# Application of Chorionic Villus Sampling to Longitudinal Studies in Pregnant Non-Human Primate Models

## TL;DR

Researchers developed a safe method to collect placental samples from pregnant monkeys using ultrasound-guided biopsies, enabling longitudinal studies without harming the animals.

## Contribution

A minimally invasive, ultrasound-guided chorionic villus sampling technique was developed for longitudinal placental research in non-human primates.

## Key findings

- Ultrasound-guided needle biopsy produced high-quality placental tissue suitable for histology and RNA analysis.
- Serial sampling did not cause injury or early pregnancy loss in rhesus macaques.
- Trophoblast organoids were successfully isolated from biopsy samples, reducing reliance on surgical delivery.

## Abstract

This study addresses the limited ability to examine placental health during an ongoing pregnancy, restricting the understanding of how complications may arise from abnormal placental development or function over time. The objective was to trial a minimally invasive method for obtaining serial placental samples from pregnant rhesus monkeys using ultrasound guidance and a fine needle biopsy technique. A realistic training simulation model was developed for personnel to practice the technique before performing sample collection from live animals. The results showed that the tissue samples obtained by needle biopsy were of sufficient quality for detailed examination, including evaluation of tissue structure and genetic material, as well as placental cell culture in a laboratory setting. Importantly, repeated sampling did not cause injury or early pregnancy loss. These findings demonstrate a safe approach for pregnancy research in a clinically relevant animal model that improves scientific knowledge and maximizes animal involvement through serially sampling across the course of pregnancy, while supporting improved outcomes for maternal and fetal health.

The rhesus macaque (Macaca mulatta) is a valuable model for pregnancy research due to its physiological similarity to humans and the ability to conduct studies in a controlled environment. Our previous work used non-invasive imaging methods to assess placental hemodynamics across gestation with correlative tissue analysis post-delivery. Here, we expand access to longitudinal timepoints from ongoing pregnancies by obtaining placental biopsies using ultrasound-guided needle aspiration. This approach aligns with New Approach Methods (NAMs) and supports animal welfare by reducing the number of animals required. We describe a chorionic villus sampling (CVS) simulation model which facilitates training to gain proficiency in technical skills prior to performing the procedure on animals. We report outcomes from three rhesus macaques that underwent CVS three times between gestational days 40 to 106 (term: 165 days). Although biopsy samples are smaller than whole placenta, tissue yields were sufficient for multiple uses. We demonstrate (1) appropriate histology from aspirated samples, (2) good RNA quality and yield, and (3) the ability to isolate trophoblast organoids, an advancement in NAMs that reduces the need for first-trimester surgical delivery. No spontaneous preterm delivery occurred following serial CVS procedures, supporting the use of this sampling method to maximize animal utilization in longitudinal pregnancy studies.