The differential regulation of placenta trophoblast bisphosphoglycerate mutase in fetal growth restriction: preclinical study in mice and observational histological study of human placenta
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Curated by eLife
eLife assessment
This study presents a valuable finding of the role of under investigated pathway associated with development of placental oxygenation during pregnancy. The evidence supporting the claims of the authors is incomplete, although inclusion of a larger number of patient samples and an animal model have strengthened the study. The work will be of interest to developmental biologists working on placental function.
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Abstract
Fetal growth restriction (FGR) is a pregnancy complication in which a newborn fails to achieve its growth potential, increasing the risk of perinatal morbidity and mortality. Chronic maternal gestational hypoxia, as well as placental insufficiency are associated with increased FGR incidence; however, the molecular mechanisms underlying FGR remain unknown.
Methods:
Pregnant mice were subjected to acute or chronic hypoxia (12.5% O 2 ) resulting in reduced fetal weight. Placenta oxygen transport was assessed by blood oxygenation level dependent (BOLD) contrast magnetic resonance imaging (MRI). The placentae were analyzed via immunohistochemistry and in situ hybridization. Human placentae were selected from FGR and matched controls and analyzed by immunohistochemistry (IHC). Maternal and cord sera were analyzed by mass spectrometry.
Results:
We show that murine acute and chronic gestational hypoxia recapitulates FGR phenotype and affects placental structure and morphology. Gestational hypoxia decreased labyrinth area, increased the incidence of red blood cells (RBCs) in the labyrinth while expanding the placental spiral arteries (SpA) diameter. Hypoxic placentae exhibited higher hemoglobin-oxygen affinity compared to the control. Placental abundance of Bisphosphoglycerate mutase (BPGM) was upregulated in the syncytiotrophoblast and spiral artery trophoblast cells (SpA TGCs) in the murine gestational hypoxia groups compared to the control. Hif1α levels were higher in the acute hypoxia group compared to the control. In contrast, human FGR placentae exhibited reduced BPGM levels in the syncytiotrophoblast layer compared to placentae from healthy uncomplicated pregnancies. Levels of 2,3 BPG, the product of BPGM, were lower in cord serum of human FGR placentae compared to control. Polar expression of BPGM was found in both human and mouse placentae syncytiotrophoblast, with higher expression facing the maternal circulation. Moreover, in the murine SpA TGCs expression of BPGM was concentrated exclusively in the apical cell side, in direct proximity to the maternal circulation.
Conclusions:
This study suggests a possible involvement of placental BPGM in maternal-fetal oxygen transfer, and in the pathophysiology of FGR.
Funding:
This work was supported by the Weizmann Krenter Foundation and the Weizmann – Ichilov (Tel Aviv Sourasky Medical Center) Collaborative Grant in Biomedical Research, by the Minerva Foundation, by the ISF KillCorona grant 3777/19.
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Author Response
Reviewer #1 (Public Review):
Here the authors sought to understand how BPGM/2,3-BPG levels are involved in adaptive responses to hypoxia and whether they are involved in fetal growth restriction. In the current state, I find the data to be confusing and lacking in mechanistic data to justify that increased BPGM is an adaptive response to hypoxia. While the authors find increased staining for the enzyme BPGM in SpA-TGCs after hypoxia, they did not assess 2,3-BPG in cord blood. This would show that increased enzymatic levels have a downstream impact. MRI experiments assessing placental and fetal haemoglobin-oxygenation, showed no differences. Human FGR samples, however, showed reduced 2,3-BPG in cord blood. Further evidence is required to show hypoxia increases BPGM as a compensatory mechanism to permit adequate 2,3-BPG …
Author Response
Reviewer #1 (Public Review):
Here the authors sought to understand how BPGM/2,3-BPG levels are involved in adaptive responses to hypoxia and whether they are involved in fetal growth restriction. In the current state, I find the data to be confusing and lacking in mechanistic data to justify that increased BPGM is an adaptive response to hypoxia. While the authors find increased staining for the enzyme BPGM in SpA-TGCs after hypoxia, they did not assess 2,3-BPG in cord blood. This would show that increased enzymatic levels have a downstream impact. MRI experiments assessing placental and fetal haemoglobin-oxygenation, showed no differences. Human FGR samples, however, showed reduced 2,3-BPG in cord blood. Further evidence is required to show hypoxia increases BPGM as a compensatory mechanism to permit adequate 2,3-BPG and placental-fetal oxygenation levels as the authors claim.
Additional experiments that demonstrate that BPGM is advantageous in the context of hypoxia would strengthen the authors arguments, and would provide a novel mechanism for adaptive responses to hypoxia in the placenta which is highly interesting.
Obtaining cord-blood from mouse embryos and analyzing its 2,3 BPG content is technically not feasible thus we concentrated on the human data only. However note that the dominant physiological effect would be on maternal blood in the placenta, where local elevation of 23BPG can aid in oxygen release.
Reviewer #2 (Public Review):
Summary:
This manuscript will be of interest for investigators in the field of development and the biology of pregnancy. The major strengths of the data are the detailed description of a hypoxia-induced mouse model of fetal growth restriction, where phenotypes, tissue histology, MRI images and metabolic analysis combine to characterize the experimental system. The data seem descriptive and preliminary, and the comparison to human pregnancy is neither supportive nor rigorous.
Strengths
• The mouse pregnancy has been used by the authors and by others as a model for placental insufficiency. The manuscript provides incremental data to characterize hypoxia- induced fetal growth restriction
• The 15.2T MR imaging technology is high quality and informative, even if the results did not reveal marked changes.
• The detailed characterization of BPGM expression in the apical mouse placental surfaces is valuable.
• The provided model may be useful for future studies by the authors.
Weaknesses
• The metabolic analysis was restricted to one enzyme and metabolite. Placental analysis of 2,3-BPG and BPGM were already published (ref 29-30). At best, if the 2,3 BPG is related to the phenotype, it night be interpreted as a part of the injury in human cases, and adaptive response in the mouse models (as the authors suggested lines 286-288 and 332-336.). However, these assumptions are not tested.
In the paper of Pritlove et al. (ref. 29) the authors demonstrated the expression of BPGM in normal human cohort. However, they did not test BPGM expression or 2,3 BPG levels in FGR placentae. In the paper of Gu et al. (ref. 30) the authors analyze murine placental BPGM expression secondary to igf2 deletion. Our study is the first to demonstrate the impact of maternal hypoxia on placental BPGM levels in murine gestational hypoxia models .
• The human cases are not very informative. The causes of FGR were not known, but clearly (Table 1) not analogous to that of the mouse model. Systemic hypoxia in humans might have been more informative. In its absence, the value of cross-species comparison is low. -
• While the provided experiments are of good quality, the approach is very descriptive and not advancing mechanistic understanding of FGR-related placental insufficiency.
The human placenta were specifically selected to exclude known causes of FGR such as heavy smoking or iron deficiency. We will work to expand the diversity of cases to test the potential role of BPGM in those cases as well.
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eLife assessment
This study presents a valuable finding of the role of under investigated pathway associated with development of placental oxygenation during pregnancy. The evidence supporting the claims of the authors is incomplete, although inclusion of a larger number of patient samples and an animal model have strengthened the study. The work will be of interest to developmental biologists working on placental function.
-
Reviewer #1 (Public Review):
Here the authors sought to understand how BPGM/2,3-BPG levels are involved in adaptive responses to hypoxia and whether they are involved in fetal growth restriction. In the current state, I find the data to be confusing and lacking in mechanistic data to justify that increased BPGM is an adaptive response to hypoxia. While the authors find increased staining for the enzyme BPGM in SpA-TGCs after hypoxia, they did not assess 2,3-BPG in cord blood. This would show that increased enzymatic levels have a downstream impact. MRI experiments assessing placental and fetal haemoglobin-oxygenation, showed no differences. Human FGR samples, however, showed reduced 2,3-BPG in cord blood. Further evidence is required to show hypoxia increases BPGM as a compensatory mechanism to permit adequate 2,3-BPG and …
Reviewer #1 (Public Review):
Here the authors sought to understand how BPGM/2,3-BPG levels are involved in adaptive responses to hypoxia and whether they are involved in fetal growth restriction. In the current state, I find the data to be confusing and lacking in mechanistic data to justify that increased BPGM is an adaptive response to hypoxia. While the authors find increased staining for the enzyme BPGM in SpA-TGCs after hypoxia, they did not assess 2,3-BPG in cord blood. This would show that increased enzymatic levels have a downstream impact. MRI experiments assessing placental and fetal haemoglobin-oxygenation, showed no differences. Human FGR samples, however, showed reduced 2,3-BPG in cord blood. Further evidence is required to show hypoxia increases BPGM as a compensatory mechanism to permit adequate 2,3-BPG and placental-fetal oxygenation levels as the authors claim. Additional experiments that demonstrate that BPGM is advantageous in the context of hypoxia would strengthen the authors arguments, and would provide a novel mechanism for adaptive responses to hypoxia in the placenta which is highly interesting.
-
Reviewer #2 (Public Review):
Summary:
This manuscript will be of interest for investigators in the field of development and the biology of pregnancy. The major strengths of the data are the detailed description of a hypoxia-induced mouse model of fetal growth restriction, where phenotypes, tissue histology, MRI images and metabolic analysis combine to characterize the experimental system. The data seem descriptive and preliminary, and the comparison to human pregnancy is neither supportive nor rigorous.
Strengths:
• The mouse pregnancy has been used by the authors and by others as a model for placental insufficiency. The manuscript provides incremental data to characterize hypoxia-induced fetal growth restriction
• The 15.2T MR imaging technology is high quality and informative, even if the results did not reveal marked changes.
• The …Reviewer #2 (Public Review):
Summary:
This manuscript will be of interest for investigators in the field of development and the biology of pregnancy. The major strengths of the data are the detailed description of a hypoxia-induced mouse model of fetal growth restriction, where phenotypes, tissue histology, MRI images and metabolic analysis combine to characterize the experimental system. The data seem descriptive and preliminary, and the comparison to human pregnancy is neither supportive nor rigorous.
Strengths:
• The mouse pregnancy has been used by the authors and by others as a model for placental insufficiency. The manuscript provides incremental data to characterize hypoxia-induced fetal growth restriction
• The 15.2T MR imaging technology is high quality and informative, even if the results did not reveal marked changes.
• The detailed characterization of BPGM expression in the apical mouse placental surfaces is valuable.
• The provided model may be useful for future studies by the authors.Weaknesses
• The metabolic analysis was restricted to one enzyme and metabolite. Placental analysis of 2,3-BPG and BPGM were already published (ref 29-30). At best, if the 2,3 BPG is related to the phenotype, it night be interpreted as a part of the injury in human cases, and adaptive response in the mouse models (as the authors suggested lines 286-288 and 332-336.). However, these assumptions are not tested.
• The human cases are not very informative. The causes of FGR were not known, but clearly (Table 1) not analogous to that of the mouse model. Systemic hypoxia in humans might have been more informative. In its absence, the value of cross-species comparison is low.
• While the provided experiments are of good quality, the approach is very descriptive and not advancing mechanistic understanding of FGR-related placental insufficiency. -
Reviewer #3 (Public Review):
The authors analyse the role of bisphosphoglycerate mutase (BPGM), an enzyme unique to erythrocytes and placental cells. The authors assess the role of BPGM in the pathogenesis of fetal growth restriction (FGR).
Strength of the work: The authors have analysed a murine model of hypoxia (acute and chronic) as well as human placental samples.
Impact of the work on the field: FGR is linked to many short- and long-term medical complications. The authors have done important efforts to understand the role of hypoxia and the placenta in the pathogenesis of FGR. The identification of BPGM as a potential link between FGR and adverse intrauterine is relatively novel.
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