The Human Placenta Project

Cervical trophoblast: A proxy of placental health and function in early pregnancy

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There is a clear gap in our ability to investigate the placenta and pregnancy risk in the first trimester of pregnancy on a cellular level.

It is well established that protein expression is different in placental cells of pregnancies that developed placental disorders including severe preeclampsia (PE). This idea exploits the fact that the placenta naturally sheds hundreds of trophoblast cells into the cervical canal from the 5th through the 20th week of gestation (1). These cells can be safely collected with a cytology brush and analyzed as an entirely new window into very early pregnancy.β€―β€―This project could develop a fast and reliable technique to screen placental trophoblasts shed into the cervical canal (specifically, cervical extravillous trophoblasts; cEVTs) for 1st trimester signatures that are warning signs of placental disorders including severe preeclampsia.

Our experience with over 3,500 enrolled patients and cEVT collections and the work of others has resulted in published data on: 1) the safety of cEVT sampling during pregnancy (2-5); 2) sampling success of hundreds of cEVTs isolated routinely across 5-20 wks of GA (6-11); 3) cervical trophoblast biology (12, 13); and 4) protein changes in abnormal pregnancies (7, 😎. Collectively, our pilot data and the work of others shows that specific placental trophoblast cells can be isolated and are available for molecular investigation.

Based on published data, trophoblast cells are a prime target to investigate cell based placental function in early pregnancy. It requires further evaluation if these cells could be widely used to understand placental biology, development and disease. We have been speer heading this notion for several years successfully and believe a more concentrated approach in this area could lead to major breakthroughs in the field.

The potential diagnostic value of analyzing isolated cEVTs to predict placental disorder development. The placenta and its various cell types are directly responsible for pregnancy success. In particular, the two main trophoblast lineages, the extra-villous (EVT) and villous trophoblasts, are crucial for proper placental function (14). EVTs are needed for successful implantation and promotion of blood flow, and are key target cells because impaired utero-placental blood flow is central to the most common placental disease (maternal-vascular malperfusion) underpinning 80% of sPE pregnancies (15). During a healthy pregnancy, EVTs invade into the uterine wall and participate in spiral artery remodeling to secure blood, nutrients and O2 for the placenta/fetus (16, 17). If unsuccessful, the villous trophoblasts responsible for fetal/maternal exchange will not work properly, resulting in chronic ischemia-reperfusion injury to the developing placental villi. Kim et al demonstrated clear lack of EVT function in sPE placentas: only 4.3% of the spiral arteries were completely remodeled in preeclamptic placental bed biopsies, compared to 59% in healthy women who delivered at term (1😎. This observation underscores the potential diagnostic value of analyzing isolated EVTs to predict the development of placental rooted preeclampsia and other disorders. Hundreds of cervical EVTs (cEVTs) can be collected in a single sample, because these cells are naturally shed in large numbers until 20 wks. The collection process is both safe and acceptable to women, who in tandem do need inspection of their cervix and a PAP test as part of their comprehensive prenatal care (6-11). Analyzing these cEVTs is the central focus of this project idea.

Understanding the biology and information carried by the cervical and other trophoblast cells can provide important information on the placenta and pregnancy health if thoroughly investigated (6-11). Including but not limited to -

1. Have a cell specific window into placental function in the first trimester and beyond.

2. Understand the impact on these cells being removed from the implantation site.

3. Provide a safe access to ex-placental material in the first trimester.

4. Allows comparisons to pregnancy outcomes and the understand the potential relevance for placental disease.

5. Can be used to support other screening methods to increase specificity in the first trimester to detect at risk pregnancies currently impossible.

6. Cell numbers are ideal for single cells and low input material for various omics approaches.

7. It targets the trophoblast cells known to be responsible for the placental dysfunction and problems during pregnancy.

Do the cells provide carry information into the cervical canal that is relevant to placental health?

The answer is: Yes ! they absolutely do. Although changes in the cells traveling through the cervical canal are likely and expected, they retain information that is relevant to placental function and pregnancy outcome as we have published in two pilot studies (7, 😎. The data shows that circulating proteins commonly used for placental assessment in second and third trimester (PLGF, PP13 and others) are already changed as early as five weeks of pregnancy, before these changes can be reliably measured in the blood. This is not surprising if the placenta is the source of these and other molecules.

Current status and challenges to detect placental disease in early pregnancy:

Limitations in 1st trimester screening methods are a major reason the root causes of sPE problems are unknown. Consequently, clinicians can only use clinical/empirical methods for risk assessment. At present they only have one tool (low-dose aspirin) to lower sPE risk, with no insight into the favorable mechanism of action (19). Besides sPE, a wide range of placenta-mediated pregnancy complications are largely undetected until overt manifestations are observed; yet, these disorders may also be amenable to trophoblast cell screening in early pregnancy. Lack of screening precision in early pregnancy is the result of current methods (ultrasound and circulating blood biomarkers) not being directly aligned to the underlying disease pathogenesis (20). This project proposes a solution to this major limitation in 1st trimester screening methods.

Ultrasound and MRI can identify placental structure and perfusion problems β€” but have major limitations in the 1st trimester. Abnormal structural (shape, form, location) and blood flow changes are measurable in later gestation by ultrasound and MRI, and can indicate potential placental problems leading to pregnancy complications (21-24). But, in the 1st trimester, resolution is a major limitation to ultrasound and MRI due to small placental size. Thus, prenatal ultrasound and MRI have limited predictive value in the 1st trimester, even when combined with blood biomarkers (20, 25, 26). CyTOF analysis of cEVTs will overcome these limitations with molecular cell-based data and opens the door for detecting warning signs of high-risk pregnancy earlier than ever before.

Biomarkers released by placental villi into the maternal blood are not informative before the 2nd trimester when utero-placental blood flow to the inter-villous space of the placenta is established. Due to reduced maternal blood supply, sPE placentas possess major gross abnormalities of the villous surface that are characterized by focal areas of trophoblast necrosis or apoptosis (27). Aberrant histology at the feto-maternal interface, and abnormal villous development, occurs in 41% and 60% of such placentas, respectively (2😎. Due to reduced blood flow, sPE placentas also show evidence of villous trophoblast hypoxia and increased production of proteins such as soluble fms-like tyrosine kinase 1 (sFLT1) and Endoglin (ENG), with repressed placental growth factor (PLGF) which is required for host adaption to pregnancy (29-31). These circulating biomarkers are easily detected in the maternal circulation, but are only empiric signals, with low screening precision and significant variation by GA (26).

Quantifying circulating placental protein biomarkers has limited predictive value in the 1st trimester because the biomarker concentrations are too low. Combined with ultrasound, biomarkers can inform about the risk of abnormal placentation and PE. However, no existing blood assay is established for screening purposes in the 1st trimester. This is because, early in pregnancy while the placenta is still small, contact to the maternal circulation is limited. This, and considerations such as BMI, greatly restrict the screening value of "downstream" biomarkers in maternal blood despite arising from placental structures. We will avoid this limitation by providing cEVT-based CyTOF data between 6-20 wks of GA.

Will the cervical environment affect cEVTs and could they produce misleading data unrelated to early signs of high-risk pregnancy? It will need to be addressed in all projects if there is the possible effect of the cervix. However, our published data shows that the cervical environment will not cause misleading results (7, 9).

References:

1. Moser G, Drewlo S, Huppertz B, Armant DR. Trophoblast retrieval and isolation from the cervix: origins of cervical trophoblasts and their potential value for risk assessment of ongoing pregnancies. Hum Reprod Update. 2018;24(4):484-96. Epub 2018/04/03. doi: 10.1093/humupd/dmy008. PubMed PMID: 29608700; PMCID: PMC6016716.

2. Orr JW, Jr., Barrett JM, Orr PF, Holloway RW, Holimon JL. The efficacy and safety of the cytobrush during pregnancy. Gynecol Oncol. 1992;44(3):260-2. Epub 1992/03/11. PubMed PMID: 1541438.

3. Rivlin ME, Woodliff JM, Bowlin RB, Moore JL, Jr., Martin RW, Grossman JH, 3rd, Morrison JC. Comparison of cytobrush and cotton swab for Papanicolaou smears in pregnancy. J Reprod Med. 1993;38(2):147-50. Epub 1993/02/01. PubMed PMID: 8445608.

4. Paraiso MF, Brady K, Helmchen R, Roat TW. Evaluation of the endocervical cytobrush and cervex-brush in pregnant women. Obstet Gynecol. 1994;84(4):539-43. Epub 1994/10/01. PubMed PMID: 8090390.

5. Foster JC, Smith HL. Use of the Cytobrush for Papanicolaou smear screens in pregnant women. J Nurse Midwifery. 1996;41(3):211-7. Epub 1996/05/01. doi: 10.1016/0091-2182(96)00013-4. PubMed PMID: 8708804.

6. Bolnick JM, Kilburn BA, Bajpayee S, Reddy N, Jeelani R, Crone B, Simmerman N, Singh M, Diamond MP, Armant DR. Trophoblast retrieval and isolation from the cervix (TRIC) for noninvasive prenatal screening at 5 to 20 weeks of gestation. Fertil Steril. 2014;102(1):135-42 e6. Epub 2014/05/16. doi: 10.1016/j.fertnstert.2014.04.008. PubMed PMID: 24825422.

7. Bolnick JM, Kohan-Ghadr HR, Fritz R, Bolnick AD, Kilburn BA, Diamond MP, Armant DR, Drewlo S. Altered Biomarkers in Trophoblast Cells Obtained Noninvasively Prior to Clinical Manifestation of Perinatal Disease. Sci Rep. 2016;6:32382. Epub 2016/09/24. doi: 10.1038/srep32382. PubMed PMID: 27660926; PMCID: PMC5034887 property that has been licensed on their behalf by Wayne State University to PerkinElmer, Inc.

8. Fritz R, Kohan-Ghadr HR, Bolnick JM, Bolnick AD, Kilburn BA, Diamond MP, Drewlo S, Armant DR. Noninvasive detection of trophoblast protein signatures linked to early pregnancy loss using transcervical retrieval and isolation from the cervix. Fertil Steril. 2015. doi: 10.1016/j.fertnstert.2015.05.010. PubMed PMID: 26051097.

9. Fritz R, Kohan-Ghadr HR, Sacher A, Bolnick AD, Kilburn BA, Bolnick JM, Diamond MP, Drewlo S, Armant DR. Trophoblast retrieval and isolation from the cervix (TRIC) is unaffected by early gestational age or maternal obesity. Prenat Diagn. 2015;35(12):1218-22. Epub 2015/08/20. doi: 10.1002/pd.4681. PubMed PMID: 26288006; PMCID: PMC4715468.

10. Imudia AN, Suzuki Y, Kilburn BA, Yelian FD, Diamond MP, Romero R, Armant DR. Retrieval of trophoblast cells from the cervical canal for prediction of abnormal pregnancy: a pilot study. Hum Reprod. 2009;24(9):2086-92. doi: 10.1093/humrep/dep206. PubMed PMID: 19497946; PMCID: 2727404.

11. Jain CV, Kadam L, van Dijk M, Kohan-Ghadr H-R, Kilburn BA, Hartman C, Mazzorana V, Visser A, Hertz M, Bolnick AD, Fritz R, Armant DR, Drewlo S. Fetal genome profiling at 5 weeks of gestation after noninvasive isolation of trophoblast cells from the endocervical canal. Science Translational Medicine. 2016;8(363):363re4-re4. doi: 10.1126/scitranslmed.aah4661.

12. Drewlo S, Armant DR. Quo vadis, trophoblast? Exploring the new ways of an old cell lineage. Placenta. 2017;60 Suppl 1:S27-S31. Epub 2017/05/10. doi: 10.1016/j.placenta.2017.04.021. PubMed PMID: 28483162.

13. Moser G, Drewlo S, Huppertz B, Armant DR. Trophoblast retrieval and isolation from the cervix: origins of cervical trophoblasts and their potential value for risk assessment of ongoing pregnancies. Human reproduction update. 2018;24(4):484-96.

14. Benirschke K, Kaufmann P, Baergen RN. Pathology of the Human Placenta. 5 ed. New York: Springer; 2006.

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17. Pijnenborg R, Bland JM, Robertson WB, Brosens I. Uteroplacental arterial changes related to interstitial trophoblast migration in early human pregnancy. Placenta. 1983;4(4):397-413. Epub 1983/10/01. doi: 10.1016/s0143-4004(83)80043-5. PubMed PMID: 6634666.

18. Kim YM, Chaiworapongsa T, Gomez R, Bujold E, Yoon BH, Rotmensch S, Thaler HT, Romero R. Failure of physiologic transformation of the spiral arteries in the placental bed in preterm premature rupture of membranes. American journal of obstetrics and gynecology. 2002;187(5):1137-42. PubMed PMID: 12439491.

19. Rolnik DL, Wright D, Poon LC, O'Gorman N, Syngelaki A, de Paco Matallana C, Akolekar R, Cicero S, Janga D, Singh M, Molina FS, Persico N, Jani JC, Plasencia W, Papaioannou G, Tenenbaum-Gavish K, Meiri H, Gizurarson S, Maclagan K, Nicolaides KH. Aspirin versus Placebo in Pregnancies at High Risk for Preterm Preeclampsia. N Engl J Med. 2017;377(7):613-22. Epub 2017/06/29. doi: 10.1056/NEJMoa1704559. PubMed PMID: 28657417.

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23. de Paula CF, Ruano R, Campos JA, Zugaib M. Quantitative analysis of placental vasculature by three-dimensional power Doppler ultrasonography in normal pregnancies from 12 to 40 weeks of gestation. Placenta. 2009;30(2):142-8. Epub 2008/12/17. doi: 10.1016/j.placenta.2008.11.010. PubMed PMID: 19073343.

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