# Pathological characterization of female reproductive organs prior to miscarriage induced by Zika virus infection in the pregnant common marmoset

## TL;DR

This study investigates how Zika virus causes miscarriage in pregnant marmosets by analyzing tissue degeneration and hormone levels before pregnancy loss.

## Contribution

The study presents a novel method for predicting and analyzing miscarriage by monitoring progesterone levels and performing pathological analysis just before pregnancy loss.

## Key findings

- Significant degeneration of trophoblast cells at the maternal-fetal interface was observed prior to miscarriage.
- ZIKV NS1 protein was detected in the endometrium and ovaries, suggesting viral impact on reproductive tissues.
- Elevated TNFα levels in amniotic fluid and cleaved-caspase indicate apoptosis contributing to miscarriage.

## Abstract

While Zika virus (ZIKV) infection in pregnant women is known to increase the risk of miscarriage and stillbirth, the mechanism by which ZIKV infection leads to the inability to continue a pregnancy is not clear. In our common marmoset models of ZIKV infection in pregnant individuals, miscarriage was observed in dams infected in the first or second trimester, and preterm delivery was observed in a dam infected in the third trimester. Serum progesterone levels were significantly lower prior to miscarriage or preterm delivery in the infected marmosets. To elucidate the pathology of the placental region just before the onset of ZIKV-induced miscarriage, we newly prepared an infected marmoset in the first trimester of pregnancy and euthanized it when the serum progesterone concentration was markedly reduced. Pathological analysis revealed significant degeneration in cells at the maternal-fetal interface, presumably trophoblasts. Cleaved-caspase was widely observed in the endometrial to placental region, and TNFα at 200 pg/mL was detected in the amniotic fluid, suggesting that apoptosis may progress in the endometrium and placenta, leading to decreased trophoblast function and miscarriage. ZIKV NS1 protein was found sporadically in the cellular degeneration area and widely in the basal layer of the endometrium. Furthermore, the viral protein was frequently detected in the follicles and corpus luteum of the ovary. The developed ZIKV infection model in pregnant marmosets would be useful not only to better understand the mechanism of ZIKV-induced miscarriage but also to analyze the effects of the viral infection on female reproductive tissues.

Although several viruses, including Zika virus (ZIKV), are known to increase the risk of miscarriage upon viral infection, the mechanism by which miscarriage is induced by viral infection is largely unknown. This is partly due to the difficulty of pathological analysis of maternal tissues in the period following viral infection and prior to miscarriage. In this study, we predicted the occurrence of miscarriage by monitoring serum progesterone levels and performed pathological analysis of peri-placental tissues at a time point assumed to be just before miscarriage. This is the first report of trophoblast degeneration prior to miscarriage, suggesting that the experimental method used here is useful for analyzing the pathogenesis of virus infection-related miscarriage. Further immunostaining revealed that ZIKV NS1 was distributed not only in the uterus but also in the ovaries, with particularly pronounced staining of oocytes. Whether ZIKV infection affects female reproductive function should be clarified in the future.