Amniotic Fluid Elicits Appetitive

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Amniotic Fluid Elicits AppetitiveResponses in Human Newborns:Fatty Acids and AppetitiveResponses

ABSTRACT: In humans, maternal cues guide newborns to the maternal breast,and transitional cues may be present in maternalfetal fluids. The aim of thepresent study was to determine the consistent presence of sensorial cues in threematernalfetal fluidsamniotic fluid, colostrum, and milkand test the ability of

these cues to produce appetitive responses in newborns. In the analytical study,gas chromatography-mass spectrometry (GC-MS) detected eight fatty acids con-sistently present in the amniotic fluid, colostrum, and milk from 12 healthy volun-teers, but we do not find a mammalian pheromone, identified in anothermammalian species (rabbits), in another 30 volunteers. In the behavioral study,we explored the ability of amniotic fluid or its fatty acids to produce appetitiveresponses in 19 human newborns


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the mammary gland to serve as a maternal pheromone

(Signoret, Levy, Nowak, Orgeur, & Schaal, 1997). One

maternal pheromone (2-methylbut-2-enal) has been

identified in the maternal milk of the rabbit (Oryctola-

gus cuniculus; Coureaud, Langlois, Sicard, & Schaal,

2004; Schaal et al., 2003), but this maternal pheromone

has not been detected in amniotic fluid or bloodfrom the same rabbit species and did not produce reac-

tions in other mammals, including mice, rats, and cats

(Schaal et al., 2003). Importantly, the presence of this

aldehyde has not been confirmed in human maternal

fluids, such as amniotic fluid, colostrum, or milk, which

appear to be essential for supporting the concept of

learning after birth in humans through this aldehyde.

In the second approach, prenatal learning appears to

follow a transnatal olfactory continuum (Coureaud,

Schaal, Hudson, Orgeur, & Coudert, 2002), in which

the fetuses are exposed to similar substances that they

will find after birth in a continuum of sensorial cuesthat leads to early learning and further recognition

through olfactory stimulation (Varendi, Christensson,

Porter, & Winberg, 1998). Rabbits are able to encode

the odor encountered in their amniotic fluid and after

birth remain able to discriminate it in milk (Coureaud

et al., 2002). In this approach, at least two relevant

aspects should be considered: (i) the existence of a

functional sensorial system and (ii) access to a sensori-

al stimulus during intrauterine development. In humans

as early as the 24th gestational week, the olfactory mu-

cosa is well developed (Nakashima, Kimmelman, &

Snow, 1985) and contains ciliated olfactory receptors

that have a mature appearance (Pyatkina, 1982). Theolfactory marker proteinan indicator of neuroreceptor

functionality (Gesteland, Yancey, & Farbman, 1982)

and connectivity with mitral cells in the main olfactory

bulb (Brunjes & Frazier, 1986)is expressed in epithe-

lia at the 28th gestational week and in the main olfacto-

ry bulb between the 32nd and 35th gestational weeks

(Chuah & Zheng, 1987). Similarly, at approximately

the 12th gestational week, the human vomeronasal or-

gan is well developed (Boehm & Gasser, 1993) and

visible in the developing fetus (Kreutzer & Jafck, 1980;

Nakashima et al., 1985; Smith et al., 1997) and new-

borns (Smith & Bhatnagar, 2000). From an anatomicalperspective, human fetuses possess a reasonably well-

developed olfactory system before birth.

Considering access to intrauterine sensorial stimula-

tion, any odoriferous compound may gain access to

fetal olfactory chemoreceptor areas through maternal

fetal circulation (Maruniak, Silver, & Moulton, 1983)

and amniotic fluid that is continuously in close proxim-

ity to developing chemosensory receptors (Logvinenko,

1990). Therefore, the developmental maturity of

the fetal olfactory system during the final trimester

of gestation suggests that the sense of smell may

even be functional in utero (Chuah & Zheng, 1987),

allowing newborns to learn early and later recognize

olfactory cues (Winberg & Porter, 1998), particularly

those related to primary functions, such as feeding

(Marlier, Schaal, & Soussignan, 1998; Schaal, Marlier,

& Soussignan, 2000).In the large white bred pig (Sus scrofa), some fatty

acids were consistently detected in amniotic fluid, colos-

trum, and milk and appeared to be a specific odoriferous

cue that guides piglets to the maternal breast (Guiraudie-

Capraz et al., 2005). A possible receptor system for

these fatty acids has been identified (Guiraudie-Capraz,

Pageat, Cain, Madec, & Nagan-Le Meillour, 2003).

However, to our knowledge, such observations of the

sensorial stimulation continuity of the olfactory system

have not been replicated in humans.

To summarize, the cues that guide newborns to the

maternal breast may emerge from two sources. Onesource may be a cue that comes from the mother that

may be produced after birth. If so, then the existence of

a maternal cue (i.e., pheromone) must be demonstrated

because it has been shown in other mammals and must

produce appetitive reactions in newborns. As another

source, if newborns follow some cue to which they

were previously exposed, its presence must be demon-

strated in maternalfetal fluids (e.g., amniotic fluid, co-

lostrum, and milk) and elicit behavioral appetitive

responses. We hypothesized that similar compounds

present in amniotic fluid, colostrum, and maternal milk

may elicit similar appetitive responses in human new-

borns. In the analytical part of the present study, weexplored the consistency of a possible maternal phero-

mone and fatty acids in amniotic fluid, colostrum, and

milk using gas chromatography-mass spectrometry

(GC-MS). In the behavioral part of the study, we tested

the effects of amniotic fluid and its identified fatty

acids on appetitive responses in newborns.

METHODS

This study was approved by the local research ethics commit-tee (Instituto de Investigaciones Biomedicas from the Univer-

sidad Nacional Autonoma de Mexico and Hospital Escuela

from the Universidad Veracruzana). During the invitation ses-

sions, all of the mother volunteers received a detailed expla-

nation of the purpose and risks of the study from two

physicians. After accepting the invitation, they signed an in-

formed consent prior to inclusion in the study. We only

obtained biological fluids by normal secretion, with no risk to

the mother or baby. We did not touch the baby at any time

during the study, with the exception of placing the baby in

the bed. Mothers were present during the behavioral tests.

222 Contreras et al. Developmental Psychobiology


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Experiment 1. Analytical Study

In the analytical study, maternal biological fluids (amniotic

fluid, colostrum, and milk) were obtained from 12 healthy

volunteers (1733 years old; n 4, first gestation; n 5,

vaginal delivery; 3741 weeks gestational age) in the

Hospital Escuela (Gynecology Obstetric services), Universi-

dad Veracruzana.To determine the presence or absence of 2-methylbut-2-

enal in maternal fluids, samples were obtained from another

30 healthy volunteers during delivery (amniotic fluid), 24

48 hr after delivery (colostrum), and 26 weeks after delivery

(maternal milk). These samples were also obtained from

healthy volunteers in the Hospital Escuela (Gynecology

Obstetric services), Universidad Veracruzana, and a local

clinical public service (Clnica Hospital ISSSTE 300400,

nursing services) from Xalapa, Veracruz, Mexico.

Analytical Test. Maternal biological fluid collection.Amniotic fluid was obtained under sterile conditions by an

obstetrics surgeon. For vaginal deliveries, after the amnioticmembrane ruptured, approximately 5 ml of fresh amniotic

fluid was collected in a sterile receptacle. For cesarean

deliveries, the surgeon collected the amniotic fluid with a

sterile syringe (5 ml, 23 gauge). Afterward, the colostrum

was collected on the 2nd day after delivery, and milk

was collected by a female pediatrician on the 7th day in a

sterile receptacle by applying soft pressure on the mammary

gland. Each sample was immediately frozen at 208C for

later analysis.

2-methylbut-2-enal. This part of the study was specificallycalibrated to only detect the presence of this aldehyde. The

analysis of fresh amniotic fluid, colostrum, and maternal milk

samples was performed with a GC (6890N) gas chromato-graph (Agilent Technologies, Santa Clara, CA) equipped with

a static head space sampler (model 7694E, Agilent Technolo-

gies). The samples were compared with a trans-2-methyl-2-

butenal (2 MB) standard (SigmaAldrich, Mexico, catalog

no. 192 619) at concentrations of 0.1 and 0.05 ng/ml. The

comparison was performed according to the standard reten-

tion time and mass spectra. An amniotic fluid, colostrum, or

maternal milk sample was introduced into a 10 ml vial that

was immediately sealed with a PTFE/Teflon cap. Each vial

was equilibrated at 858C for 20 min in the static head space

sampler. Each vial was pressurized with carrier gas for 6 s,

and the sample was injected into a DB-5 capillary column

(60 m

0.25 mm

0.25 mm film thickness; J&W Scientif-ic, Folson, CA). The injector temperature was set at 2508C,

and helium (1.0 ml/min) was the carrier gas. The oven tem-

perature was maintained at 408C for 9 min and programmed

to 608C at 108/min and then 2808C at 308/min for 3 min. The

mass spectrometer operated in the electron impact ionization

mode (70 eV). The ion source temperature was set at 2808C.

Fatty acids and esterification. From 1 ml of each bio-logical sample (amniotic fluid, colostrum, and milk), we twice

performed a v/v extraction with hexane [high-performance

liquid chromatography (HPLC) grade], followed by alkaline

hydrolysis using 1.0 ml of a solution of 0.5 M NaOH (HPLC

grade) at 808C for 15 min with constant agitation. We then

added 1.0 ml of 14% BF3/MeOH at 808C for 15 min, also

with constant agitation. Methyl esters were extracted using

1.0 ml hexane (HPLC grade), and the hexanic extract was

dried with NaSO4 and filtered for later injection into the gas

chromatograph.

The GC-MS analyses of methyl esters consisted of esterifi-

cation and obtaining a hexanic extract. One microliter of the

samples was injected. The compounds were separated using a

GC-MS Agilent Technologies model 6890N Net Work GC

system equipped with a DB-5 5%-phenyl-methyl polysiloxane

column (Agilent Technologies), 60 m long with a 0.25-mm

internal diameter and 0.25-mm film thickness, with the fol-

lowing temperature program: maintained at 1508C for 5 min,

heated to 2108C at 308C/min, heated from 2108C to 2138C at

18C/min, heated from 2138C to 2258C a t 2 08C/min, and

maintained at 2258C for 40 min, with helium as the carrier

gas at a pressure of 24.91 psi. The total running time was 50

60 min. The temperature of the injector was 2508C, with an

injection split ratio of 50:1.Mass spectra were recorded in electronic impact mode

(70 eV) with a mass range of 50550 amu using an Agilent

Mass Selective Detector 5975 inert XL series. All compounds

were initially identified based on a NIST 05 mass spectral

search program, version 2.0d. For quantitative analysis, we

used a FAME mix standard (C:8-C:22, catalog no. 18920-

lamp, SigmaAldrich, St. Louis, MO) as the external stan-

dard, which was analyzed under the same conditions as

above.

Preparation of solutions for behavioral test. The ana-lytical data from amniotic fluid were used for preparing a

vehicle. Half of the previously obtained amniotic fluid samplewas centrifuged at 3,500 rpm for 10 min in a Sol-Bat J-600

centrifuge (Veracruz, Mexico). The supernatant liquid (ap-

proximately 1 ml) was obtained with a pipette while avoiding

touching the lower portion of the assay tube. For this centri-

fuged amniotic fluid, the fatty acid content was measured by

GC-MS as described above.

Statistical Analysis. For the analytical part of the study,we used two-way repeated-measures analysis of variance

(RM-ANOVA), with fluid (amniotic fluid, colostrum, and ma-

ternal milk) as the first factor and fatty acids as the second

factor. Values of p < 0.05 were considered statistically signif-

icant, in which case the Holm-Sidak post hoc test was applied(SigmaStat 3.1). An analysis of the content profile of fatty

acids in each fluid was also performed by pooling individual

amounts of fatty acids from the 12 volunteers included in this

part of the study. The fatty acid content of fresh amniotic

fluid and centrifuged amniotic fluid, determined by GC-MS,

was compared as described above, the results of which were

compared by two-way ANOVA, with the procedure (fresh or

centrifuged) as the first factor and fatty acid content as the

second factor. Significant results in the ANOVA (p 0.05)

were followed by the Holm-Sidak post hoc test. The results

are expressed as mean SE.

Developmental Psychobiology Possible Role of Fatty Acids 223


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Experiment 2. Behavioral Test

Mother Volunteers and Newborns. We obtained written con-

sent from 19 mothers to include their babies in the study. The

mean age of the mothers was 23.9 years (1.25 years; range,

1537 years), and all participants were in optimal health. A

brief interview was conducted to exclude participants previ-

ously diagnosed with any psychiatric or neurologic pathology.The participants were right-handed, and none were smokers.

Regarding deliveries, seven were by cesarean section, and 12

were by the vaginal route.

The newborn infants included in the behavioral study

(n 19) were selected using the following criteria: gestation-

al age !37 weeks, birth weight appropriate for the gestational

age (median birth weight, 3,390 81.4 g; range, 2,700

4,550 g), Apgar score 8.3 0.12 to 9.1 0.08 immediately

and 5 min after delivery, respectively, no need for resuscita-

tion or intensive care, the absence of neonatal pathologies or

congenital malformations, a Silverman score of zero, and an

average Caopurro score of 39.5 0.16. Newborns were test-

ed approximately 22.0 h after delivery (1.21 h; range, 18

30 h). After colostrum secretion was visually confirmed by a

pediatrician, all newborns fed from the maternal breast

15 min before the behavioral test. None of the newborns re-

ceived any artificial milk formulation or bottle feeding before

the tests.

In addition to the baby and the mother, three researchers

were in the examination room. One researcher was tasked

with applying the stimuli, another researcher performed the

videorecording, and a third researcher coordinated the activi-

ties (i.e., the sequence of stimuli and explaining the proce-

dures to the mother). Only one of the researchers was near

the baby, at a distance necessary to apply the stimuli while

not touching the baby. The other researchers and mother were

at least 2 m away from the baby, remaining as silent and sta-tionary as possible.

Olfactory Stimuli. The main olfactory stimulus was fresh

amniotic fluid obtained as described above, with each new-

born receiving its own fresh amniotic fluid. Based on the

results from the analytical study of amniotic fluids, we pre-

pared an artificial mixture of fatty acids (all analytical grade

fatty acids; SigmaAldrich) in 100 ml of vehicle (96% pro-

pylene glycol and 4% ethanol) at a temperature


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statistical analyses. Categories of positive and negative facial

reactions have been previously described by others (Schaal

et al., 2000) who used similar experimental designs.

The AUs that reflected negative facial actions included

AU4 (brow lowering), AU9 (nose wrinkling), AU10 (upper

lip raising), AU15 (lip corner depressing), AU20 (lip stretch-

ing), and AU26/AU27 (gaping). Acceptance mouthing (posi-

tive facial actions; that is, appetitive reactions; e.g., sucking,

licking, munching, and chewing) included the following cod-

ed facial reactions: AU18 (lip puckering), AU19 (tongue

showing), AU25 (lips parting), and AU28 (lips sucking).

Statistical Analysis. The duration of positive and negative fa-

cial reactions during the 30-s period were summed. We

used two-way RM-ANOVA, with the coded facial reaction

(positive or negative) as the first factor and fluid (fresh amni-

otic fluid, centrifuged amniotic fluid, fatty acid mixture, or

propylene glycol) as the second factor. Values of p 0.05

were considered statistically significant, in which case the

Holm-Sidak post hoc test was applied. The results are

expressed as mean SE of the mean.

RESULTS

Experiment 1. Analytical Study

GC-MS Results. 2-methylbut-2-enal. Using GC-MS,

we did not detect any trace of 2-methylbut-2-enal in

the analyzed fluids. With the standard, we detected a

peak retention time of approximately 10.4 s at a con-

centration around 0.05 ng/ml. This peak verified the

identification of 2-methylbut-2-enal using the GC-MS

library. Therefore, this substance was discarded for thebehavioral tests.

Fatty acids. We detected approximately 20 fatty acids,

but only eight were consistently present in the three

analyzed fluids (Tab. 1): lauric acid (C12:0), myristic

acid (C14:0), palmitic acid (C16:0), palmitoleic acid

(C16:1), stearic acid (C18:0), elaidic acid (C18:1 trans),

oleic acid (C18:1cis), and linoleic acid (C18:2).

The two-way RM-ANOVA of the fluid factor

revealed that the total amount of the eight consistently

present fatty acids was different among the three fluids

(F2,154 22.212, p

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Amniotic Fluid Elicits Appetitive