discovery in the 1930s, the effects of Rh phenotype on human health and wellbeing, with the exception of the effects of Rh-negativity of a mother on the risk of hemolytic anemia of Rh-positive children, has only rarely been studied. In the last few years, however, several studies have shown that Rh-negative subjects have worse health and performance in certain tests than their Rh-positive peers. Nothing is known about the effect of Rh phenotype on the quality of life of subjects as measured by a standard instrument.

Methods

We hereby analyzed the data of 1768 male (24% Rh-negative) and 3759 female participants (23% Rh-negative) of an anonymous internet study using the partial Kendall test with the age and the population of the hometown of subjects controlled.

Results

The results showed that the Rh-negative women, but not men, scored worse in wellbeing measured with the WHO-BREFF. The Rh-negative men scored worse in mental health-related variables and in their reported economic situation and the Rh-negative women scored better in physical health-related variables. Both the Rh-negative men and women reported higher sexual activity than their Rh-positive peers.

Conclusions

The effects of the Rh phenotype were significant after the correction for multiple tests. However, they were usually weaker and less numerous than those of smoking, consuming alcohol, and high body mass index, which were used as a sort of internal control.

1 Introduction

About 16% of the population in Europe (5% in Africa and 1% in Asia) are Rh-negative, i.e., they carry two copies of the allele of the RHD gene with large deletion, the d allele [1,2]. The gene codes a part of ammonium or CO2 pump on the surface of erythrocytes; however, the biological function of this pump is unknown [3,4]. The protein coded by the functional allele D possesses an extremely strongly immunogenic epitope. Therefore, the Rh-positive progeny of Rh-negative mothers often died due to hemolytic anemia of newborns before the introducing of prophylactic treatment with anti-D immunoglobulins during pregnancy [5,6]. It has been suggested that this selection pressure is (or was) counterbalanced by another selection, for example, by selection in favor of heterozygotes [7] or in favor of Rh-negative subjects during the Paleolithic period [8]. With the exception of this immunological effect and sporadic reports about the possible association of Rh negativity with certain personality traits [9], no effects of the Rh genotype on human phenotype have been described. Recently, however, several studies suggested the existence of specific effects of Rh phenotype and genotype on human psychomotor performance [1014] and health [1517]. Generally, Rh-negative subjects have worse health, score worse in some performance tests, and express higher sensitivity to various negative environmental factors, such as Toxoplasma infection and smoking. Rh-positive heterozygotes were found likeliest to have the best health and performance relative to all other subjects, suggesting that the Rh-polymorphism is sustained in the population by balancing selection in favor of heterozygotes [16]. Until now, no data has been published regarding the effect of Rh phenotype on quality of life, for example, on wellbeing, measured with a standard psychological instrument.

In the present cross-sectional study, we search for the possible effects of Rh negativity on wellbeing, physical and mental health, fecundity, and sexual desire and behavior of 5,527 subjects, 3,759 women and 1,768 men. These subjects participated in a large internet study primarily dealing with the effect of the Toxoplasma infection on physical and mental health. To enable assessment of the relative strength of the observed effects, we also computed the effects of smoking, alcohol consumption, and being overweight (body mass index) on the same set of health- and wellbeing-related variables.

2 Material & methods

2.1 Subjects

The internet questionnaire was distributed as a Qualtrics survey. Subjects were invited to participate in the study using a Facebook-based snowball method [18]. Potential volunteers, mostly members of the “Lab Bunnies” community, an 18,000-member group of Czech and Slovak nationals willing to take part in evolutionary psychology experiments, and their Facebook friends, were invited (using about 10 different posts on the Lab bunnies timeline) to participate in an anonymous study about “magical thinking, superstitions, prejudices, religion and the relation between various environmental factors, and health and wellbeing.” The questionnaire was also promoted in various electronic and printed media and TV–always without mentioning RhD, rhesus factor, or blood groups. Responders were not paid for their participation in the study; however, after finishing the 80-minute questionnaire, they were provided information about the results of related studies and their own results of several tests that were part of the questionnaire. At the first screen of the survey, the participants were given the following information and were asked to provide their informed consent to participate in the study: “The study is anonymous and obtained data will be used exclusively for scientific purposes. Your cooperation in the project is voluntary, and you can terminate it at any time by closing this web page. You can also skip any uncomfortable questions; however, complete data is most valuable. If you agree to participate in the research press the ‘Next’ button”. Only the subjects who provided their informed consent by pressing the button could participate in the study. Some pages of the questionnaire contained the Facebook share and like buttons. These buttons were pressed by more than 1,600 participants, which resulted in obtaining data from 12,600 responders in total between 27th May 2016 and 29th June 2018. The project, including the method of obtaining electronic informed consent to participate in this anonymous study from all participants, was approved by the IRB of the Faculty of Science, Charles University (“Komise pro práci s lidmi a lidským materiálem Přírodovědecké Fakulty Univerzity Karlovy”)—No. 2015/07.

2.2 Questionnaires

The electronic survey consisted of several parts that concerned various unrelated projects on evolutionary psychology and psychiatry. In the present study, we inspected and analyzed only responses to the questions concerning health, wellbeing, number of children (a proxy of biological fitness), sexuality, and Rh phenotype. The responders were asked about their biological sex, age, body height and weight, and the size of the communities where they currently live (ordinal variable urbanization‒ 0: less than 1000 inhabitants, 1: 1‒5 thousand inhabitants, 2: 5‒50 thousand inhabitants, 3: 50‒100 thousand inhabitants, 4: 100‒500 thousand inhabitants, 5: more than 500 thousand inhabitants). They were asked about their ABO blood group (not used in the present study) and in the next question about their Rh factor. The possible responses were “I do not know, I am not sure”, “negative (the rarer variant)”, or “positive (the more frequent variant)”; the answer “I do not know, I am not sure” was a priori checked. As a benchmark for the relative importance of Rh phenotype on the health, wellbeing, and biological fitness, we looked for the associations of health, wellbeing, and fitness with three unrelated but well-known risk factors: body mass index (BMI) calculated from body height and body weight, frequency of smoking, i.e., how many cigarettes they smoke a day (ordinal scale 0: 0, 1: 00.1, 2: 0.1‒1, 3: 1.1‒3, 4: 3.1‒10, 5: 11‒20, 6: 21‒40, 7: more than 40), and frequency of alcohol consumption “not to be allowed to drive a car for a while for this reason” (ordinal scale: 0- never, 1- maximally 1× a month, 2- maximally 2× a month, 3- maximally 4× a month, 4- maximally 2× a week, 5- every second day, 6- every day, 7- nearly all the time; in Czech, no measurable level of alcohol in blood is tolerated in drivers).

In another part of the questionnaire, the information concerning the following outcome health-related variables was collected from the responders: How they rate their physical health status in comparison with other people of the same age (subjective physical health problems: six points scale, anchored with 0: definitively better status, 5: definitively worse status), how they rate their mental health status in comparison with other people of the same age (subjective mental health problems: the same scale), how they rate their family situation, e.g., the quality of emotional support they can receive (0: poor, 5: excellent), and how they rate the economic situation of their family (0: poor, 5: excellent). To obtain more objective and concrete information on the health status of responders, they were also asked the following questions: how many kinds of drugs prescribed by a medical doctor were they currently taking, how many kinds of drugs non-prescribed by a doctor were they currently taking (“how many different herbs, food supplements, multivitamins, superfoods, etc. do you currently take per day”), how many times they visited their primary care doctor in the past 365 days (“not for prevention”), how many times they used antibiotics in the past 365 days, and how many different medical specialists they visited (“not for prevention”) in the past 5 years. The physical health problems score was calculated as a mean of Z-scores of the last five variables. The responders were also requested to rate how much they suffer from anxieties, phobias, depression, mania, obsessions, auditory hallucinations, visual hallucinations, and headaches using eight 0‒100 scales. The number of diagnosed and the number of undiagnosed mental health disorders of responders, both checked on a list of 25 mental health disorders and epilepsy, was counted. The mental health problems score was calculated as a mean of Z-scores of the last 10 variables. Using a 0‒100 scale the responders also answered the questions ‒ how intensely they are sexually attracted to men, how intensely they are sexually attracted to women, (Z-score of the higher of these two responses was considered as the intensity of sexual desire). They were also asked with how many men (women) they already had sex (“vaginal, oral or anal”). To respond to these two questions, the participants used a 0‒9 ordinal scale (0: 0, 1: 1, 2: 2, 3: 3, 4: 4, 5: 5‒6, 6: 7‒9, 7: 10‒19, 8: 20 and more). The higher of these two responses (men vs. women) was considered to reflect the number of preferred sex-sexual partners. Similarly, the participants were asked with how many men (women) they already exchanged in French kissing (the same ordinal scale as previous questions)–the higher of these two responses (men vs. women) was considered to reflect the number of preferred sex-French kissing-partners. In the other part of the questionnaire, the responders were asked to estimate how many minutes daily they spend engaged in various activities. The list of 22 activities also contained “any form of sex, including consuming pornography”. A score of sexual activity was computed as the mean of Z-scores of the number of preferred sex sexual partners, preferred sex French kissing partners, and minutes spent engaged in any form of sex per day. The number of biological children was used as a proxy for biological fitness.

The Czech version of the standard 24-item WHOQOL-BREF (The World Health Organisation Quality of Life Assessment Instrument–the abbreviated version of the WHOQOL-100) [19] was used for the assessment of the quality of life of responders. This instrument was translated into Czech and standardized to the Czech population [20]. It monitors the general quality of life and its four specific domains: Physical health (the activity of daily living, dependence on medical substances and aids, energy and fatigue, mobility, pain and discomfort, sleep and rest, work capacity), Psychological (bodily image and appearance, negative feelings, positive feelings, self-esteem, spirituality/religion/personal beliefs, thinking, learning, memory, and concentration), Social relationships (personal relationships, social support, sexual activity), and Environment (financial resources, freedom, physical safety and security, health and social care: accessibility and quality, home environment, the opportunity of acquiring new information and skills, participation in and opportunity for recreation/leisure activities, physical environment ‒ pollution/noise/traffic/climate, transport).

2.3 Statistical analyses

Before any analyses, records of all subjects who did not answer the question about Rh factor and of about 2% of subjects who provided a suspicious combination of answers to other questions (too high/low body height, weight, age, an unrealistically high number of neuropsychiatric disorders, who answered all or nearly all questions by the same code, etc.) were filtered out.

The final set contained data from 6,602 subjects. However, not all of them responded to all questions concerning their health and quality of life. The distribution of all relevant (semi-continuous and ordinal) variables was visually checked and then all secondary indices, see above, were computed. Distributions of some variables were asymmetric and the number of subjects in the RhD-negative subgroup was lower than in RhD-positive subgroup (app. 23%). For this reason, we used the partial Kendall test to search for the effect of RhD phenotype on all variables. It is a nonparametric test insensitive to the character of data distribution, which can therefore be used to analyze imbalanced data sets. We used R v. 3.3.1 [21], package ppcor v. 1.1 [22] for computing the partial Kendall correlation. The correction for multiple tests was done using the Benjamini-Hochberg procedure with the false discovery rate pre-set to 0.20 [23].

All relevant data are available at Figshare https://doi.org/10.6084/m9.figshare.8216276 [24].

3 Results

The final data set contained responses from 425 (24.0%) Rh-negative men (mean age 35.9, SD 12.25), 1,343 Rh-positive men (mean age 36.2, SD 12.5), 864 (23.0%) Rh-negative women (mean age 36.7, SD 13.0), and 2,895 Rh-positive women (mean age 36.5, SD 12.8). No difference in age between men and women, or Rh-negative and Rh-positive subjects was significant (p > 0.76).

Many examined health and wellbeing-related variables had asymmetrical or multimodal distribution, and most of them correlated with age and the population of the hometown of the responders. Therefore, we analyzed the effects of Rh negativity, smoking, alcohol consumption, and high body mass index (BMI) with nonparametric partial Kendall correlation with age and the population of hometown as covariates. Table 1 shows that Rh-negative women reported worse quality of life as measured with WHOQOL-BREF, worse economic situations, and better physical health while the Rh-negative men reported worse economic situations, worse mental health and lower number of children. Both the Rh-negative men and women also reported higher sexual activity. Table 1, however, also shows that the effects of the other three variables, namely smoking, alcohol consumption, and BMI, were stronger and more numerous. Table 2 shows the effect of Rh-negativity, smoking, alcohol consumption, and BMI on wellbeing subscales and the individual variables used for computing mental health problems score, physical health score, sexual activity score, and sexual desire.

Table 1. Relationship between Rh-negativity and other factors with wellbeing and various health-related variables.

wellbeing (WHOQOL-BREF) no. of children family situation economic situation mental health problems score physical health problems score subject. mental health problems subject. physical health problems sexual activity sexual desire
Men
Age 0.080 0.529 0.082 0.077 -0.203 0.035 -0.063 -0.040 0.217 0.062
Urbanization -0.020 -0.152 -0.053 -0.018 0.069 0.034 0.033 0.024 0.068 -0.013
Rh-negativity 0.001 -0.033 -0.017 -0.045 0.036 -0.003 0.033 -0.004 0.036 -0.024
Smoking -0.052 -0.006 -0.011 -0.081 0.073 0.005 0.038 0.084 0.216 -0.031
Alcohol consumption 0.016 0.000 0.031 0.043 0.023 -0.063 0.006 -0.061 0.072 0.026
Overweight (BMI) -0.103 0.094 -0.013 0.002 0.034 0.076 0.058 0.198 0.047 0.044
Women
Age -0.005 0.549 -0.041 0.028 -0.142 0.085 -0.082 -0.088 0.108 -0.046
Urbanization -0.004 -0.196 -0.033 -0.015 0.068 -0.007 0.028 0.014 0.012 0.014
Rh-negativity -0.031 -0.011 -0.004 -0.024 -0.002 -0.024 -0.001 -0.005 0.030 0.002
Smoking -0.046 -0.055 -0.089 -0.114 0.080 0.003 0.028 0.041 0.238 0.018
Alcohol consumption 0.014 -0.090 -0.022 -0.021 0.062 -0.018 0.030 -0.046 0.129 0.014
Overweight (BMI) -0.074 0.055 -0.026 -0.054 0.032 0.094 0.036 0.247 0.012 0.014

The table shows Taus (for the factors age and urbanization) and partial Taus with age and urbanization controlled (for other factors). The sign and size of the Tau reflect the direction and strength of the associations between the factors listed in the headers of rows and the responses listed in the headers of columns. A negative partial Tau indicates a negative correlation between the factor and the response, e.g., worse wellbeing in older subjects, subjects living in larger settlements, Rh-negative subjects, smokers, alcohol-consuming subjects and subjects with a larger body mass index. Associations that remained significant after the correction for multiple tests are printed in bold.

Table 2. Relationship between Rh-negativity and other factors with the variables used for computing wellbeing, mental health, physical health, and sexual activity scores.

Men Women
  Rh-neg. smoking alcohol BMI Rh-neg. smoking alcohol BMI
Physical health wellbeing 0.008 -0.051 0.018 -0.098 -0.021 -0.010 0.033 -0.083
Psychological wellbeing 0.002 -0.039 -0.013 -0.076 -0.033 -0.043 -0.039 -0.055
Social relationships wellbeing 0.018 -0.011 0.001 -0.054 -0.025 -0.045 -0.002 -0.042
Environment wellbeing -0.019 -0.049 0.037 -0.084 -0.012 -0.045 0.030 -0.030
Drugs prescribed -0.001 0.021 -0.066 0.135 0.001 -0.026 -0.056 0.132
Drugs non-prescribed 0.003 -0.011 -0.018 0.004 -0.046 -0.020 -0.002 -0.021
Practical doctor (365 days) -0.029 -0.007 -0.038 0.055 -0.009 0.003 -0.016 0.089
Antibiotics (365 days) -0.011 -0.018 -0.037 0.066 -0.008 0.029 0.007 0.055
Medical specialists (five years) 0.023 -0.005 -0.047 0.044 -0.012 -0.008 -0.002 0.062
Anxiety 0.026 0.055 0.025 0.013 0.014 0.060 0.073 0.002
Phobia 0.026 0.013 0.045 0.019 0.002 0.032 0.026 0.038
Depression 0.033 0.085 0.056 0.051 0.001 0.073 0.088 0.032
Mania 0.035 0.096 0.055 -0.027 0.004 0.093 0.085 0.012
Obsession 0.013 0.031 0.049 -0.020 -0.024 0.022 0.056 -0.010
Auditory hallucinations -0.004 0.030 0.028 -0.010 0.002 0.041 0.025 -0.012
Visual hallucination 0.015 0.023 0.026 -0.002 0.016 0.033 0.011 0.011
Headache 0.024 -0.015 -0.004 0.058 -0.022 0.020 0.015 0.016
Diagnosed mental health dis. 0.052 0.089 -0.046 0.059 -0.002 0.098 -0.020 0.067
Undiagnosed mental health dis. 0.011 0.099 0.036 0.044 -0.017 0.063 0.039 0.034
Preferred sex sexual partners 0.032 0.203 0.053 0.063 0.032 0.234 0.112 0.021
Preferred sex French kissing partners 0.032 0.224 0.093 0.025 0.042 0.247 0.145 0.004
Time spent by sex 0.024 0.057 0.055 0.062 -0.020 0.052 0.079 -0.009

The table shows partial Taus with age and urbanization controlled (see the legend for Table 1). Significant associations are printed in bold; no correction for multiple tests was done in this exploratory part of the study.

4 Discussion

The present study showed that Rh negativity was associated with the impaired economic situations, and wellbeing and improved physical health of women and with the impaired economic situations and mental health of men. The strength of the observed associations was relatively low, especially in comparison with other negative factors such as smoking, consuming alcohol, and high BMI. Surprisingly, we also found a relatively strong positive association of Rh-negativity with the sexual activity of male and female responders. In Rh-negative men, the higher sexual activity (higher number of past sexual partners, past French-kissing partners, and larger part of a day usually spent by sexual activities, including consuming pornography) contrasted with the trend for their lower sexual desire (Tau = -0.024, p = 0.187); no such trend for sexual desire was observed in Rh-negative women (Tau = 0.002, p = 0.865). It can be only speculated whether the higher sexual activity of Rh-negative subjects is part of the transition from a slow to a faster life strategy in individuals as a result of worse health status and correspondingly lower life-expectancy [25,26].

4.1 Unanticipated results of the explorative part of the study

The Czech and Slovak populations contain about 16% of Rh-negative subjects. The frequency of such subjects, however, was much higher (about 23%) among the participants of the present study and even higher (25%) among participants of other internet studies, for example among the participants of our previous study about the sexual behavior of the Czech population [27]. It can be speculated that a fraction of subjects intentionally or unintentionally misreports their Rh phenotype. If this fraction is similar in Rh-negative and Rh-positive subjects (for example about 7%) it could result in a seemingly higher representation of the rarer phenotype, here the Rh-negative phenotype. Our records show that the prevalence of Rh-negative subjects in 2,611 participants of our experiments whose Rh phenotype have been examined serologically in our lab during the past 15 years is 22.2%. Therefore, the more probable explanation is that the personality of Rh-negative and Rh-positive subjects differ and the Rh-negative subjects are more interested in participating in our internet, as well as non-internet studies. It is worth mentioning that overrepresentation of RhD-negative subjects can be observed in practically all our studies, including the oldest one that investigates the effect of toxoplasmosis on psychomotor performance of blood donors at the blood transfusion department of the Institute of Hematology and Blood Transfusion, Prague. In that study, the prevalence of RhD-negative subjects was 27.2% (33.3% in men and 17.8% in women) [10].

The participants who reported consuming alcohol more frequently reported worse mental health; however, they also reported better physical health than other people. Either the alcohol consumption in moderate amount has a positive effect on physical health, or it is more likely that people with physical health problems consume alcohol less frequently or at least are reluctant to report that they consume alcohol even in an anonymous internet study.

All negative factors under study, possibly except BMI in women, had positive effects on sexual activity reported by participants. This phenomenon deserves future attention.

4.2 Limitations of the study

As discussed above (section 4.1), RhD-negative subjects were overrepresented among participants of the present study as well as in all past studies (both internet and non-internet) we had performed on self-recruited populations. The most likely explanation of this phenomenon is that the personality profile and therefore also willingness voluntarily participate in research differs between RhD-negative and RhD-positive subjects. Such differences could influence the results of all questionnaire studies: one can never be certain whether the observed effects of the RhD phenotype are due to a real difference in wellbeing and health or just a consequence of inclination to report a worse quality of life. To address this important issue, a personality questionnaire ought to be included in future internet questionnaires and the relation between wellbeing/health, personality, and RhD phenotype should be analyzed using a structural modelling technique, such as path analysis.

It must be emphasized, however, that the responders were blind to group assignment because the question about Rh factor was ‘hidden’ among 600 other questions (the time needed to complete the questionnaire was about 80 minutes) and the Rh-negative and the Rh-positive participants had no reason to respond to questions regarding their health and wellbeing differently unless they really felt differently about those issues.

It ought to be also noted that possible effects of personality on self-selection is a challenge not only for internet studies but for all studies, including those performed on so-called ‘representative’ samples. All studies performed in the past forty years are (or should be) based on self-selected populations, because participants must sign an informed consent form before starting their participation. In this consent form, they must be clearly informed that their participation is voluntarily and they can terminate their involvement in it without giving any reason and at any time. It is highly probable that the personality profile of consenting and not-consenting population differs.

This study, together with nearly all studies that started before the year 2018, was not pre-registered. The authors of non-pre-registered studies could be suspected of publishing only a subset of results, here, for example, the results for the variables showing the significant association with Rh phenotype. However, the authors has already published or pre-registered several studies [17,2830] https://osf.io/y5z64/ in which the effects of other factors on the same or a very similar set of wellbeing- and health-related variables were analyzed.

The effect of Rh negativity on the output variables was analyzed by comparing the value of output variables in Rh-negative subjects and Rh-positive subjects. However, Rh-positive subjects represent a mixture of Rh-positive homozygotes and heterozygotes. It was shown that the health of Rh-positive heterozygotes, but often not Rh-positive homozygotes, is better than that of Rh-negative homozygotes [16]. It would be important to repeat the study on an Rh-genotyped population in the future in order to avoid underestimating the strength of the effect of Rh negativity.

Participants of the study could represent a nontypical subpopulation because they knew their Rh phenotype and were willing to participate even in absence of any material compensation in a relatively time-consuming anonymous study. They could be more inquisitive or perhaps more altruistic than typical members of the general population. It must be emphasized, however, that both the cases (Rh-negatives) and the controls (Rh-positives) were recruited from the same population of inquisitive and altruistic subjects. Any possible untypicality of the sample therefore could not affect the main result of the study. Still, the study should be repeated with other populations in the future and one ought to be careful regarding the generalization potential of results of the present study.

The present study analyzed the information provided by the participants of the study. Both the information about their Rh phenotype and about their health status may be invalid for some, possibly many, people. As was confirmed by Monte-Carlo modeling [31], a stochastic noise that is inevitably present in the internet studies data would be able to cause only the false-negative result of studies (non-detection of existing associations) but not the false-positive results of a study (detection of non-existent associations). However, specific subpopulations of people, e.g., intentionally inaccurate responders, may have characteristic responses to the questions of their Rh phenotype as well as their wellbeing and health. Existence of such populations can also result in false-positive results of a study. It will be therefore necessary to repeat the study on the population of people with empirically examined Rh phenotype, or preferably genotype. Such studies have been already done for other traits demonstrating, for example, the association between Rh phenotype and performance in weight-holding and handgrip tests [14], intelligence [13], psychomotor performance [1012], and several personality traits [32]. It must be mentioned, however, that a large binational cohort study found no difference in mortality of Rh-positive and Rh-negative subjects [33].

The prevalence of RhD-negative subjects differs between European, Asian, and African populations and even between populations of various European countries. One could thus speculate that ethnic difference between RhD-negative and RhD-positive participants of the study might be responsible for the observed differences. On the other hand, the ethnic composition of Czech population is extremely homogenous and the questionnaire was written in Czech, a difficult Slavic language relatively easily learned only by Slovaks, since Slovak is a closely related language. Among about 15,000 of members of the Lab Bunnies community (a group of volunteers willing to participate in questionnaire studies) there are practically no foreign surnames. It is highly probable therefore that more over 99% of participants of the present study are ethnic Czechs or Slovaks.

4.3 Strengths of the study

A relatively large number of participants.

Absence of a priori knowledge of the participants about the subject of the study (the effect of Rh phenotype).

Standard questionnaire WHOQOL-BREF used to assess the quality of life of the responders.

5 Conclusions

The present study performed on a self-selected internet population had shown that the Rh-negative subjects report worse quality of life than the Rh-positive subjects. It also has been confirmed that they had worse mental health (men), better physical health (women), and worse economic situations. Rh-negative subjects also reported higher sexual activity than the Rh-positive subjects. The effects of Rh-negativity were less numerous and weaker than the effects of smoking, alcohol consumption, and high BMI. In fact, the partial Tau 2.4–3.6 correspond to Cohen f 3.8–5.7, which fall within a range of small (but not negligible) effects according to the Cohen classification [34]. It must be remembered, however, that total number of Rh-negative subjects in the human population is large and that Rh-negative subjects stay affected by they Rh negativity permanently. Therefore, if the effects observed in our sample are present in the general population, their total impact on public health could be larger than that of some formally stronger effects, such as the impact of alcohol consumption.

Acknowledgments

We thank Charles Lotterman for the final revisions of our text.

Data Availability

All relevant data are available at Figshare https://doi.org/10.6084/m9.figshare.8216276 [24].

Funding Statement

JF: 18-13692S Czech Science Foundation The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

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Decision Letter 0

Calogero Caruso

8 Jun 2020

PONE-D-20-09062

Rhesus-minus phenotype as a predictor of sexual desire and behavior, wellbeing, mental health, and fecundity

PLOS ONE

Dear Dr. Flegr,

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Reviewer #1: Partly

**********

2. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: No

**********

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The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: Yes

**********

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Reviewer #1: Yes

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Reviewer #1: Like many other studies on the influence of blood group antigens on general health outcomes, it is a cross-sectional study.

The main weakness of the study is the low and most probably biased proportion of individuals reporting their Rh phenotype. The study was an online study, and the analysis is based on the self-disclosed Rh phenotype of the participants. The authors state that there were 12600 responders, 2% were filtered out due to suspicious answering pattern. Non-suspicious responders who disclosed their Rh phenotype numbered 6602. From these numbers, I calculate that only about 47% of participants did disclose their Rh phenotype. Was this as random sample? I do not believe. In the Czech population, the prevalence of the Rh negative phenotype is 16%, in the study sample the frequency was 23%. The authors note this discrepancy and describe that the fraction of Rh negative is in a similar range in their other studies even if checked by serology. They conclude that Rh-negative subjects are more interested in participating in their internet as well as non-internet studies. This assumption is probably correct, because the Flegr laboratory has high regard in the Czech and Slowak population for “explaining the function of Rh”. Assuming that (i) the participation in an internet survey depends on the economic and psychological situation and (ii) the likelihood to participate is heavily confounded by the Rh phenotype, any effect observed may as well be due to the selection bias.

Surprisingly, the authors mention this observation but did not include it in “limitations of the study”. In my opinion, a highly biased selection process is a major limitation.

A second weakness not considered by the authors is the possible association of Rh phenotype and ethnicity. The Rh negative phenotype is much more frequent in Europe than in Asia and Afrika. Immigrants will most likely be overrepresented in the Rh positive group and might differ in psychoeconomic factors due to their cultural background and possible discrimination. However, to my knowledge the prevalence of immigrants in the Czech republic is low and I would have expected a worse economic situation of (Rh positive) immigrants, just the opposite of the reported effect.

Apart from this likely bias, the authors did their best in the analysis of the obtained set of data.

I disagree with the conclusions. The statement “The present study has shown, probably for the first time, that the Rh-negative subjects have, or at least report, worse quality of life than the Rh-positive subjects” cannot be derived from data established ina highly biased collective.

A minor comment:

Ref 9: The citation is not correct, the reference is Cattle RB, Hundleby JD, Blood groups and personality traits, Am J Hum Genet 1972; 24(4): 485-486. It is a two pages letter in which the second page carries the name of the second author and the references. In addition, I cannot find a statement on the association of Rh negativity with personality traits as suggested by the authors' citation (it just states that these possible confounders were excluded in an older study)

**********

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PLoS One. 2020 Jul 20;15(7):e0236134. doi: 10.1371/journal.pone.0236134.r002

Author response to Decision Letter 0


17 Jun 2020

Formated version of the folloving text has been also uploaded as .doc file

Reviewer #1: Like many other studies on the influence of blood group antigens on general health outcomes, it is a cross-sectional study.

The main weakness of the study is the low and most probably biased proportion of individuals reporting their Rh phenotype.

The biased proportion of individuals (overrepresentation of Rh-negative subjects) could be a strength, not weakness, of this study. Ideal for a study such as ours would be a case-control design: a comparison between a group of Rh-negative subjects and a group of Rh-positive controls of the same size. We used a suboptimal design, which admittedly could miss some week effects of the Rh but – more importantly – could not identify nonexistent effects, for pragmatic reasons: the main subject of the research questionnaire were the effects of latent infection of Toxoplasma gondii infection, that is, not Rh.

We included following paragraph into the Material and Methods section of the manuscript:

“Distributions of some variables were asymmetric and the number of subjects in the RhD-negative subgroup was lower than in RhD-positive subgroup (app. 23%). For this reason, we used the partial Kendall test to search for the effect of RhD phenotype on all variables. It is a nonparametric test insensitive to the character of data distribution, which can therefore be used to analyze imbalanced data sets.”

The study was an online study, and the analysis is based on the self-disclosed Rh phenotype of the participants. The authors state that there were 12600 responders, 2% were filtered out due to suspicious answering pattern. Non-suspicious responders who disclosed their Rh phenotype numbered 6602. From these numbers, I calculate that only about 47% of participants did disclose their Rh phenotype. Was this as random sample? I do not believe.

We absolutely agree. We now include in the Limitations section of the manuscript the following:

“Participants of the study could represent a nontypical subpopulation because they knew their Rh phenotype and were willing to participate even in absence of any material compensation in a relatively time-consuming anonymous study. They could be more inquisitive or perhaps more altruistic than typical members of the general population. It must be emphasized, however, that both the cases (Rh-negatives) and the controls (Rh-positives) were recruited from the same population of inquisitive and altruistic subjects. Any possible untypicality of the sample therefore could not affect the main result of the study. Still, the study should be repeated with other populations in the future and one ought to be careful regarding the generalization potential of results of the present study.”

In the Czech population, the prevalence of the Rh-negative phenotype is 16%, in the study sample the frequency was 23%. The authors note this discrepancy and describe that the fraction of Rh negative is in a similar range in their other studies even if checked by serology. They conclude that Rh-negative subjects are more interested in participating in their internet as well as non-internet studies. This assumption is probably correct, because the Flegr laboratory has high regard in the Czech and Slowak population for “explaining the function of Rh”.

Our laboratory is known for a study of the effects of Toxoplasma on human personality and behavior that has been going on for 25 years. We started studying the effects of Rh factors on human health only seven years ago and five years ago, we started to publish the first results of these studies in international journals (in English). Over ninety percent of our responders do not know that Rh is one of the many subjects our research team investigates. In fact, we already ran eight large internet studies and only one (not the present one) was promoted as a study of the effects of Rh factor on human personality, performance, and health. We have observed an overrepresentation of Rh-negative subjects in all the abovementioned studies, including a popular study on sexual behavior. About 60,000 subjects took this long questionnaire (that took over 100 minutes) and only a small fraction of them ever heard about Flegr and his research.

In the amended version of the manuscript, we included the following information:

“The questionnaire was also promoted in various electronic and printed media and TV – always without mentioning RhD, rhesus factor, or blood groups.”

We also wrote:

“It is worth mentioning that overrepresentation of RhD-negative subjects can be observed in practically all our studies, including the oldest one that investigates the effect of toxoplasmosis on psychomotor performance of blood donors at the blood transfusion department of the Institute of Hematology and Blood Transfusion, Prague. In that study, the prevalence of RhD-negative subjects was 27.2% (33.3% in men and 17.8% in women) (Novotna et al. 2008).”

Assuming that (i) the participation in an internet survey depends on the economic and psychological situation and (ii) the likelihood to participate is heavily confounded by the Rh phenotype, any effect observed may as well be due to the selection bias.

Surprisingly, the authors mention this observation but did not include it in “limitations of the study”. In my opinion, a highly biased selection process is a major limitation.

We have now included in the Limitations section the following paragraph:

“As discussed above (section 4.1), RhD-negative subjects were overrepresented among participants of the present study as well as in all past studies (both internet and non-internet) we had performed on self-recruited populations. The most likely explanation of this phenomenon is that the personality profile and therefore also willingness voluntarily participate in research differs between RhD-negative and RhD-positive subjects. Such differences could influence the results of all questionnaire studies: one can never be certain whether the observed effects of the RhD phenotype are due to a real difference in wellbeing and health or just a consequence of inclination to report a worse quality of life. To address this important issue, a personality questionnaire ought to be included in future internet questionnaires and the relation between wellbeing/health, personality, and RhD phenotype should be analyzed using a structural modelling technique, such as path analysis.”

We also added:

“It ought to be noted that possible effects of personality on self-selection is a challenge not only for internet studies but for all studies, including those performed on so-called ‘representative’ samples. All studies performed in the past forty years are (or should be) based on self-selected populations, because participants must sign an informed consent form before starting their participation. In this consent form, they must be clearly informed that their participation is voluntarily and they can terminate their involvement in it without giving any reason and at any time. It is highly probable that the personality profile of consenting and not-consenting population differs.”

Moreover, we also added:

“It must be emphasized, however, that the responders were blind to group assignment because the question about Rh factor was ‘hidden’ among 600 other questions (the time needed to complete the questionnaire was about 80 minutes) and the Rh-negative and the Rh-positive participants had no reason to respond to questions regarding their health and wellbeing differently unless they really felt differently about those issues.”

A second weakness not considered by the authors is the possible association of Rh phenotype and ethnicity. The Rh negative phenotype is much more frequent in Europe than in Asia and Afrika. Immigrants will most likely be overrepresented in the Rh positive group and might differ in psychoeconomic factors due to their cultural background and possible discrimination. However, to my knowledge the prevalence of immigrants in the Czech republic is low and I would have expected a worse economic situation of (Rh positive) immigrants, just the opposite of the reported effect.

The referee is right. The ethnic composition of Czech population is rather homogenous due to the fact that in the past century and more, people tended to emigrated from Czechia rather than immigrate here. Moreover, the questionnaire was written in Czech, which is a difficult Slavic language spoken by few people who do not reside here for a long time. It is, however, closely related to Slovak. In future, Czech citizens of Vietnamese origin (and there is a sizeable community here) could bring some additional factors to control for similar studies. Unfortunately, Vietnamese surnames are currently almost absent in the database of our registered volunteers. We are therefore certain that over 99% of participants of the present study are either Czech or Slovak.

We included the following paragraph into the Discussion section:

“The prevalence of RhD-negative subjects differs between European, Asian, and African populations and even between populations of various European countries. One could thus speculate that ethnic difference between RhD-negative and RhD-positive participants of the study might be responsible for the observed differences. On the other hand, the ethnic composition of Czech population is extremely homogenous and the questionnaire was written in Czech, a difficult Slavic language relatively easily learned only by Slovaks, since Slovak is a closely related language. Among about 15,000 of members of the Lab Bunnies community (a group of volunteers willing to participate in questionnaire studies) there are practically no foreign surnames. It is highly probable therefore that more over 99% of participants of the present study are ethnic Czechs or Slovaks.”

Apart from this likely bias, the authors did their best in the analysis of the obtained set of data.

Thank you.

I disagree with the conclusions. The statement “The present study has shown, probably for the first time, that the Rh-negative subjects have, or at least report, worse quality of life than the Rh-positive subjects” cannot be derived from data established in a highly biased collective.

We toned down the conclusion. Now we write:

“The present study performed on a self-selected internet population had shown that the Rh-negative subjects report worse quality of life than the Rh-positive subjects.”

We also substituted:

“Therefore, the total impact of these effects on public health could be larger than formally stronger effects of, e.g., alcohol.”

with

“Therefore, if the effects observed in our sample are present in the general population, their total impact on public health could be larger than that of some formally stronger effects, such as the impact of alcohol consumption.”

A minor comment:

Ref 9: The citation is not correct, the reference is Cattle RB, Hundleby JD, Blood groups and personality traits, Am J Hum Genet 1972; 24(4): 485-486. It is a two pages letter in which the second page carries the name of the second author and the references. In addition, I cannot find a statement on the association of Rh negativity with personality traits as suggested by the authors' citation (it just states that these possible confounders were excluded in an older study)

Thank you for finding this error in our bibliographic database. Now we included the correct citation:

Cattell, R.B., Young, H.B., Hundleby, J.D. Blood groups and personality traits. Am J Hum Genet 16(4), 1964: 397-402.

In their paper, the authors described a method of Rh assay and how they included Rh into their statistical models. In their results, they unfortunately only state that “Associations with Rh type are not included in the present report.”

Attachment

Submitted filename: listOfChangess2.docx

Decision Letter 1

Calogero Caruso

30 Jun 2020

Rhesus-minus phenotype as a predictor of sexual desire and behavior, wellbeing, mental health, and fecundity

PONE-D-20-09062R1

Dear Dr. Flegr,

We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements.

Within one week, you’ll receive an e-mail detailing the required amendments. When these have been addressed, you’ll receive a formal acceptance letter and your manuscript will be scheduled for publication.

An invoice for payment will follow shortly after the formal acceptance. To ensure an efficient process, please log into Editorial Manager at http://www.editorialmanager.com/pone/, click the 'Update My Information' link at the top of the page, and double check that your user information is up-to-date. If you have any billing related questions, please contact our Author Billing department directly at authorbilling@plos.org.

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Kind regards,

Calogero Caruso, MD

Academic Editor

PLOS ONE

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Comments to the Author

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Reviewer #1: All comments have been addressed

**********

2. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: Yes

**********

3. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: Yes

**********

4. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: Yes

**********

5. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #1: Yes

**********

6. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: I thank the authors very much for considering my arguments and toning down some conclusions. I am still puzzled with the RhD neg overhang among the study participants, however, the authors could convince me that it is very unlikely to be due to a special stimulus on RhD neg subjects to participate in an important "RhD function laboratory study". The argument with the overrepresantation of RhD neg individuals even before the group turned to Rh is convincing in this respect.

**********

7. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

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Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.

Reviewer #1: No

Acceptance letter

Calogero Caruso

6 Jul 2020

PONE-D-20-09062R1

Rhesus-minus phenotype as a predictor of sexual desire and behavior, wellbeing, mental health, and fecundity

Dear Dr. Flegr:

I'm pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now with our production department.

If your institution or institutions have a press office, please let them know about your upcoming paper now to help maximize its impact. If they'll be preparing press materials, please inform our press team within the next 48 hours. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information please contact onepress@plos.org.

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