This is an open-access article distributed under the terms of the Creative Commons Attribution License (https://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work, first published in JMIR Research Protocols, is properly cited. The complete bibliographic information, a link to the original publication on https://www.researchprotocols.org, as well as this copyright and license information must be included.
The ratio of the second finger length to the fourth finger length (2D:4D ratio) is considered to be negatively correlated with prenatal androgen exposure (PAE) and positively correlated with prenatal estrogen. Coincidentally, various brain regions are sensitive to PAE, and their functions in adults may be influenced by the prenatal actions of sex hormones.
This study aims to assess the relationship between PAE (indicated by the 2D:4D ratio) and various physiological (sex hormone levels and sleep-wake parameters), psychological (mental health), and sexual parameters in healthy young adults.
This study consists of two phases. In phase 1, we will conduct a survey-based study and anthropometric assessments (including 2D:4D ratio and BMI) in healthy young adults. Using validated questionnaires, we will collect self-reported data on sleep quality, sexual function, sleep chronotype, anxiety, and depressive symptoms. In phase 2, a subsample of phase 1 will undergo polysomnography and physiological and genetic assessments. Sleep architecture data will be obtained using portable polysomnography. The levels of testosterone, estradiol, progesterone, luteinizing hormone, follicle-stimulating hormone, prolactin, melatonin, and circadian regulatory proteins (circadian locomotor output cycles kaput [CLOCK], timeless [TIM], and period [PER]) and the expression levels of some miRNAs will be measured using blood samples. The rest and activity cycle will be monitored using actigraphy for a 7-day period.
In Poland, 720 participants were recruited for phase 1. Among these, 140 completed anthropometric measurements. In addition, 25 participants joined and completed phase 2 data collection. Recruitment from other sites will follow.
Findings from our study may help to better understand the plausible role of PAE in sleep physiology, mental health, and sexual quality of life in young adults.
DERR1-10.2196/29199
Sexual differentiation of the brain largely occurs before birth, and this process primarily occurs during short periods when the brain is most sensitive to hormones. In humans, this happens in midpregnancy and the first 3 months after birth [
The exposure to androgens (eg, testosterone) before birth also influences the digit ratio (DR) or the ratio of finger lengths of the second and fourth digits (2D:4D) [
Given the early event of sexual differentiation in the brain, various studies have shown that PAE, using DR as a marker, is associated with psychological conditions, including anxiety [
PAE may also influence sleep function in adults, but the evidence is minimal. Studies in rodents [
Verster et al [
Sleep is a state of unconsciousness, mandatory for everyone, that can be influenced by external auditory, sensory, or other stimuli. The role of sleep in general health has not been fully examined. Researchers connect sleep deprivation, especially chronic sleep loss, with numerous health problems such as obesity [
A healthy individual should spend approximately one-third of their day sleeping, stressing the importance of the process. The requisite sleep quality and quantity can change, but there is no specific boundary concerning sleep duration. This parameter varies both intra- and interindividually; thus, many factors determine the required amount of sleep. The American Academy of Sleep Medicine and Sleep Research Society, in their recent recommendation [
Studies have shown that, besides age, sex is an important factor influencing the quantity of sleep [
Sleep or circadian rhythms (CRs) can be examined using subjective and objective methods. The first method includes questionnaires. Examples of commonly used validated scales include the Pittsburgh Sleep Quality Index (PSQI) for assessing sleep quality measurement and the Morningness-Eveningness Questionnaire (MEQ) for measuring chronotype. Both are designed as self-assessment questionnaires [
Actigraphy is another tool frequently used to objectively evaluate the sleep-wake parameters. The American Academy of Sleep Medicine recommends its use for examining people with sleep-wake disorders or sleep-disordered breathing [
PSG, however, is commonly used as the gold standard for sleep examinations. If multiple channels are available, PSG can be coupled with EEG, electrooculogram, electromyogram, electrocardiogram, airflow, and oxygen saturation. Using PSG, every sleep phase could be distinguished and examined. An individual, when examined with EEG, electrooculogram, and electromyogram, presents three states: vigilance, nREM, and REM sleep [
Sleep modulation and CR depend on a variety of factors. These concern both neuroanatomical structures (mainly suprachiasmatic nucleus [SCN]) and sleep-modulatory molecules (such as gamma amino butyric acid [GABA], adenosine, acetylcholine, and serotonin) [
SCN releases the neurotransmitter GABA, which promotes sleep [
Therefore, GABA plays a key role in sleep promotion. The level of GABA is increased during both nREM and REM sleep in comparison with waking values [
The rhythmic secretion of most hormonal factors is governed by the internal biological clock and sleep state. Hormones can be divided into sleep-dependent hormones, such as growth hormone, prolactin, thyroid-stimulating hormone, and renin, or determined by CR-dependent hormones such as adrenocorticotropic hormone (ACTH), cortisol, and melatonin [
In utero, gonadal embryogenesis occurs through certain genes (eg,
In adults, the plasma levels of various hormones vary according to the sleep stage or CR [
The gonadotrophin-releasing hormone is synthesized in neurons within the hypothalamus. Its main function is to stimulate the anterior pituitary gland to release follicle-stimulating hormone (FSH) and luteinizing hormone (LH) [
In males, LH binds to receptors on Leydig cells and stimulates them to produce testosterone. In contrast, FSH acts on Sertoli cells to help promote spermatogenesis. Testosterone can be further converted into two forms: estradiol or 5α-dihydrotestosterone. Its production is implicated in the development of the male phenotype in embryos, providing sexual maturity at puberty and sexual function. In addition, both testosterone and estradiol inhibit the release of gonadotrophin-releasing hormone by the process of negative feedback [
In females, LH and FSH stimulate ovarian production of estradiol and progesterone, which have an impact on sleep changes during the menstrual cycle. There is a pulsatile rise in their levels at sleep onset in both sexes. During prepuberty and puberty, their levels increase during sleep. In addition, hormonal fluctuations during the menstrual cycle have an impact on sleep, for example, by decreasing the REM stage in the luteal phase and increasing sleep stage 2 in the midluteal phase [
The association between reproductive hormones and sleep has been studied previously; however, further research is needed. One evidence comes from the fact that hormonal profiles vary between sexes, and there are several sex differences in sleep patterns. Women generally need more sleep, spend more time in bed, and sleep longer. They also report more sleep difficulties, but by actigraphy, women have better sleep quality compared with men [
Several studies have explored the relationship between sleep and reproductive hormone secretion [
Various genes may also influence sleep and CR. For example, CR relies on the functioning of the
The exact roles of the aforementioned gene products are still not well defined. However, studies have shown that PER plays an important role in maintaining appropriate circadian timing [
In peripheral blood, products of circadian genes expression can be found at a concentration similar to that of SCN (obtained from mononuclear cells).
Epigenetics provides new insights into the circadian clock. The commonly known types of epigenetic changes involve the expression of noncoding RNAs, such as circulating miRNAs [
Due to the global obesity problem, an understanding of the factors significantly related to body weight and composition is needed. Obesity is currently one of the most common causes of health problems in developed countries, including Poland, New Zealand, Japan, and the United Kingdom [
A variety of factors, such as physical activity, exposure to stress, and eating habits as well as sleep quality, may affect body composition. Sleep is an essential element in the regeneration of the body and provides appropriate management of energy resources [
Another goal of this study is to explore the association between sleep quality and sexual function. Sleep problems are common in many populations [
Sexual inactivity is also becoming common in some societies [
Several preclinical studies have explored the impact of sleep deprivation on male sexual behavior. However, these studies mainly used the same sleep deprivation paradigm (ie, 96 hours of REM sleep deprivation). The findings from these studies were inconsistent, including no effect [
Human studies have also documented an association between sleep deprivation and impaired sexual function. For example, Seehuus and Pigeon [
Currently, however, there is little information on how objective sleep parameters (eg, sleep stages, sleep or wake latencies, and number of awakenings) are linked to sexual function in healthy populations. However, determining this would have clinical relevance as it may help indicate which sleep-related outcomes need to be improved to increase sexual function.
In this study, we will conduct both subjective and objective assessments of sleep as well as collect sexual function data using validated questionnaires, with the aim of finding associations between sleep and sexual parameters. Subjective sleep quality will be measured using the PSQI [
Sexual function will be assessed using the Arizona Sexual Experience Scale, which is a brief five-item scale measuring self-reported information on the strength of sex drive, how easy it is to be sexually aroused, to get and maintain an erection, to reach an orgasm, and orgasm satisfaction [
Some data support the Organizational and Activational Hypothesis on human sexuality, but these studies have limitations. For example, the majority (93%) of 46XY individuals with androgen insensitivity syndrome are androphilic, that is, attracted to men [
Many studies have explored the association between DR and sexual orientation. Breedlove and his team [
Overall, studies on the relationship between DR and sexual orientation remain controversial [
Several factors have been discussed regarding the limitations of assessing DR. One aspect, which is often noted, is the fact that the effect size is not large, and there is no cut-off indicating male or female pattern of DR; thus, there is a large overlap in the distribution of DR between sexes. This is also true for studies exploring differences in DRs of people with various sexual orientations. In addition, many studies that attempted to find an association between DR and sexual orientation have collected DR data through indirect methods, such as from photocopies or scans. However, it is now known that there is a potential discrepancy between direct and indirect measurements of DR [
In addition, studies on sexual orientation and DR have rarely considered two important factors: (1) handedness and (2) sexual attraction (how attracted one is to the same and opposite sex)
Finally, we are not aware of any previous or pending studies that have associated DR with sexual function
We will explore the association between PAE as indicated by the DR and sleep function in young adults. This research will have two phases to address this objective. In phase 1, sleep parameters will be assessed using a validated questionnaire (subjective measure). In phase 2, we will determine if PAE is related to objective sleep measures (using actigraphy and portable PSG), along with sleep-related correlates such as hormones, circadian regulatory proteins, and body composition.
Here, we will explore if sleep quality is associated with sexual functions. We will collect both objective and subjective sleep parameters. Sexual function data will be assessed using validated questionnaires. In addition, as noted above, we will investigate the extent of how PAE is associated with sexual function in young adults.
Finally, we will determine the association between PAE (indicated by DR) and sexual attraction, while taking into account the participants’ handedness information. Our finding may help show whether handedness information (which is associated with hormones and brain lateralization) can help explain the inconsistencies in past findings of DR and sexual orientation studies.
This research will be undertaken in two phases, with parallel recruitments in 4 different countries. Five institutions (Medical University of Lodz, Poland; University of Lodz, Poland; University of Otago, New Zealand; Aichi Medical University, Japan; and Swansea University, United Kingdom) are participating in this study.
As shown on
Flowchart of the study. CLOCK: circadian locomotor output cycles kaput; PER: period; TIM: timeless; 2D:4D ratio: ratio of the second digit length to the fourth digit length.
A full understanding of the study rules by the participant was confirmed by written informed consent to participate in the study
Age range: 18-30 years
Diagnosis of chronic, hormonal, and mental health conditions
Pregnancy and lactation
Taking long-term medicines (including hormonal contraception)
Injuries to the fingers of the upper limbs
Deformation of the fingers of the upper limbs
Diseases leading to deformation of the fingers of the upper limbs
Lack of consent or inability to follow recommendations related to participation in the study
Upon consenting, participants will need to do the following:
Complete questionnaires on demographics (eg, age, ethnicity, sex, gender, sexual attraction using the Kinsey Scale, sexual orientation, and handedness); PSQI; MEQ; Arizona Sexual Experience Scale; Sexual Satisfaction Scale; Generalized Anxiety Disorder screener; Mood Disorder Questionnaire; Patient Health Questionnaire-9, short-form health survey; and SapioQ.
Have anthropometric measurements (body weight, height, BMI, finger length: index and ring fingers, waist, hips, and neck circumference) recorded.
Body composition analysis (InBody 270) will be used to determine fat and muscle mass. We include this analysis because obesity is linked to sleep-related breathing disorders as well as insomnia. Noninvasive, easy, and common body impedance analysis method is based on measuring the electrical impedance in various body tissues, that is, the sum of geometric resistance (active resistance) and reactance (passive resistance). Bioelectrical impedance analysis is used to assess the values of the body components, such as fat-free mass (%), fat mass (%), muscle mass (%), and total body water (%). In addition, segmental lean and fat distribution can be measured to assess tissue distribution in each quarter of the body. The basic metabolic rate is calculated. Before each measurement, foot, feet, and hands must be wiped using wet wipes to increase electrical conductivity. During the measurement, the individual should be in an upright position [
When phase 1 is completed, we will follow up with those interested in phase 2. For this phase, we estimate that we will need at least 50 male and 50 female participants, regardless of sexual orientation. Using reference data from the PSQI, we estimate that, for each sex, we will need 25 participants with DR<1.0 (indicative of high PAE) and 25 participants with DR>1.0 (indicative of low PAE) to detect a 50% difference in sleep quality, with an α level of .05 and a power of 0.8.
Once consented, each participant will need to do the following:
A 1-week quantitative daily and nocturnal activity recording using portable actigraphy. On the basis of the actigraphic record, it will be possible to evaluate parameters such as the average level of activity during the day and at night, the amount of time spent actively and inactively during the day and night, average sleep time, sleep continuity, number of awakenings during sleep, and number of naps during the day. On the last day, one-night PSG data (total sleep time, duration of REM and nREM phases, apnea-hypopnea index, arousal index, and saturation) will be captured with a Nox A1 (ResMed). During 1-week actigraphy, all participants will have to agree to sexual abstinence because of blood samples before and just after PSG.
Blood collection, before and after the polysomnographic night for assessment of various hormones (testosterone, estrogen, estradiol, progesterone, LH, FSH, and prolactin) and circadian regulatory proteins (eg, CLOCK, PER, and TIM) as well as selected miRNAs.
The levels of the investigated proteins will be assessed using enzyme-linked immunosorbent assay kits. Each assay will include six standard concentrations, and every sample will be run in duplicate. The concentration will be assessed using a microplate reader to measure the absorbance at 450 nm. The standard curves will be created using the four-parameter logistic method. All samples should be within the assay range in accordance with the information supplied by the kit manufacturer. An interassay coefficient of variation of less than 15% is acceptable. The intra-assay coefficient of variation values were less than 10%.
The common miRNA procedure includes the following steps: RNA isolation, reverse transcription, and quantitative polymerase chain reaction using appropriate starters for each miRNA. An RNA/miRNA purification kit is needed to isolate miRNAs. A reverse transcription kit was used to perform reverse transcription. The analyses will be performed according to the protocol supplied by the manufacturer, using an appropriate amount of RNA in each sample. The reactions will be performed in a thermocycler according to the reaction parameters supplied by the manufacturer. Furthermore, we will dilute the obtained cDNA according to a protocol using RNAse-free and DNAse-free water. The final step will be quantitative polymerase chain reaction analysis for each sample in duplicate and each miRNA (using proper starters) using a real-time polymerase chain reaction machine. The obtained results will be analyzed using the software that calculated the cycle threshold (Ct) for each sample. The samples over Ct=39 cycles will be excluded from further analyses. The spike in the kit is used to normalize the expression levels of the obtained miRNAs. The delta cycle Ct method will be used to make a final calculation using the following formula: 2(mean CtmiR − mean Ct of reference) [
Demographic data will be summarized with descriptive statistics.
Subjective sleep measures and sexual data from phase 1 as well as objective sleep measures and its (hormonal, molecular, and psychological) correlates from phase 2 will be compared between participants with DRs<1.0 (indicative of high PAE) and participants with DR>1.0 (indicative of low PAE) for each biological sex, while taking into account their handedness.
Multiple regression analyses will be performed to determine the relationship between sleep and sexual parameters while controlling for age, BMI, psychological health (anxiety and depression), chronotype, and sexual orientation.
For each sex, DR data will be categorized by handedness and correlated with sexual attraction (measured using the Kinsey Scale).
In 2020, we started the first and second stages of our study in Poland. In phase 1, we recruited 720 participants, of whom 140 underwent anthropometric measurements. The second stage started in February 2021, and since then, 25 participants have completed the data collection for phase 2. We expect to complete the data collection for phase 2 in 2022.
Data from this study will provide some information on how PAE is associated with sleep and sexual functions as well as sexual attraction. Many factors may contribute to sleep and sexual problems. However, our research should provide further insight from a biological perspective on how early-life hormonal factors can have a long-term impact on these functions. Furthermore, we will obtain information on the role of PAE in sexual attraction.
Peer-reviewer report from the National Science Centre, Poland.
adrenocorticotropic hormone
brain and muscle ARNT-Like 1
circadian locomotor output cycles kaput
circadian rhythm
cycle threshold
digit ratio
electroencephalogram
follicle-stimulating hormone
gamma amino butyric acid
luteinizing hormone
Morningness-Eveningness Questionnaire
nonrapid eye movement
prenatal androgen exposure
period
polysomnography
Pittsburgh Sleep Quality Index
rapid eye movement
suprachiasmatic nucleus
timeless
This study is conducted in accordance with the amended Declaration of Helsinki, and the Ethics Committee of the Medical University of Lodz approved the study protocol (RNN/394/19/KE); Miniatura 4, National Science Centre—no 2020/04/X/NZ4/00564.
The contribution of WK and EW is equivalent and accounts for 80% of the contribution to this research.
None declared.