Hypoxia-inducible factor 1 signaling drives placental aging and can provoke preterm labor

Reviewer #1 (Public Review):

Ciampa et al. investigated the role of the hypoxia-inducible factor 1 (HIF-1) pathway in placental aging. They performed transcriptomic analysis of prior data of placental gene expression over serial timepoints throughout gestation in a mouse model and identified increased expression of senescence and HIF-1 pathways and decreased expression of cell cycle and mitochondrial transcripts with advancing gestational age. These findings were confirmed by RT-PCR, Western blot, and mitochondrial assessment from mouse placental tissues from late gestation time points. Studies of human placental samples at similar late gestational ages showed similar trends in increased HIF-1 targets and decreased mitochondrial abundance with increasing gestation, but were not significantly significant due to the limited availability of uncomplicated preterm placenta samples. The authors demonstrated that stabilization of HIF-1 in vitro using primary trophoblasts and choriocarcinoma cell lines recapitulated the gene and mitochondrial dysfunction seen in the placental tissues and were consistent with senescence. Interestingly, cell-conditioned media from HIF-1 stabilized placenta cell lines induced myometrial cell contractions in vitro and correspondingly, induction of HIF-1 in pregnant mice was associated with preterm labor in vivo. These data support the role of the HIF-1 pathway in the process of placental senescence with increasing gestational age and highlight this pathway as a potentially important contributor to gestational length and a potential target for therapeutics to reduce preterm birth.

Overall, the conclusions of this study are mostly well supported by the data. The concept of placental aging has been controversial, with several prior studies with conflicting viewpoints on whether placental aging occurs at all, is a normal process during gestation, or rather only a pathologic phenomenon in abnormal pregnancies. This has been rather difficult to study given the difficulty of obtaining serial placental samples in late gestation. The authors used both a mouse model of serial placental sampling and human placental samples obtained at preterm, but non-pathologic deliveries, which is an impressive accomplishment as it provides insight into a previously poorly understood timepoint of pregnancy. The data clearly demonstrate changes in the HIF-1 pathway and cellular senescence at increasing gestational ages in the third trimester, which is consistent with the process of aging in other tissues.

Weaknesses of this study are that although the authors attribute alterations in HIF-1 pathways in advanced gestation to hypoxia, there are no experiments directly assessing whether the changes in HIF-1 pathways are due to hypoxia in either in vitro or in vivo experiments. HIF-1 has both oxygen-dependent and oxygen-independent regulation, so it is unclear which pathways contribute to placental HIF-1 activity during late gestation, especially since the third-trimester placenta is exposed to significantly higher oxygen levels compared to the early pregnancy environment. Additionally, the placenta is in close proximity to the maternal decidua, which consists of immune and stromal cells, which are also significantly affected by HIF-1. Although the in vitro experimental data in this study demonstrate that HIF-1 induction leads to a placenta senescence phenotype, it is unclear whether the in vivo treatment with HIF-1 induction acts directly on the placenta or rather on uterine myometrium or decidua, which could also contribute to the initiation of preterm labor.

Reviewer #2 (Public Review):

The authors sought to characterize normal placental aging to better understand how the molecular and cellular events that trigger the labor process. An understanding of these mechanisms would not only provide insight into term labor, but also potential triggers of preterm labor, a common pregnancy complication with no effective intervention. Using bulk transcriptomic analysis of mouse and human placenta at different gestational timepoints, the authors determined that stabilization of HIF-1 signaling accompanied by mitochondrial dysfunction and cellular senescence are molecular signatures of term placenta. They also used in vitro trophoblast (choriocarcinoma) and a uterine myocyte culture system to further validate their findings. Lastly, using chemically induced HIF-1 induction in vivo in mice, the authors showed that stabilization of HIF-1 protein in the placenta reduced the gestational length significantly.

The major strength of this study is the use of multiple model systems to address the question at hand. The consistency of findings between mouse and human placenta, and the validation of mechanisms in vitro and in vivo modeling are strong support for their conclusions. The rationale for studying the term placentas to understand the abnormal process of preterm birth is clearly explained. Although the idea that hypoxic stress and placental senescence are triggers for labor is not novel, the comprehensiveness of the approach and idea to study the normal aging process are appreciated.

There are some areas of the manuscript that lack clarity and weaknesses in the methodology worth noting. The rationale for focusing on senescence and HIF-1 is not clearly given that other pathways were more significantly altered in the WGCNA analysis. The placental gene expression data were from bulk transcriptomic analyses, yet the authors do not explicitly discuss the limitations of this approach. Although the reader can assume that the authors attribute the mRNA signature of aging to trophoblasts - of which, there are different types - clarification regarding their interpretation of the data and the relevant cell types would strengthen the paper. Additionally, while the inclusion of human placenta data is a major strength, the differences between mouse and human placental structure and cell types make highlighting the specific cells of interest even more important; although there are correlations between mouse and human placenta, there are also many differences, and the comparison is further limited when considering the whole placenta rather than specific cell populations.

Additional details regarding methods and the reasons for choosing certain readouts are needed. Trophoblasts are sensitive to oxygen tension which varies according to gestational age, and it is unclear if this variable was taken into consideration in this study. Many of the cellular processes examined are well characterized in the literature yet the rationale for choosing certain markers is unclear (e.g., Glb1 for senescence; the transcripts selected as representative of the senescence-associated secretory phenotype; mtDNA lesion rate).

Overall, the findings presented are a valuable contribution to the field. The authors provide a thoughtful discussion that places their findings in the context of current literature and poses interesting questions for future pursuit. Their efforts to be comprehensive in the characterization of placental aging is a major strength; few placental studies attempt to integrate mouse and human data to this extent, and the validation and presentation of a potential mechanism by which fetal trophoblasts signal to maternal uterine myocytes are exciting. Nevertheless, a clear discussion of the methodologic limitations of the study would strengthen the manuscript.

Reviewer #3 (Public Review):

In this study, Ciampa and colleagues demonstrate that HIF-1α activity is increased with gestation in humans and mice placentas and use several in vitro models to indicate that HIF activation in trophoblasts may release factors (yet to be identified) which promote myometrial contraction. Previous studies have linked placental factors to the preparation of the myometrium for labour (e.g. prostaglandins), but HIF-1α has not been implicated. Due to several issues regarding the experimental design, the results do not currently support the conclusions.

Major concerns:

1. The hypothesis states that placental aging promotes parturition via HIF-1a activation, the study does not provide any evidence of an aged placenta. Aging is considered a progressive and irreversible loss of functional capacity, inability to maintain homeostasis, and decreased ability to repair the damage. The placenta retains all these abilities throughout pregnancy [PMID: 9462184], and there's no evidence that the placenta functionally declines between 35-39 weeks, otherwise, it wouldn't be able to support fetal development. However, there is evidence of a functional decline in post-term placentas (i.e. >40 weeks in humans) but the authors compare preterm placentas with E17.5 mice placentas or 39-week human placentas, both these gestational periods are prior to the onset of parturition in most pregnancies (human = 40wkGA, mice=E18.5).

2. While the authors provide evidence that HIF-1α activity increases in both the human and mice placenta as gestation progresses, the mechanistic link between placental HIF-1α and parturition is not strongly supported. For example, most of the evidence is based on in vitro studies showing that conditioned media from trophoblasts treated with CoCl2 increased the contraction of myometrial cells. The specific factor responsible was not identified but the authors allude to pro-inflammatory factors such as cytokines. It was therefore interesting to note that the conditioned media had undergone a filtration step that removes all substances >10kDa, which includes the majority of cytokines and hormones.

3. An alternative explanation is that CoCl2 treatment-induced trophoblast differentiation and the effects on myometrial contraction may be related to differences in secreted factors produced by cytotrophoblasts versus syncytiotrophoblast. Although JAR cells do not spontaneously differentiate, they can be induced to syncytialise upon cAMP stimulation. Ref 39 the authors cite shows this. Indeed, the morphology of the cells in Fig5F that were exposed to CoCl2 indicates that they may be syncytialised. Syncytialised trophoblasts also express markers of senescence including increased SA-β-gal activity and reductions in mitochondrial activity.

4. The in vivo experiment showing reduced gestation length in pregnant mice receiving DMOG injection is interesting. However, we cannot exclude the effects of DMOG on non-placental tissues (both maternal and fetal) or the non-specific effects of DMOG (i.e. HIF-1α independent). Furthermore, previous studies using a more direct approach to alter HIF-1α activity in the placenta using trophoblast-specific overexpression of HIF-1α in mice do not lead to changes in gestation length [PMID: 30808910].

5. Lack of appropriate experimental models. E.g. JAR choriocarcinomas are not an ideal model of the human trophoblast as they are malignant. Much better models are available e.g. primary human trophoblasts from term placentas or human trophoblast stem cells from first-trimester placentas. Similarly, the mouse model is also not specific as discussed above.

6. Lack of cohesion between the different experimental models. E.g. CoCl2 was used to induce hypoxia/HIF1α in mouse TBs, but DMOG was used in vivo in mice. SA-β Gal staining was carried out in cells but not in mouse or human tissues.

7. Evidence of senescence and mitochondrial abundance could be strengthened by providing additional markers. E.g. only GLB1 mRNA expression is provided as evidence of senescence, and COX IV protein for mitochondrial abundance in mouse and human placentas.

8. Given that the main goal of this study was to investigate the role of hypoxia, hypoxia (i.e. low oxygen) was never directly induced and the results were based on chemical inducers of HIF-1α which have multiple off-target effects.