Quantification of spiral artery remodelling using an Adobe Photoshop-based technique

in Reproduction
Correspondence should be addressed to J N Bulmer; Email: judith.bulmer@ncl.ac.uk

(G E Lash is now at Guangzhou Institute of Pediatrics, Guangzhou Women and Children’s Medical Center, Guangzhou, China)

(B Schiessl is now at Praxis für Pränatalmedizin, Munchen, Germany)

*(E Ball is now at Obstetrics and Gynaecology, Barts NHS trust, London, UK)

Restricted access

Uterine spiral arteries undergo remodelling in normal pregnancy, with replacement of the musculoelastic arterial media by fibrinoid containing extravillous trophoblast cells. Deficient spiral artery remodelling is associated with several adverse pregnancy outcomes. Although there are distinct components of spiral artery remodelling, assessment is subjective and often based on an overall impression of morphology. We aimed to develop a quantitative approach for assessment of uterine spiral artery remodelling. Placental bed biopsies were immunostained using smooth muscle markers, digital images of spiral arteries were captured and Adobe Photoshop was used to analyse positive immunostaining. The method was then used to investigate variation in the same vessel at different levels within a paraffin block, and the effect of parity, pre-eclampsia or miscarriage on vascular smooth muscle cell content. Results were also compared with a more subjective morphology-based assessment system. There was good intra- and interobserver agreement and the method correlated well with the more subjective assessment system. There was an overall reduction in vascular smooth muscle, as detected by caldesmon 1 (h-caldesmon) immunopositivity, with increasing gestational age from 8 weeks to term. A previous pregnancy did not affect the amount of spiral artery smooth muscle. Comparison of pre-eclampsia and late miscarriage samples with controls of the appropriate gestational age demonstrated increased medial smooth muscle in pathological samples. This technique provides a simple, rapid, reproducible and inexpensive approach to quantitative assessment of spiral artery remodelling in normal and pathological human pregnancy, a process which although fundamental for successful pregnancy, is still incompletely understood.

 

    Society for Reproduction and Fertility

Related Articles

Article Information

Metrics

All Time Past Year Past 30 Days
Abstract Views 72 72 72
Full Text Views 15 15 15
PDF Downloads 14 14 14

Altmetrics

Figures

  • View in gallery

    Schematic illustrating the photoshop method. (A) Section of placental bed biopsy immunostained for H-caldesmon and counterstained with PAS showing four cross-sections of a partially remodelled SpA. (B) Immunopositive SpA cross-sections were selected using the magnetic lasso tool and pasted into a new layer to give the total pixel number for each SpA wall cross section (numbers in parentheses). (C) The threshold intensity of immunostaining was identified using the magic wand tool, similar areas were selected and copied into a new layer to give the number of immunopositive pixels (numbers in parentheses). (D) Table showing the total and positive pixel numbers and calculation of % positive pixels.

  • View in gallery

    Serial sections of a partially remodelled SpA (magnification ×400) showing immunostaining with: (A) h-caldesmon (h-cal); (B) myosin heavy chain (MyHC); (C) α-smooth muscle actin (αSMA); (D) desmin. (E) Analysis of spiral artery smooth muscle immunostaining with h-cal, MyHC, αSMA and desmin. Data are expressed as the percentage of positive pixels in the vessel wall image for each antibody (n = 25 SpAs from five placental bed samples).

  • View in gallery

    (A and B) Linear plot (A) and scatter plot (B) showing the percentage pixel counts for analyses carried out by two independent investigators (n = 10 spiral arteries in seven placental bed biopsies). (C) Variation in positive staining between 3 μm step (1st, 5th, 10th, 15th, 20th, 25th) sections of individual spiral arteries in five different placental bed samples.

  • View in gallery

    (A) Effect of gestational age on spiral artery vascular smooth muscle. Each data point represents the mean % positive pixels calculated for each placental bed sample and bars show the median percentage of positive pixels. (B) Effect of parity on spiral artery smooth muscle stained for h-cal, and elastin detected with Elastic van Gieson stain and anti-elastin. Data are expressed as the percentage of positive pixels in the image of the vessel wall.

  • View in gallery

    (A) Percentage positive staining for h-cal in non-transformed vessels (N) compared with partially transformed (P1 and P2) and completely transformed (C) vessels. (B and C) Comparison of positive staining in spiral arteries from (B) 3rd trimester normal pregnancies and those complicated by pre-eclampsia; and (C) gestational age matched normal pregnancy and late miscarriage. *P < 0.05.

References

  • AbbertonKMTaylorNHHealyDLRogersPA 1996 Vascular smooth muscle alpha-actin distribution around endometrial arterioles during the menstrual cycle: increased expression during the perimenopause and lack of correlation with menorrhagia. Human Reproduction 204211. (https://doi.org/10.1093/oxfordjournals.humrep.a019020)

    • Search Google Scholar
    • Export Citation
  • AbbertonKMHealyDLRogersPA 1999 Smooth muscle alpha actin and myosin heavy chain expression in the vascular smooth muscle cells surrounding human endometrial arterioles. Human Reproduction 30953100. (https://doi.org/10.1093/humrep/14.12.3095)

    • Search Google Scholar
    • Export Citation
  • BallEBulmerJNAyisSLyallFRobsonSC 2006a Late sporadic miscarriage is associated with abnormalities in spiral artery transformation and trophoblast invasion. Journal of Pathology 535542. (https://doi.org/10.1002/path.1927)

    • Search Google Scholar
    • Export Citation
  • BallERobsonSCAyisSLyallFBulmerJN 2006b Early embryonic demise: no evidence of abnormal spiral artery transformation or trophoblast invasion. Journal of Pathology 528534. (https://doi.org/10.1002/path.1926)

    • Search Google Scholar
    • Export Citation
  • CeballosKMNielsenGPSeligMKO’ConnellJX 2000 Is anti-h-caldesmon useful for distinguishing smooth muscle and myofibroblastic tumors? An immunohistochemical study. American Journal of Clinical Pathology 746753. (https://doi.org/10.1309/K5JP-A9EN-UWN7-B5GG)

    • PubMed
    • Search Google Scholar
    • Export Citation
  • ChesleyLC 1984 History and epidemiology of preeclampsia-eclampsia. Clinical Obstetrics and Gynecology 801820. (https://doi.org/10.1097/00003081-198412000-00004)

    • PubMed
    • Search Google Scholar
    • Export Citation
  • CravenCMMorganTWardK 1998 Decidual spiral artery remodelling begins before cellular interaction with cytotrophoblasts. Placenta 241252. (https://doi.org/10.1016/S0143-4004(98)90055-8)

    • PubMed
    • Search Google Scholar
    • Export Citation
  • KhongTYDe WolfFRobertsonWBBrosensI 1986 Inadequate maternal vascular response to placentation in pregnancies complicated by pre-eclampsia and by small-for-gestational age infants. British Journal of Obstetrics and Gynaecology 10491059. (https://doi.org/10.1111/j.1471-0528.1986.tb07830.x)

    • PubMed
    • Search Google Scholar
    • Export Citation
  • KhongTYAdemaEDErwichJJ 2003 On an anatomical basis for the increase in birth weight in second and subsequent born children. Placenta 348353. (https://doi.org/10.1053/plac.2002.0922)

    • PubMed
    • Search Google Scholar
    • Export Citation
  • KimYMBujoldEChaiworapongsaTGomezRYoonBHThalerHTRotmenschSRomeroR 2003 Failure of physiologic transformation of the spiral arteries in patients with preterm labor and intact membranes. American Journal of Obstetrics and Gynecology 10631069. (https://doi.org/10.1067/s0002-9378(03)00838-x)

    • PubMed
    • Search Google Scholar
    • Export Citation
  • LashGEPitmanHMorganHLInnesBAAgwuCNBulmerJN 2016 Decidual macrophages: key regulators of vascular remodeling in human pregnancy. Journal of Leukocyte Biology 315325. (https://doi.org/10.1189/jlb.1A0815-351R)

    • PubMed
    • Search Google Scholar
    • Export Citation
  • LehrHAJacobsTWYazijiHSchnittSJGownAM 2001 Quantitative evaluation of HER-2/neu status in breast cancer by fluorescence in situ hybridization and by immunohistochemistry with image analysis. American Journal of Clinical Pathology 814822. (https://doi.org/10.1309/AJ84-50AK-1X1B-1Q4C)

    • PubMed
    • Search Google Scholar
    • Export Citation
  • LyallFSimpsonHBulmerJNBarberARobsonSC 2001 Transforming growth factor beta expression in human placenta and placental bed in the third trimester normal pregnancy, preeclampsia, and fetal growth restriction. American Journal of Pathology 18271838. (https://doi.org/10.1016/s0002-9440(10)63029-5)

    • Search Google Scholar
    • Export Citation
  • LyallFRobsonSCBulmerJN 2013 Spiral artery remodeling and trophoblast invasion in preeclampsia and fetal growth restriction: relationship to clinical outcome. Hypertension 10461054. (https://doi.org/10.1161/HYPERTENSIONAHA.113.01892)

    • PubMed
    • Search Google Scholar
    • Export Citation
  • MacGillivrayI 1958 Some observations on the incidence of pre-eclampsia. Journal of Obstetrics and Gynaecology for the British Empire 536539. (https://doi.org/10.1111/j.1471-0528.1958.tb08557.x)

    • Search Google Scholar
    • Export Citation
  • MatkowskyjKACoxRJensenRTBenyaRV 2003 Quantitative immunohistochemistry by measuring cumulative signal strength accurately measures receptor number. Journal of Histochemistry and Cytochemistry 205214. (https://doi.org/10.1177/002215540305100209)

    • Search Google Scholar
    • Export Citation
  • MeekinsJWPijnenborgRHanssensMMcFadyenIRvan AssheA 1994 A study of placental bed spiral arteries and trophoblast invasion in normal and severe pre-eclamptic pregnancies. British Journal of Obstetrics and Gynaecology 669674. (https://doi.org/10.1111/j.1471-0528.1994.tb13182.x)

    • PubMed
    • Search Google Scholar
    • Export Citation
  • MorganTCravenCLalouelJMWardK 1999 Angiotensinogen Thr235 variant is associated with abnormal physiologic change of the uterine spiral arteries in first-trimester decidua. American Journal of Obstetrics and Gynecology 95102. (https://doi.org/10.1016/s0002-9378(99)70156-0)

    • PubMed
    • Search Google Scholar
    • Export Citation
  • NucciMRO'ConnellJTHuettnerPCCvikoASunDQuadeBJ 2001 h-Caldesmon expression effectively distinguishes endometrial stromal tumors from uterine smooth muscle tumors. American Journal of Surgical Pathology 455463. (https://doi.org/10.1097/00000478-200104000-00004)

    • Search Google Scholar
    • Export Citation
  • OwensGK 1995 Regulation of differentiation of vascular smooth muscle cells. Physiological Reviews 487517. (https://doi.org/10.1152/physrev.1995.75.3.487)

    • PubMed
    • Search Google Scholar
    • Export Citation
  • PijnenborgRAnthonyJDaveyDAReesATiltmanAVercruysseLvan AsscheA 1991 Placental bed spiral arteries in the hypertensive disorders of pregnancy. British Journal of Obstetrics and Gynaecology 648655. (https://doi.org/10.1111/j.1471-0528.1991.tb13450.x)

    • PubMed
    • Search Google Scholar
    • Export Citation
  • PijnenborgRVercruysseLHanssensM 2006 The uterine spiral arteries in human pregnancy: facts and controversies. Placenta 939958. (https://doi.org/10.1016/j.placenta.2005.12.006)

    • PubMed
    • Search Google Scholar
    • Export Citation
  • PrefumoFBhideASairamSPennaLHollisBThilaganathanB 2004 Effect of parity on second-trimester uterine artery Doppler flow velocity and waveforms. Ultrasound in Obstetrics and Gynecology 4649. (https://doi.org/10.1002/uog.908)

    • Search Google Scholar
    • Export Citation
  • PrefumoFGanapathyRThilaganathanBSebireNJ 2006 Influence of parity on first trimester endovascular trophoblast invasion. Fertility and Sterility 10321036. (https://doi.org/10.1016/j.fertnstert.2005.09.055)

    • PubMed
    • Search Google Scholar
    • Export Citation
  • PriceRJOwensGKSkalakTC 1994 Immunohistochemical identification of arteriolar development using markers of smooth muscle differentiation. Evidence that capillary arterialization proceeds from terminal arterioles. Circulation Research 520527. (https://doi.org/10.1161/01.res.75.3.520)

    • PubMed
    • Search Google Scholar
    • Export Citation
  • RobsonSCSimpsonHBallELyallFBulmerJN 2002 Punch biopsy of the human placental bed. American Journal of Obstetrics and Gynecology 13491355. (https://doi.org/10.1067/mob.2002.126866)

    • PubMed
    • Search Google Scholar
    • Export Citation
  • RogersPAAbbertonKM 2003 Endometrial arteriogenesis: vascular smooth muscle cell proliferation and differentiation during the menstrual cycle and changes associated with endometrial bleeding disorders. Microscopy Research and Technique 412419. (https://doi.org/10.1002/jemt.10279)

    • PubMed
    • Search Google Scholar
    • Export Citation
  • SkalliORoprazPTrzeciakABenzonanaGGillessenDGabbianiG 1986 A monoclonal antibody against alpha-smooth muscle actin: a new probe for smooth muscle differentiation. Journal of Cell Biology 27872796. (https://doi.org/10.1083/jcb.103.6.2787)

    • Search Google Scholar
    • Export Citation
  • SmithSDDunkCEAplinJDHarrisLKJonesRL 2009 Evidence for immune cell involvement in decidual spiral arteriole remodeling in early human pregnancy. American Journal of Pathology 19591971. (https://doi.org/10.2353/ajpath.2009.080995)

    • Search Google Scholar
    • Export Citation
  • StarzykKASalafiaCMPezzulloJCLageJMParkashVVercruysseLHanssensMPijnenborgR 1997 Quantitative differences in arterial morphometry define the placental bed in pre-eclampsia. Human Pathology 353358. (https://doi.org/10.1016/s0046-8177(97)90135-0)

    • PubMed
    • Search Google Scholar
    • Export Citation

PubMed

Google Scholar