Effect of a High Estrogen Level in Early Pregnancy on the Development and Behavior of Marmoset Offspring

Abstract

The use of assisted reproductive technology (ART) has risen steadily worldwide over the past 3 decades and helps many infertile families. However, ART treatments lead to an abnormal internal environment in the uterus, which may increase the risks of health problems for the offspring. Higher maternal estradiol (E₂) is a notable feature in women who use ART treatments, and this has been suggested as a key factor for the risk of diseases in the offspring. In the current study, we have established a marmoset model with a high E₂ level in early pregnancy to examine its potential risk to the development and behavior of the offspring. In comparison with the normal group, babies of the high E₂ group exhibited lower average survival rates and birth weights. However, those who survived in the high E₂ group demonstrated normal vocal production with rich call repertoires, normal speed during locomotion, and normal behaviors in the home cage. In contrast to the normal group, surviving babies of the high E₂ group spent more time sleeping during development without signs of sleep disorders. In summary, our study revealed that high estrogen in early pregnancy may cause low survival rates and birth weights of the offspring, though the surviving infants did not show obvious behavioral deficiencies during development. The current study is a valuable and highly important non-human primate study for evaluating the safety of ART treatments. However, it is worth noting that some results did not reach the significant level, which may be due to the small sample size caused by animal shortage stemming from the COVID-19 epidemic.

Figure 1

Establishment of the marmoset model with a high E₂ level at early pregnancy. (A) Experiment design for the establishment of the high E₂ marmoset model at early pregnancy. Female marmosets were administered with 800 μg/kg of estradiol valerate per day for 45 days starting from 10 to 20 days after pregnancy (E10–20). If the high E₂ treatment was started at embryonic day 10 (E10), it was finished at embryonic day 55 (E55). The gestation period of the common marmoset is generally 145 days. E145, embryonic day 145. (B) Average serum E₂ levels during early pregnancy in the high E₂ group (high E₂, n = 3 animals) and the normal group (normal, n = 5 animals). Results are expressed as means ± SE. (P = 0.0005, rank-sum test). ***P < 0.001. (C) Head restraint device for blood collection from awake adult marmosets.

Figure 2

Physical measurements of baby marmosets produced by normal and high E₂ females. (A) Birth weight of marmoset triplets produced by female marmosets in normal and high E₂ groups (babies of normal females, n = 21 and babies of high E₂ females, n = 3), respectively. (B) Body length of newborn marmosets produced by normal and high E₂ females (babies of normal females, n = 5 and babies of high E₂ females, n = 3). (C) Head circumference of newborn marmosets produced by normal and high E₂ females (babies of normal females, n = 5 and babies of high E₂ females, n = 3). (D) Crown–rump length of newborn marmosets produced by normal and high E₂ females (babies of normal females, n = 5 and babies of high E₂ females, n = 3). The data in B–D were collected from the same animals. (E) Body weight of marmoset babies (1 month old) produced by female marmosets in normal and high E₂ groups (babies of normal females, n = 12 and babies of high E₂ females, n = 3). (F) Body weight of marmoset babies (3 months old) produced by female marmosets in normal and high E₂ groups (babies of normal females, n = 4 and babies of high E₂ females, n = 3). Results are expressed as means ± SE. *P < 0.05.

Figure 3

Vocal production of the offspring produced by normal and high E₂ female marmosets. (A) Different types of marmoset calls recorded from newborn marmosets at postnatal day one (P1). M, L, O, S, T, U, AH, and AK are animal IDs. Phee, trill-phee, twitter, trill, tsik, ekk, cry, compound-cry, and sub-harmonics are different types of marmoset calls. √ indicates that this type of call was recorded from the animal listed. Blank indicates that the animal did not produce this type of call during vocal recordings. (B) Spectrograms of marmoset calls (trill, phee, trill-phee, and twitter) recorded from newborn offspring of normal and high E₂ females. Left panel, calls produced by the offspring of normal female (normal); right panel, calls produced by the offspring of high E₂ female (high E₂). (C) Average number of call types emitted by offspring of normal and high E₂ females (offspring of normal females, n = 6 and offspring of high E₂ females, n = 2).

Figure 4

Free movement in the home cage recorded from the offspring of normal and high E₂ female marmosets. (A) Locomotion trajectories (blue traces) of marmoset offspring (8 months old) produced by normal and high E₂ females. The videos were captured using a high-definition video camera, which was positioned in front of the animal cage to record the animal’s daily activities in the cage without external disturbance. For each animal, the recording was performed for 10 min per day for 5 times. (B) Average speed of marmoset offspring (8 months old) produced by normal and high E₂ females (offspring of normal females, n = 3 and offspring of high E₂ females, n = 3). For each animal, the locomotion speed was calculated by the distance traveled per second and averaged from 5 day recordings. (C) Time staying on the top half of the animal cage in marmoset offspring (8 months old) produced by normal and high E₂ females (offspring of normal females, n = 3 and offspring of high E₂ females, n = 3). For each animal, the time staying on the top half of the cage was averaged from 5 day recordings. The staying time on the top was usually used as one behavioral index of marmosets for stress. (B,C) were calculated from the same video recordings. Results are expressed as means ± SE.

Figure 5

The offspring of the high E₂ females slept longer and displayed more naps. (A) Examples showing the daily activity and sleep patterns of the offspring produced by the normal (Left) and high E₂ female marmosets (Right) captured using a Mini ActiWatch. The data were continuously collected for 10–14 days from animals at the age of 9–12 months old. The sleep phase is indicated by a black bar and the awake phase is indicated by a white bar. Daytime: 8 AM to 8 PM, orange bar. Nighttime: 8 PM to 8 AM, blue bar. (B) Average sleep time per day for the offspring of normal and high E₂ marmosets (offspring of normal females, n = 6 and offspring of high E₂ females, n = 3). For each animal, the sleep time was defined as the total sleep time in 24 h and was averaged over 10–14 days during recordings. (C) Sleep bouts during the night time (8 PM to 8 AM) for the offspring of normal and high E₂ marmosets (offspring of normal females, n = 6 and offspring of high E₂ females, n = 3). (D) Sleep efficiency of the offspring produced by normal and high E₂ marmosets (offspring of normal females, n = 6 and offspring of high E₂ females, n = 3). Sleep efficiency is defined as the proportion (%) of time that animals spent sleep during the night. (E) Naps during daytime for the offspring of normal and high E₂ marmosets (offspring of normal females, n = 6 and offspring of high E₂ females, n = 3). (F) Interdaily stability showing the variation in the sleep time and sleep phase across the recording days (10–14 days) for the offspring of normal and high E₂ marmosets (offspring of normal females, n = 6 and offspring of high E₂ females, n = 3). (G) Fragmentation index showing the restlessness state of the offspring of normal and high E₂ marmosets (offspring of normal females, n = 6 and offspring of high E₂ females, n = 3). B–G was calculated from the same sleep data set. Results in B–G are expressed as means ± SE. *P < 0.05.