Stability and inter-family associations of hair endocannabinoid and N-acyl-ethanolamines across the perinatal period in mothers, fathers, and children

doi:10.21203/rs.3.rs-3787391/v1

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Stability and inter-family associations of hair endocannabinoid and N-acyl-ethanolamines across the perinatal period in mothers, fathers, and children

https://doi.org/10.21203/rs.3.rs-3787391/v1

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Analysis of endocannabinoids (ECs) and endocannabinoid-related compounds (ERCs) in hair is assumed to retrospectively assess long-term EC/ERC concentrations. To inform their use, this study investigated stability of EC/ERC hair concentrations in mothers, fathers, and their children across the perinatal period as well as associations between family members. In a prospective cohort study, EC (AEA, 1-AG/2-AG) and ERC (SEA, PEA, OEA) levels were quantified in hair samples taken four times in mothers (n =336) and their partners (n = 225) from pregnancy to two years postpartum and in offspring (n = 319) from shortly after birth to two years postpartum. Across the perinatal period, maternal and paternal hair ECs/ERCs showed fair-to-good multiple-test consistency and variable relative stability, and inconsistent absolute stability for mothers. Regarding children, hair ECs/ERCs evidenced poor multiple-test consistency, no absolute stability, and either no or variable relative stability. Hair ECs/ERCs were significantly related across the perinatal period within couples and parent-child dyads. Findings suggest hair ECs/ERCs during the perinatal period possess some trait-like stability in adults, highlighting the utility of hair analysis. The first two years of life may be a dynamic phase for the endocannabinoid system in children, potentially characterized by endocannabinoid system synchrony within the family.

Biological sciences/Developmental biology

Biological sciences/Psychology

Health sciences/Biomarkers

The analysis and quantification of hormones and ligands in hair has emerged as an important methodological addition to the psychoneuroendocrine research toolbox^1,2. In particular, hair cortisol concentrations (HCC) represent the most established hair-based marker to date, showing utility for clinical diagnostics, therapy monitoring, and mental health and stress research¹. It is assumed that hormones and ligands are incorporated in growing hair and consequently hair concentrations retrospectively reflect cumulated secretion of the respective hormone or ligand over several months³. An implicit assumption underlying hair analysis is that in the absence of major life events, hair concentrations remain largely stable within an individual². HCC have been intensively investigated and validated, with studies documenting a considerable degree of intraindividual stability of HCC in community samples^4–6 and postpartum in mothers and infants⁷, but not in expectant mothers across pregnancy⁸. Taken together, findings underscore HCC as a useful trait estimate of long-term cortisol secretion and a key variable in chronic stress studies. However, evidence for other hair-based biomarkers remains limited in this respect.

This is particularly relevant for the ligands of the endocannabinoid system (ECS), the endocannabinoids (ECs), which have recently been quantified in human hair⁹. The ECS is a neuromodulatory system which regulates multiple physiological functions in the body ranging from the central nervous system to the reproductive system^10,11. Amongst these, the ECS has been shown to interact with the hypothalamus-pituitary-adrenal (HPA) axis, assuming an important regulatory function under basal and stressful conditions¹². ECs are lipid signalling molecules synthesized mainly on demand that act primarily as retrograde messengers via presynaptic cannabinoid receptors (CB1 and CB2)¹³. The main ECs and best studied in terms of their biologic functions are anandamide (AEA¹⁴) and 2-arachidonoylglycerol (2-AG¹⁵), while additional endocannabinoid-related compounds (ERCs) exist, among others stearoylethanolamide (SEA), palmitoylethanolamide (PEA), and oleoylethanolamide (OEA), that exert their respective biological activities through different receptors^16,17.

Measurement of ECs/ERCs to assess EC signalling in humans represents an important step to better understand and examine ECs/ERCs in health and disease¹⁸. Most frequently, ECs/ERCs have been measured in the circulation via blood plasma and serum^19–21 and less frequently in other specimens such as saliva²², urine²³, or breast milk²⁴. These measurements represent EC/ERC levels acutely (e.g., blood, saliva) or cumulated over a short time period (e.g., urine) and may be influenced by situational factors, such as food intake, time of day, sleep, and activity level, thereby hampering their usefulness to assess overall state of EC/ERC signalling¹⁸.

In this regard, the recent development of quantifying ECs/ERCs in human hair may hold important advantages as it has been suggested to provide a retrospective picture of long-term peripheral EC/ERC levels over several months in addition to its non-invasive and stress-free sampling process^9,25. Similar to HCC, the most common hypothesis for incorporation of ECs/ERCs in hair is based on the multi-compartment model²⁶. Incorporation is suggested to occur mainly 1) during hair shaft formation via diffusion from blood circulation, 2) after formation via sweat and sebum glands or from surrounding tissues, and also 3) after hair has emerged from the skin via the external environment²⁷. Basic lipophilic compounds, such as AEA and ERCs, are assumed to mainly be incorporated into hair via the bloodstream, whereas 2-AG, with its neutral chemical structure, is likely incorporated via sweat and sebum²⁸.

Several studies have employed hair analyses of ECs/ERCs to examine their role in the pathophysiology of (stress-related) mental disorders, such as depression²⁹, (childbirth-related) posttraumatic stress disorder^30,31, anorexia nervosa³², anxiety, and burnout⁹. In these studies, hair ECs/ERCs were employed to assess ECS activity over an extended time up to 3 months, implicitly assuming that in the absence of major life events hair EC/ERC concentrations are somewhat stable within an individual, i.e., partly reflect a trait factor. However, this assumption has been insufficiently validated to date. Only one study systematically examined this for hair ECs/ERCs, finding high intra-individual stability of hair ECs/ERCs in a community sample of 100 female adults assessed across 2.5 years³³. These initial results suggest ECs/ERCs measured in hair may represent trait-like biomarkers that remain largely stable across time. However, this study is not generalizable to males or children and did not actively control for major life events during the 2.5-year study period.

Hence it remains to be tested whether hair ECs/ERCs show intraindividual stability and represent trait-like biomarkers also in the presence of a major life event. One possibility to examine this is to look at how the experience of a particular major life event, such as pregnancy and childbirth, affects intraindividual stability of hair ECs/ERCs in mothers and fathers. It should be considered that for (expectant) mothers, pregnancy and childbirth represent not only significant (potentially stressful) life experiences³⁴, but also go hand in hand with major physiological changes³⁵, including changes to endocannabinoid and cortisol signalling³⁶ and HPA axis functioning³⁷. Some physiological changes have also been observed in (expectant) fathers (e.g., decline in testosterone^38–40). Furthermore, while evidence regarding a role for the ECS in child development is accumulating^41,42, only one study has investigated hair ECs/ERCs in children aged up to one year⁴³. They found no association between child hair ECs/ERCs in utero (i.e., assessed via hair taken shortly after birth) and at one year after birth, indicating low stability and further reported an increase in 1-AG/2-AG and a decrease in ERCs in this time. Regarding child HCC, the effect of age has been mixed⁴⁴, but studies did find that HCC correlated positively across time up to the age of 8 years^45,46. These HCC findings indicate a stable component, consistent with findings of intraindividual stability of salivary cortisol in the first year of life⁴⁷. Hence, investigating whether hair EC/ERCs also show stability, particularly from birth onwards, is warranted to stimulate developmental research and inform interpretation of findings.

This study therefore primarily aimed to investigate multiple-test consistency, relative stability, and absolute stability for maternal, paternal, and child hair ECs/ERCs across the perinatal period. Regarding parents, we examined these measures of stability when a major life event (i.e., pregnancy and childbirth) was included and when it was not (i.e., examining only the later postpartum period from 14 to 24 months postpartum) and expected for mothers that multiple test consistency and relative and absolute stability in hair ECs/ERCs would differ between these two conditions, as previous evidence suggests alterations in hair ECs/ERCs during pregnancy in (expectant) mothers³⁶. Due to scarce research on paternal hair ECs/ERCs during the perinatal period, no specific predictions were formulated for fathers. We further expected parental hair EC/ERCs to show at least moderate multiple-test consistency and test-retest stability, but as findings regarding absolute stability have been inconsistent³³, no specific hypotheses were formulated. Furthermore, due to insufficient evidence amongst children’s hair ECs/ERCs, no specific hypotheses regarding their stability were posed.

Finally, only two studies have to date examined the relation between hair ECs/ERCs in children and maternal hair ECs/ERCs sampled shortly after birth⁴⁸ and at one year postpartum⁴³, finding no consistent relationships between maternal and child hair ECs/ERCs. Findings regarding a more established biomarker in hair (i.e., HCC) indicate a positive relationship that attenuates with increasing age^49–51, but see^7,52 for no significant relationship. Consequently, we aimed at replicating previous findings and investigate how maternal and child hair EC/ERC concentrations relate to one another up to two years after birth. Furthermore, to expand on prior research we also examined relationships between children’s and fathers’ and fathers’ and mothers’ hair EC/ERC concentrations as a positive relationship has been reported regarding HCC⁵³.

2.1 Study design and participants

Participants of this study took part in the Dresden Study on Parenting, Work, and Mental Health (DREAM) and its endocrine sub-study DREAM_HAIR⁵⁴, a large prospective cohort study approved by the ethics committee of the Technische Universität Dresden (EK 278062015). The study was conducted in accordance with the Declaration of Helsinki. DREAM_HAIR is a multi-method study investigating long-term biological determinants of perinatal stress and mental health-related outcomes in mothers, their partners, and their offspring across five measurement points from birth to 4.5 years postpartum (T1 DREAM_HAIR – T6 DREAM_HAIR, see Fig. 1).

Recruitment of expectant parents in the Dresden area began in June 2017 and ended in December 2020. T1 DREAM was completed during pregnancy and participants were recruited for DREAM_HAIR via a telephone screening if they completed the T1 DREAM questionnaires at least four weeks prior to their anticipated birth date. Informed consent was obtained from all participants and for the children from their legal guardians. DREAM_HAIR specific questionnaires and hair samples were taken 4±2 weeks before the anticipated birth date for T1, for T2 approximately eight weeks after the anticipated birth date, for T3 at 14 months after birth, and for T4 at 24 months after birth (see Fig. 1). Hair samples of children were collected at the same time as parental samples, except for T1, which took place approximately 10 days after birth. The current investigation used data from T1 DREAM for sociodemographic information (i.e., age, academic degree, and parity) as well as T1 DREAM_HAIR through T4 DREAM_HAIR for hair samples of mothers (N = 336), fathers (N = 225), and children (N = 319) (see Fig. 2 for inclusion criteria).

2.2 Hair endocannabinoid and N-acylethanolamines analyses

Hair strands (length ≥ 2 cm; collective diameter 3 mm with exception of child hair samples at T1 and T2) were taken scalp-near from a posterior vertex position either by study employees at T1 or by participants themselves after having been provided with instructive materials (i.e., checklist, video link). Samples were stored at room temperature in aluminum foil in dry and dark surroundings until sent to the Kirschbaum laboratory at the Technische Universität Dresden in five batches. AEA, 1-AG/2-AG, SEA, PEA, and OEA were quantified in the scalp-near 2 cm segment following a validated protocol⁹, assumed to reflect EC/ERC secretion of two months before hair sampling. As 2-AG isomerizes rapidly to 1-AG in biological matrices, a sum of the two was used, which is consistent with the fact that both isomers activate CB1 receptors⁵⁵. Also, as analysis in different batches as well as storage time and stressful life circumstances have been shown to influence hair biomarkers^8,56, their effects were controlled for in absolute stability analyses (see 2.3).

2.3 Statistical analyses

Analyses were conducted using IBM® SPSS® version 29 and R version 4.2.2⁵⁷. This study was preregistered (https://osf.io/tc276/?view_only=4e326d2c62144d1d8ef1873f9c50d548). Statistical significance was determined using a critical p-value of .05. Correlational analysis p-values were FDR-corrected for multiple testing⁵⁸. Due to their significantly positively skewed distribution, hair ECs/ERCs were log-transformed to reduce skewness. Apart from visualisations and descriptive statistics, all further analyses were conducted with log-transformed data. Extreme outliers in ECs and ECRs were defined as ± 3 SDs from the mean using log-transformed data. Extreme outliers were replaced with the highest and lowest measured value within ± 3 SDs at each time point for each sample respectively⁵⁹ and non-detectable values, extremely low values below the detection limit⁶⁰, were replaced with the lowest measurable value for each EC/ERC at each measurement point divided by two⁶¹ (AEA = 0.5% – 15.2%; PEA = 0.5%; OEA = 0.9%).

This study examined stability, i.e., the extent to which measurements are similar within an individual and across time, via three indicators. First, multiple-test consistency was examined via the intraclass correlation coefficient (ICC) as an index reflecting how much of the variance in a variable (i.e., hair EC/ERC concentrations) is attributable to constant mean differences between individuals, such that a high ICC indicated high similarity between measurements of the same person, thereby reflecting consistency across multiple tests (ICC: < 0.4 poor, 0.4–0.59 fair, 0.6–0.74 good, > 0.75 excellent consistency⁶²). The ICC was calculated in SPSS RELIABILITY using a two-way mixed-effects model assessing consistency both for a single measurement (ICC_SINGLE), i.e., reliability if a single hair sample was used, and for the mean value (ICC_MEAN), i.e., reliability when the average of all four hair samples is used⁶³. We also report ICC_SINGLE from a fixed linear time random intercept model from SPSS MIXED as this analysis can handle missing data using restricted maximum likelihood (REML⁶⁴), to examine whether the ICC remained similar despite the increase in sample size. Secondly, test-retest stability was calculated as the Pearson correlation between hair EC/ERC measurements at two time points, reflecting relative stability across measurements (r < .30 (weak), r = 0.3–0.59 (moderate), r > .59 (high) relative stability⁶). Finally, we assessed absolute stability, providing information as to whether the average hair endocannabinoid concentration across all participants changes significantly across time. A linear mixed model (LMM) with a fixed effect of time controlling for fixed effects of batch, storage time, and extraordinary burden for each of the five hair ECs and ERCs for mothers, fathers, and the children separately was conducted (p > .05 for time as a predictor indicating absolute stability⁶). See supplementary materials for descriptive statistics for batch and storage time. Post-hoc tests with Sidak correction were conducted. As assumptions (i.e., normality and homoscedasticity of residuals) of LMM for AEA were only met when excluding non-detectable values from the analyses, results for hair AEA are reported without imputed non-detectable values and if results differed this is mentioned (see supplementary materials for results with non-detectable values). For mothers and fathers, multiple-test consistency analyses were conducted using measurement for all four time points (T1–T4) and were repeated to include only T3–T4 to contrast stability levels with/without the major life event of pregnancy and childbirth. To explore the relationship between family members’ hair ECs/ERCs across the perinatal period, Pearson correlations were estimated between child and parental hair concentrations at each measurement occasion.

3.1 Descriptive statistics

Table 1 shows descriptive statistics for the three samples. Most mothers were primiparous and about 66% of mothers and fathers had an academic degree. Amongst the children, 53% were female and 47% male. Overall, the range of raw EC/ERC values was rather large, indicating a wide physiological range of EC/ERC concentrations in hair.

Table 1

*Demographic, hair-related, and raw hair EC/ERC concentrations of the study participants*
Variables	Sample of Mothers (N = 336)				Sample of Fathers (N = 225)				Sample of Children (N = 319)
Time points	T₁ (N = 299)	T₂ (N = 288)	T₃ (N = 261)	T₄ (N = 175)	T₁ (N = 189)	T₂ (N = 188)	T₃ (N = 188)	T₄ (N = 165)	T₁ (N = 207)	T₂ (N = 252)	T₃ (N = 257)	T₄ (N = 241)
Demographic characteristics
Academic degree (n, %)	222 (66.7)				146 (66.1)
Primiparous (n, %)	285 (84.8)
Age in ^ayears, ^bweeks, ^cmonths, M (SD)	30.30^a (3.93)				32.64^a (5.10)				0.98^b (0.81)	8.48^b (1.40)	13.86^c (0.57)	23.90^c (0.62)
Medication intake (n, %)
Antibiotics	5 (1.7)	14 (4.9)	9 (3.4)	4 (2.3)	6 (3.2)	2 (1.1)	7 (3.7)	2 (1.2)	5 (2.4)	6 (2.4)	13 (5.1)	6 (2.5)
Glucocorticoid-containing medication	5 (1.7)	1 (0.3)	3 (1.1)	0 (0.0)	5 (2.6)	2 (1.1)	1 (0.5)	1 (0.6)	0 (0.0)	0 (0.0)	3 (1.2)	3 (1.2)
Antidepressant medication	2 (0.7)	2 (0.7)	2 (0.8)	1 (0.6)	1 (0.5)	2 (1.1)	2 (1.1)	2 (1.2)	N/A	N/A	N/A	N/A
Oral contraceptives	0 (0.0)	28 (9.8)	43 (16.7)	37 (21.6)	N/A	N/A	N/A	N/A	N/A	N/A	N/A	N/A
Hair endocannabinoids and N-acyl-ethanolamines
AEA, M (SD)	1.24 (1.80)	1.51 (2.52)	2.09 (2.10)	3.05 (2.64)	1.05 (0.96)	1.08 (1.21)	1.56 (1.17)	1.89 (1.41)	2.50 (1.86)	2.07 (2.09)	0.85 (0.71)	0.92 (0.66)
2-AG/1-AG, M (SD)	42.02 (31.47)	44.14 (32.84)	66.85 (46.56)	82.06 (46.36)	62.56 (48.75)	62.02 (44.71)	74.64 (46.17)	78.08 (36.04)	131.93 (126.54)	294.82 (362.45)	93.01 (62.85)	103.03 (52.1)
SEA, M (SD)	1052.93 (1036.48)	1079.55 (1370.02)	981.45 (1067.06)	792.53 (797.16)	880.51 (693.93)	843.11 (664.92)	760.59 (532.49)	601.00 (340.57)	305.40 (272.61)	872.85 (775.69)	1054.87 (650.10)	761.45 505.93)
PEA, M (SD)	3811.10 (4892.80)	3912.25 (5348.57)	2567.28 (3034.85)	2279.98 (2701.83)	2695.28 (2533.96)	2322.33 (1943.53)	1912.17 (1787.80)	1465.37 (905.57)	507.01 (529.43)	1666.02 (1819.07)	1537.88 (1049.43)	1124.45 (700.52)
OEA, M (SD)	3233.87 (4866.80)	3543.21 (6200.86)	2242.16 (1145.52)	2278.43 (3975.58)	2147.11 (2560.64)	1738.90 (1844.36)	1560.20 (1776.55)	1291.93 (1030.98)	399.42 (593.53)	1384.87 (2055.98)	1674.42 (1326.20)	1347.11 (942.53)
Note. ^a in years. ^b in weeks, ^c in months.

3.2 Stability of hair ECs/ERCs

3.2.1 Multiple-test consistency

Regarding mothers, multiple-test consistency (Table 2) was good for ECRs and 1-AG/2-AG (ICC_MEAN = .62 – .73) and fair for AEA (ICC_MEAN = .51) regarding the mean measure and poor to fair for all ECs/ERCs regarding the single measure (ICC_SINGLE = .21 – .40) from T1 to T4 for mothers. When examining only the later postpartum period (T3–T4), consistency of the mean measure was slightly lower compared to T1–T4, but single measure consistency increased for all ECs/ERCs except AEA (ICC_SINGLE = .38 – .43).

Regarding fathers, multiple-test consistency was good for PEA and OEA (ICC = .68 – .69) and fair for AEA, 1-AG/2-AG, and SEA (ICC_MEAN = .41 – .59) in the mean measure and poor for all ECs/ERCs in the single measure (ICC_SINGLE = .15 – .36) from T1 to T4. In the later postpartum period (T3–T4), multiple-test consistency in the mean measure decreased for ECs and ERCs, but single measure consistency increased compared to T1–T4 for all ECs/ERCs except AEA (ICC_SINGLE = .38 – .46).

Children showed poor multiple-test consistency from T1 to T4 for all ECs/ERCs in the mean (ICC_MEAN = .09 – .36) and single (ICC_SINGLE = .02 – .12) measure. Overall, results remained largely consistent in linear mixed models (LMM), except for ICC of ERCs of children increasing noticeably in LMM.

Table 2

*Multiple test consistency of hair ECs/ERCs*
	Single measure		Mean measure		From LMM
Mothers	ICC_SINGLE	95% CI	ICC_MEAN	95% CI	ICC_SINGLE
From T1 to T4	(n = 129)		(n = 129)		(n = 336)
AEA	.21	[.12, .30]	.51	[.36, .64]	.20
2-AG/1-AG	.29	[.20, .39]	.62	[.50, .72]	.30
SEA	.35	[.26, .45]	.69	[.59, .77]	.32
PEA	.38	[.29, .47]	.71	[.62, .78]	.35
OEA	.40	[.31, .50]	.73	[.64, .80]	.35
From T3 to T4	(n = 158)		(n = 158)		(n = 278)
AEA	− .01	[-.17, .14]	− .03	[-.41, .25]	.00
2-AG/1-AG	.41	[.27. .53]	.58	[.42, .69]	.39
SEA	.43	[.29, .53]	.60	[.45, .71]	.42
PEA	.42	[.28, .54]	.59	[.44, .70]	.43
OEA	.38	[.23, .50]	.55	[.38, .67]	.37
Fathers	ICC_SINGLE	95% CI	ICC_MEAN	95% CI	ICC_SINGLE
From T1 to T4	(n = 112)		(n = 112)		(n = 225)
AEA	.15	[.06, .25]	.41	[.21, .57]	.16
2-AG/1-AG	.27	[.17, .37]	.59	[.45, .70]	.27
SEA	.26	[.17, .37]	.58	[.44, .70]	.30
PEA	.35	[.25, .45]	.68	[.57, .77]	.36
OEA	.36	[.26, .47]	.69	[.59, .78]	.36
From T3 to T4	(n = 151)		(n = 151)		(n = 202)
AEA	.10	[-.06, .25]	.18	[-.13, .40]	.11
2-AG/1-AG	.38	[.24, .51]	.55	[.38, .68]	.38
SEA	.43	[.29, .55]	.60	[.44, .71]	.42
PEA	.46	[.32, .57]	.63	[.49, .73]	.45
OEA	.46	[.32, .57]	.63	[.49, .73]	.45
Children	ICC_SINGLE	95% CI	ICC_MEAN	95% CI	ICC_SINGLE
From T1 to T4	(n = 123)		(n = 123)		(n = 319)
AEA	.07	[-.001, .16]	.24	[-.01, .44]	.08
2-AG/1-AG	.04	[-.03, .13]	.15	[-.12, .38]	.04
SEA	.02	[-.04, .11]	.09	[-.20, .33]	.08
PEA	.07	[-.01, .15]	.22	[-.03, .42]	.13
OEA	.12	[.04, .22]	.36	[.15, .53]	.19
Note. ICC = Intraclass Correlation Coefficient. ICC_SINGLE = Single Measures Intraclass Correlation Coefficient. ICC_MEAN = Mean Measures Intraclass Correlation Coefficient. 95% CI = 95% Confidence Interval. LMM = Linear mixed model.

3.2.2 Relative stability

For mothers, associations between hair EC/ERC levels across time points were strongest T1–T2. Weak to moderate relative stability was found regarding maternal hair ERC assessments across all measurement occasions. Regarding AEA and 1-AG/2-AG, significant weak to moderate associations were present across most time points, except for associations at T1–T4 and T2–T4 and only regarding AEA for T3–T4 (Table 3 for full results).

Fathers showed moderate to high relative stability for T1–T2 for hair ECs/ERCs. Regarding the ERCs PEA and OEA, weak to moderate relative stability was present across time points except for T2–T4 for PEA. AEA showed no relative stability apart from T1–T2 and 1-AG/2-AG and SEA lacked relative stability at T1–T3, T1–T4, T2–T3, and T2–T4 (only SEA).

Regarding children, weak to moderate relative stability of hair ECs/ERCs was present for all at T1–T2 and for all except AEA and OEA at T3–T4. Otherwise, children’s hair ECs/ERCs were inconsistently and sometimes negatively associated with one another across time (Table 3).

Table 3

*Test-retest relative stability of hair endocannabinoids and endocannabinoid-related compounds for mothers, fathers, and children*
		T1–T2	T1–T3	T1–T4	T2–T3	T2–T4	T3–T4
		r	r	r	r	r	r
Mothers	AEA	.44^***	.21^*	− .03	.24^**	.11	− .02
	2-AG/1-AG	.50^***	.16^*	.07	.31^***	.17	.41^***
	SEA	.53^***	.20^**	.37^***	.23^**	.19^*	.43^***
	PEA	.56^***	.24^***	.32^***	.30^***	.22^*	.42^***
	OEA	.54^***	.25^***	.37^***	.36^***	.23^**	.38^***
		(n = 258)	(n = 234)	(n = 158)	(n = 233)	(n = 155)	(n = 158)
Fathers	AEA	.48^***	.10	− .02	.15	.06	.10
	2-AG/1-AG	.59^***	.11	.18	.08	.23^*	.39^***
	SEA	.63^***	.16	.17	.16	.14	.43^***
	PEA	.61^***	.24^**	.32^***	.26^***	.18	.47^***
	OEA	.62^***	.18^*	.27^**	.31^***	.25^**	.46^***
		(n = 155)	(n = 160)	(n = 137)	(n = 164)	(n = 143)	(n = 151)
Children	AEA	.23^**	.14	− .06	.22^**	− .11	.07
	2-AG/1-AG	.41^***	− .23^**	− .12	− .05	− .14	.20^*
	SEA	.44^***	.07	− .16	− .11	− .27^***	.26^***
	PEA	.46^***	-01	− .03	− .08	.01	.23^***
	OEA	.52^***	.11	.03	.00	.13	.14
		(n = 182)	(n = 169)	(n = 158)	(n = 203)	(n = 185)	(n = 210)
Note. ^* p < .05, ^ p < .01, ^* p < .001, FDR-corrected p-values. r = Pearson correlation coefficient.

3.2.3 Absolute stability

Random intercept LMM adjusting for batch, storage time, and extraordinary burden were conducted to test the effect of time on hair EC/ERCs (see Fig. 3).

In mothers, time (T1–T4) was a significant predictor of maternal AEA (F (3, 422.29) = 4.91, p = .002), 1-AG/2-AG (F (3, 477.72) = 4.33, p = .005), and PEA (F (3, 447.24) = 3.08, p = .027), but not SEA and OEA (all p > .121). Post-hoc tests showed significant differences between T1–T2 (AEA), T1–T3 (1-AG/2-AG, PEA), T1–T4 (AEA), T2–T3 (1-AG/2-AG, PEA) and T2–T4 (1-AG/2-AG), with earlier assessments showing lower levels of the ECs and higher levels of PEA (Table 4).

Regarding fathers, time (T1–T4) did not significantly predict hair EC/ERC levels (p’s > .093; Table 4 for pairwise comparisons).

Regarding children, time (T1–T4) was a significant predictor of all hair ECs/ERCs (AEA: F (3, 417.79) = 89.77, p < .001; 1-AG/2-AG: F (3, 479.05) = 97.53, p < .001; SEA: F (3, 509.27) = 185.90, p < .001; PEA: F (3, 529.53) = 129.71, p < .001; OEA: F (3, 495.29) = 127.25, p < .001). Post-hoc tests showed that hair EC/ERC levels differed significantly between all time points (p < .001), except between T3 and T4 (all ECs/ERCs: p > .086), between T2 and T3 (PEA: p = .071), and between T2 and T4 (PEA: p = .680; Table 4). Results were the same when calculated separately for child sex, apart from no significant AEA level difference T1–T2 for female children (p = .065; supplementary materials).

Table 4

*Pairwise comparisons of hair EC/ERC levels with Sidak correction for mothers, fathers, and children*
	AEA^{a b}		1-AG/2-AG^c		SEA^c		PEA^c		OEA^c
	M_DIFF	p	M_DIFF	p	M_DIFF	p	M_DIFF	p	M_DIFF	p
Mothers
T1–T2	− .09	.003^d	.00	1.00	.04	.474	.01	1.00	.03	.958
T1–T3	− .08	.115	− .08	.017	.02	.986	.10	.032	.11	.104
T1–T4	− .12	.022	− .09	.098	.02	.996	.10	.282	.09	.618
T2–T3	.01	.999	− .08	.005	− .02	.983	.09	.038	.08	.382
T2–T4	− .03	.956^e	− .09	.043	− .02	.997	.09	.295	.06	.886
T3–T4	− .04	.601^e	.01	1.00	− .00	1.00	− .00	1.00	− .02	1.00
Fathers
T1–T2	− .02	.995	.01	.989	.03	.875	.03	.777	.05	.440
T1–T3	.00	1.00	.03	.937	.04	.717	.08	.085	.06	.559
T1–T4	− .01	1.00	.05	.728	.04	.886	.09	.114	.06	.845
T2–T3	.02	.997	.01	.999	.01	.996	.05	.467	.01	1.00
T2–T4	.01	1.00	.03	.905	.01	.999	.06	.420	.00	1.00
T3–T4	− .01	1.00	.02	.934	.00	1.00	.01	.993	− .00	1.00
Children
T1–T2	.11	< .001	− .29	< .001	− .47	< .001	− .57	< .001	− .60	< .001
T1–T3	.51	< .001	.21	< .001	− .63	< .001	− .63	< .001	− .81	< .001
T1–T4	.43	< .001	.18	< .001	− .64	< .001	− .61	< .001	− .82	< .001
T2–T3	.40	< .001	.50	< .001	− .16	< .001	− .06	.071	− .21	< .001
T2–T4	.32	< .001	.48	< .001	− .17	< .001	− .04	.680	− .22	< .001
T3–T4	− .07	.082	− .03	.747	− .01	.977	.02	.641	− .00	1.000
Note. M_DIFF = Mean difference. Numbers in bold = p < .05. ^a mothers n = 331, fathers n = 225, children n = 315. ^b calculated when excluding non-detectable values. ^c mothers: n = 336, fathers n = 225, children n = 318. ^d This comparison was not significant when including non-detectable values. ^e This comparison was significant when including non-detectable values.

3.3 Parent-child associations in hair ECs/ERCs across the perinatal period

Within mother-child and father-child dyads, all ECs and ERCs showed statistically significant small-to-medium positive correlations at almost all measurement points, apart from 1-AG/2-AG at T4 in mother-child dyads and AEA and OEA at T1 in father-child dyads (Table 5). The strength of correlations was highest at T4 for mother-child dyads (except for 1-AG/2-AG), whereas for father-child dyads this was the case at T3.

Within couples, ECs/ERCs again were statistically significantly positively correlated at all measurement occasions (with the exception of AEA and OEA at T1 and T4) in the small to medium range. Strength of inter-parental associations seemed to increase from T1, reaching a peak at T3 and lower values at T4.

Table 5

*Parent-child associations of hair endocannabinoids and N-acylethanolamines.*
		T1	T2	T3	T4
		r	r	r	r
Mother-Child ^a	AEA	.21^*	.34^***	.21^**	.39^***
	2-AG/1-AG	.40^***	.40^***	.23^**	.04
	SEA	.27^***	.22^**	.25^**	.27^**
	PEA	.18^*	.17^*	.21^**	.33^**
	OEA	.19^*	.18^*	.18^*	.38^***
Father-Child ^b	AEA	.02	.20^*	.33^***	.20^*
	2-AG/1-AG	.40^***	.39^***	.50^***	.24^*
	SEA	.33^**	.33^***	.44^***	.35^***
	PEA	.26^*	.40^***	.42^***	.44^***
	OEA	.21	.39^***	.41^***	.35^***
Mother-Father ^c	AEA	.13	.24^*	.39^***	.19
	2-AG/1-AG	.52^***	.44^***	.53^***	.32^**
	SEA	.28^**	.29^**	.36^***	.27^*
	PEA	.24^**	.28^**	.38^***	.25^*
	OEA	.17	.27^**	.38^***	.23
Notes. ^* p < .05, ^ p < .01, ^* p < .001, FDR-corrected p-values. r = Pearson correlation coefficient. ^a T1 n = 183; T2 n = 214–215; T3 n = 212; T4 n = 153. ^b T1 n = 117; T2 n = 141–142; T3 n = 154; T4 n = 146. ^c T1 n = 167; T2 n = 160; T3 n = 153; T4 n = 98.

This prospective investigation examined whether a core assumption of hair EC/ERC analysis, namely that hair EC/ERC concentrations are largely stable within an individual, also holds true in the perinatal period in mothers, fathers, and their children and also when experiencing the major life event of pregnancy and childbirth in mothers and fathers. As a secondary aim, we investigated how hair ECs/ERCs are associated within the family unit across the first two years after birth.

Descriptive classification of hair EC/ERC levels across the perinatal period

In general, the physiological range of hair ECs/ERCs varied widely in both adults and children, consistent with prior research on ECs/ERCs in serum⁶⁵ and hair^33,43. Regarding mothers, hair AEA concentrations were higher than in women within⁴³ and outside the perinatal period^29,33. In contrast, 1-AG/2-AG levels were lower at T1, and increased at T4 to a level comparable to women outside the perinatal period³³. ERC levels at T1 and T3 were comparable to previous research with postpartum mothers^43,48 and were higher than in women outside the perinatal period³³, but decreased to comparable levels by T4. Fathers’ hair AEA and 1-AG/2-AG across all time points were higher than reported outside the perinatal period⁹, whereas hair ERC concentrations were elevated at T1, but decreased to levels similar to outside the perinatal period by T4⁹. Regarding children, concentrations can be compared with Hitzler et al.⁴³, who assessed hair ECs/ERCs in mothers and children shortly after birth and at one year postpartum. Shortly after birth, hair 1-AG/2-AG and SEA levels were lower and hair PEA and OEA higher compared to Hitzler et al.⁴³, whereas all hair ECs/ERCs were higher in this study at 14 months after birth compared to their levels at twelve months. Overall, comparison of hair EC/ERC levels between studies is hampered by differences in hair segment length being analyzed, variations due to continuously evolving laboratory techniques, and different hair sample storage times.

Stability of parental hair ECs/ERCs across the perinatal period

Regarding the stability of hair ECs/ERCs of mothers and fathers across the perinatal period (T1 – T4), consistent with our hypothesis, this study found fair-to-good multiple-test consistency regarding all ECs/ERCs on the mean measure, but poor multiple-test consistency on the single measure. Our finding suggests that between 41% and 73% (when taking the average of several hair samples) and between 15% and 40% (when using a single hair sample) of the variance in hair ECs/ERCs across the perinatal period in mothers and fathers may be attributed to between-person differences, i.e., individual traits. It should be noted that multiple-test consistency was lowest for AEA in the present study and that consistency of hair ECs/ERCs was overall lower compared to Gao et al.’s³³ investigation of 100 women, which may be attributable to unique stressors and changes during the perinatal period.

Regarding the effect of the major life event of pregnancy and childbirth on stability of parental hair ECs/ERCs, findings showed that while mean multiple-test consistency was higher, the single measure was slightly lower when including pregnancy and childbirth for both mothers and fathers. This finding indicates that hair ECs/ERCs continue to show stability also in the presence of a major life event and can represent trait-like biomarkers for parents in this time. It should be noted that higher estimates of multiple test-consistency when using measures from all four time points may be due to the higher numbers of included measurements, compared to estimates based on T3 and T4 only. Thus, comparisons of the single measure are more meaningful, suggesting that consistency of a single hair sample taken in the later postpartum period may reflect slightly more trait components than when taken at any time in the perinatal period. Future studies would benefit from investigating other major life events (e.g., graduation, relocation) to corroborate our finding.

Furthermore, results indicated consistently weak to moderate test-retest relative stability for SEA, PEA, and OEA for mothers and PEA and OEA for fathers. Regarding AEA and 1-AG/2-AG, test-retest stability was present for T1 to T2 for both parents, but less consistently at later time-points. As the time between T1 and T2 (about 3 months) was shorter than between assessments from T2 onwards (10 to 24 months), this could explain why associations were particularly strong from T1 to T2 for all hair ECs/ERCs and may indicate that for mothers and fathers AEA and 1-AG/2-AG show short-term stability but may vary more across time compared to ERCs. Our findings align with Hitzler et al.⁴³, who also reported significant associations between maternal hair ERCs assessed shortly after birth and 12 months after birth, but found no significant relations regarding AEA and 1-AG/2-AG. However, Gao et al.³³ demonstrated moderate-to-high associations across six six-month intervals for all hair ECs/ERCs, suggesting that time period between assessments and the unique circumstances of the perinatal period may influence the strength of associations.

Finally, results showed that across the perinatal period SEA and OEA in mothers and all hair ECs/ERCs in fathers demonstrated absolute stability. This suggests that hair EC/ERC concentrations averaged across all fathers in this study did not change significantly across the perinatal period, while this was not the case for mothers. Given that pregnancy, childbirth, and the early postpartum period entail major physiological changes in mothers³⁵, including changes to the ECS and HPA axis^36,66, it fits that mean levels AEA, 1-AG/2-AG, and PEA were less stable for mothers than fathers. Specifically, maternal hair AEA during the third pregnancy trimester was significantly lower than at 8 weeks and at two years after birth, partly aligning with findings by Kumbholz et al.³⁶ who also found an increase from third trimester to nine weeks after birth. 1-AG/2-AG during pregnancy and 8 weeks after birth was significantly lower than at 14 months, consistent with prior research finding maternal 1-AG/2-AG increases from pregnancy to 12 months after birth⁴³. Finally, PEA levels were higher during pregnancy and eight weeks after birth than at 14 months, which was descriptively also the case in Hitzler et al.⁴³, albeit not significant. Results indicate that regulation of the maternal ECS may change during pregnancy and postpartum, with potential implications for glucocorticoid regulation and immune system functioning in this time. Both AEA and 1-AG/2-AG have been characterized as HPA axis regulators⁶⁷, with AEA correlating negatively with cortisol^29,30,36, and PEA has been attributed anti-inflammatory, analgesic, and neuroprotective properties⁶⁸. Thus, changes in these ECs/ERCs may reflect adaptive responses to regain homeostasis in the context of cortisol increases over pregnancy⁶⁹ and an inflammation-immunosuppression-inflammation phenotype during pregnancy⁷⁰. It remains for future research to disentangle the exact timeline of EC/ERC secretion patterns across the perinatal period to understand their contribution and interplay.

Stability of child hair ECs/ERCs across the perinatal period

Results further demonstrated poor multiple-test consistency for child hair ECs/ERCs taken shortly after birth up to two years of age, indicating a low trait component in this time, and no absolute stability. Furthermore, findings indicated that while all hair ECs/ERCs of children were significantly positively correlated in the early postpartum period (T1–T2), relative stability was subsequently only consistently present from 14 to 24 months after birth for 1-AG/2-AG, SEA, and PEA. This pattern could indicate that the first two years of life represent a particularly sensitive and dynamic developmental period for the ECS⁷¹ during which marked ECS changes occur. This is further underscored by negative associations of child hair ECs/ERCs at T1 or T2 with those at T3 or T4, suggesting that higher levels in infancy may be associated with reduced levels in toddlerhood and vice versa. Our findings of low stability for children align with the only other study assessing EC/ERC levels in child hair in the perinatal period up to one year after birth⁴³ and low intra-individual stability in salivary cortisol measured in 5 to 8 month old infants⁷².

Similar to Hitzler et al.⁴³, children’s hair 1-AG/2-AG and AEA levels decreased from shortly after birth to 14 months. However, in our study 1-AG/2-AG concentrations increased markedly in hair samples taken 8 weeks after birth, reflecting the early postnatal period. This aligns with animal research showing a peak in 2-AG levels in the rat brain during early postnatal development and subsequent decreases^73,74. It has been suggested that elevated 2-AG during early development may function to induce suckling behavior and support (neuro)development and growth⁷⁵. Decreases in hair AEA contrasts rat studies showing that AEA levels in the brain increase from birth into adulthood^73,76. Furthermore, research suggests that in neonates and infants there is a greater relative expression of AEA degrading (i.e., FAAH) compared to synthesizing (i.e., NAPE-PLD) enzymes in the human dorsolateral prefrontal cortex (DLPFC), which reduces with increasing age and switches around toddlerhood in favor of AEA synthesizing enzymes⁷¹. These findings are consistent with observed increases in AEA and 1-AG/2-AG from T3 to T4. Our findings should be interpreted considering we measured peripheral EC/ERC levels and the ECS is widespread throughout the body, thereby hampering comparability to region-specific findings⁹.

Regarding child ERCs, findings align with Hitzler et al.⁴³ showing increased hair ERC levels at 14 months after birth compared to T1 shortly after birth, where ERC levels were markedly below parental levels. These ERC patterns align with findings regarding AEA outlined above, which is biologically similar to ERCs, showing that in rats, AEA increases across infancy⁷³ and adolescence⁷⁷. Importantly, sensitivity analyses by child sex indicated no major differences between male and female children regarding hair EC/ERC trajectories up to 24 months after birth, contrasting research indicating sex-divergent ECS functioning due to crosstalk with sex steroid hormones^41,78. Finally, results showed no significant differences between 14 and 24 months of age in any hair EC/ERC levels in children, possibly indicating that the ECS may be more stable in this developmental period. However, further research investigating children’s hair EC/ERC levels across childhood and adolescence is needed to better understand developmental trajectories. This is particularly relevant when considering research showing that the ECS becomes functionally active already during early developmental stages in humans and rodents and its expression patterns in the brain implicates a role also in neurodevelopment⁴¹.

Inter-family associations of hair ECs/ERCs

Findings indicated that maternal, paternal, and child hair ECs/ERCs were significantly positively cross-sectionally associated with one another across the perinatal period. While these findings contradict previous research in a comparably small sample on ECs/ERCs^43,48, they are in line with HCC research, where positive associations between all family members at child age 6 years were found⁵³ as well as positive maternal-child associations reported at various child ages (9–12 months⁵⁰; 2 years⁷⁹; 4–5 years⁸⁰; 5–14 years⁵¹; 7–14 years⁴⁹; 14 years⁸¹; but see^45,52 for no associations at 9–12 months and shortly after birth, respectively). The observed synchrony may be explained by an interplay of genetic and environmental factors. Associations in the mother-child dyad at T1 may partly reflect intrauterine interactions of maternal and child ECS via the fetoplacental unit⁸². Research indicates that endocannabinoid receptors (CB1, CB2) and synthesizing and degrading enzymes (DAGL, FAAH) are expressed in the placenta and that the placenta regulates local endocannabinoid tonus, with implications for correct placental functioning and labor^41,66,82. Yet, our finding that positive associations persist beyond the intrauterine period and are present also in father-child dyads, even at T1, suggest additional mechanisms. The finding of hair EC/ERC mother-child and father-child synchrony implicates the ECS as a heritable familial trait. This is supported by recent twin-study research suggesting heritability in endocannabinoid signaling⁸³ and showing that in 138 twins (45 monozygotic; 24 dizygotic) serum endocannabinoid ligands had a certain degree of heritability (AEA 46%; OEA 35%; PEA 39%; SEA 34%), but were also markedly influenced by the environment⁸⁴. A role for environmental factors also seems particularly plausible considering we found significant maternal-paternal associations. While positive assortative mating, a preference for individuals similar to oneself, could partly explain these findings⁸⁵, environmental factors including various psychological and lifestyle factors shared or similar within the family context also seem plausible. For instance, nutrition and food intake, stress exposure, sleeping pattern, or physical activity level have all been linked to the ECS^41,86–88 and could contribute to ECS synchrony. Furthermore, considering transfer of maternal EC lipids, particularly 2-AG, to the child via breast milk has been suggested^24,89, maternal-child synchrony in the postpartum period could partly be attributable to breastfeeding practices. However, the fact that we found paternal-child associations of similar or greater strength in the postpartum period suggests that genetics and other shared environmental influences are equally important. Considering this study covered the first 24 months after birth, contact intensity and frequency likely declines with time hereafter and future research should examine trajectories of inter-family synchrony in hair ECs/ERCs as the child ages to see whether a decline in synchrony can be detected similar to findings with HCC⁵¹. Taken together, our results suggest inter-family synchrony in hair ECs/ERCs across the perinatal period, however more research is needed to tease apart genetic and environmental contributions.

Strengths and Limitations

Key strengths include the prospective design, our large sample size, our focus specifically on the perinatal period, and including all family members. However, limitations exist. Firstly, the operationalization of pregnancy and childbirth as a major life event was done post-hoc and not during the design stage of the study. Moreover, as the present investigation is longitudinal, batch effects present a complication⁹¹ that may also affect biomarker concentrations in hair samples⁸, which we statistically controlled for in this study. Furthermore, it remains to be determined what peripheral EC/ERC levels measured in hair samples exactly reflect in terms of ECS functioning, as a first investigation found no significant link with blood and urine EC/ERC levels⁹². Finally, it should be noted that the present sample involved a community with above-average health and education status⁵⁴. Hence, investigations in more heterogeneous samples are needed to evaluate generalizability of findings.

Overall, the present investigation revealed that parental hair ECs/ERCs represent some trait-like stability across the perinatal period, even in the presence of a major life event (i.e., pregnancy and childbirth), but that absolute stability is consistently present only in fathers in this time. Our findings also suggest hair ECs/ERCs are not stable across time within children in the first two years of life, indicating a developmental window. These discoveries provide important insights into the analysis of hair ECs/ERCs, indicating that this method could be well-suited for research applications seeking to obtain stable estimates of EC/ERC secretion in adults. Finally, this study highlights for the first time the family as a unit that may exert significant effects on ECS functioning in the period from late pregnancy to two years after birth, thus highlighting the need for future research to further examine how this relates to parental psychological and child developmental outcomes.

Data availability statement

The datasets analysed during the current study are not publicly available due legal and ethical constraints (i.e., public sharing of participant data was not included in the informed consent of the study) but are available from the corresponding author on reasonable request.

Funding details

This work was supported by the German Research Foundation (“Deutsche Forschungsgemeinschaft”) under grant numbers GA 2287/4-1 and GA 2287/4-2.

Disclosure statement

The authors report there are no competing interests to declare.

Acknowledgements

We want to thank all (expectant) parents and their children for supporting our project. Furthermore, we want to thank all cooperating clinics and midwives for providing access to potential participants as well as all students who performed the recruitment. We also want to especially thank Professor Clemens Kirschbaum and his laboratory for their support. Data were collected and managed using Research Electronic Data Capture (Harris et al., 2009, 2019). REDCap is a secure, web-based application designed to support data capture for research studies, hosted at the “Koordinierungszentrum für Klinische Studien” at the Faculty of Medicine of the Technische Universität Dresden, Germany.

Author Contributions

LB: Conceptualisation, Investigation, Data Curation, Formal Analysis, Writing – Original Draft, Writing – Review & Editing, Visualisation. SS-S: Conceptualisation, Resources, Supervision, Writing – Review & Editing, Project administration. MK: Investigation, Data Curation, Writing – Review & Editing. IJ: Investigation, Data Curation, Writing – Review & Editing. TvS: Formal Analysis, Writing – Review & Editing. WG: Resources, Writing – Review & Editing. SG-N: Conceptualisation, Resources, Supervision, Writing – Review & Editing, Project administration, Funding acquisition.

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Stability and inter-family associations of hair endocannabinoid and N-acyl-ethanolamines across the perinatal period in mothers, fathers, and children

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Abstract

Figures

1 Introduction

2 Methods

2.1 Study design and participants

2.2 Hair endocannabinoid and N-acylethanolamines analyses

2.3 Statistical analyses

3 Results

3.1 Descriptive statistics

3.2 Stability of hair ECs/ERCs

3.2.1 Multiple-test consistency

3.2.2 Relative stability

3.2.3 Absolute stability

3.3 Parent-child associations in hair ECs/ERCs across the perinatal period

4 Discussion

Conclusions

Declarations

References

Additional Declarations

Supplementary Files

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