Micro-anatomic alterations of the placenta in a non-human primate model of gestational protein-restriction

Objectives: Maternal protein malnutrition is associated with
impaired fetal growth, and lifetime consequences for the offspring. Our
group has previously developed a model of protein-restriction in the
non-human primate, which was associated with fetal growth restriction,
stillbirth, decreased placental perfusion, and evidence of fetal hypoxia,
suggesting perturbed vascular development. Previous assessments
of placental vasculature have relied upon stereological or
vascular casting methods, but these methods have limitations. Our
objective was to histologically characterize the micro-anatomic
alterations associated with adverse pregnancy outcomes using a newer
method that permits investigation of the 3D vascular structure and
surrounding histology. Methods: Rhesus macaques were assigned in
the pre-gestational period to a control diet that contained 26% protein,
or study diet containing 13% protein (50% PR diet). Placental tissue was
collected at delivery and processed using a clarification,
immunohistochemistry, and confocal microscopy protocol published
previously by our group. 3-dimensional reconstructions and quantitative
analysis of the vascular micro-anatomy was performed using analysis
software (Imaris®) and statistical analysis incorporated maternal,
pregnancy, and perinatal outcomes. Results: In unadjusted
analysis, when comparing those pregnancies on a 50% PR diet (n=4) with
those on a control diet (n=4), protein-restriction diet was associated
with decreased maternal pre-pregnancy weight (difference of -1.975kg, 95%
CI -3.267 to -0.6826).  When controlling for maternal
pre-pregnancy weight, fetal sex, and latency from tissue collection to
imaging, a gestational protein-restriction diet was associated with
decreases in total vascular length, total vascular surface area, total
vascular volume, and vascular density. Conclusion: In this pilot
study, a gestational protein-restriction diet was associated with changes
in the placental micro-vasculature, which may be related to the observed
adverse pregnancy outcomes and perturbed placental perfusion demonstrated
in this model.

研究目的：母体蛋白质营养不良与胎儿生长受损及子代终生健康结局密切相关。本团队此前已构建非人灵长类（non-human primate）蛋白质限制模型，该模型可出现胎儿生长受限、死胎、胎盘灌注降低及胎儿缺氧征象，提示血管发育异常。既往对胎盘脉管系统的评估多依赖立体学（stereological）或血管铸型方法，但此类方法存在局限性。本研究旨在采用一种可同时观测三维血管结构及周围组织学特征的新型方法，对不良妊娠结局相关的显微解剖改变进行组织学表征。

研究方法：妊娠前将恒河猴（Rhesus macaques）随机分为两组，对照组给予蛋白质含量26%的常规饮食，干预组给予蛋白质含量13%的饮食（即50%蛋白质限制(protein-restriction, PR)饮食）。分娩时采集胎盘组织，采用本团队此前发表的澄清处理、免疫组织化学（immunohistochemistry）及共聚焦显微镜（confocal microscopy）方案进行样本制备。使用分析软件Imaris®完成三维重建及血管显微解剖结构的定量分析，并结合产妇、妊娠及围产期结局数据进行统计学分析。

研究结果：未校正分析中，对比50% PR饮食组（n=4）与对照组（n=4），蛋白质限制饮食与产妇孕前体重降低相关（体重差异为-1.975kg，95%置信区间(CI)：-3.267至-0.6826）。在校正产妇孕前体重、胎儿性别及组织采集至成像的间隔时间后，妊娠期蛋白质限制饮食与总血管长度、总血管表面积、总血管体积及血管密度降低显著相关。

研究结论：本预实验（pilot study）结果显示，妊娠期蛋白质限制饮食可引起胎盘微血管系统改变，这可能与本模型中观察到的不良妊娠结局及胎盘灌注异常相关。