Haem oxygenase-1 dictates intrauterine fetal survival in mice via carbon monoxide

ORIGINAL PAPERJournal of PathologyJ Pathol 2011; 225: 293–304Published online 8 July 2011 in Wiley Online Library(wileyonlinelibrary.com) DOI: 10.1002/path.2946

Haem oxygenase-1 dictates intrauterine fetal survival in mice viacarbon monoxideMaria Laura Zenclussen,1 Pablo Ariel Casalis,2 Tarek El-Mousleh,1 Sofia Rebelo,3 Stefanie Langwisch,1Nadja Linzke,1 Hans-Dieter Volk,4,5 Stefan Fest,6 Miguel Parreira Soares3 and Ana Claudia Zenclussen1*

1 Department of Experimental Obstetrics and Gynecology, Medical Faculty, Otto-von-Guericke University, Gerhart-Hauptmann-Strasse 35,39108, Magdeburg, Germany2 Department of Neurosurgery, Charite, Universitatsmedizin Berlin, Campus Virchow Klinikum, Augustenburger Platz 1, 13353, Berlin, Germany3 Instituto Gulbenkian de Ciencia, Rua da Quinta Grande 6, P2780-156, Oeiras, Portugal4 Institute of Medical Immunology, Charite Universitatsmedizin, Chariteplatz 1, 10115 Berlin, Germany5 Berlin-Brandenburg Center for Regenerative Therapies, Augustenburgerplatz 1, 13353, Berlin, Germany6 Pediatric Immunotherapies, Department of Pediatrics and Institute of Molecular and Clinical Immunology, Medical Faculty, Otto-von-GuerickeUniversity, Leipziger Strasse 44, 39120, Magdeburg, Germany

*Correspondence to: Ana Claudia Zenclussen, Department of Experimental Obstetrics and Gynecology, Medical Faculty, Otto-von-GuerickeUniversity, Gerhart-Hauptmann-Strasse 35, 39108, Magdeburg, Germany. e-mail: [email protected]

AbstractPregnancy establishment implies the existence of a highly vascularized and transient organ, the placenta, whichensures oxygen supply to the fetus via haemoproteins. Haem metabolism, including its catabolism by haemoxygenase-1 (HO-1), should be of importance in maintaining the homeostasis of haemoproteins and controllingthe deleterious effects associated with haem release from maternal or fetal haemoglobins, thus ensuring placentalfunction and fetal development. We demonstrate that HO-1 expression is essential to promote placental functionand fetal development, thus determining the success of pregnancy. Hmox1 deletion in mice has pathologicalconsequences for pregnancy, namely suboptimal placentation followed by intrauterine fetal growth restriction(IUGR) and fetal lethality. These pathological effects can be mimicked by administration of exogenous haemin wild-type mice. Fetal and maternal HO-1 is required to prevent post-implantation fetal loss through amechanism that acts independently of maternal adaptive immunity and hormones. The protective HO-1 effectson placentation and fetal growth can be mimicked by the exogenous administration of carbon monoxide (CO), aproduct of haem catabolism by HO-1 that restores placentation and fetal growth. In a clinical relevant model ofIUGR, CO reduces the levels of free haem in circulation and prevents fetal death. We unravel a novel physiologicalrole for HO-1/CO in sustaining pregnancy which aids in understanding the biology of pregnancy and reveals apromising therapeutic application in the treatment of pregnancy pathologies.Copyright 2011 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Keywords: haem; pregnancy; haem oxygenase; carbon monoxide; placentation; intrauterine growth restriction

Received 13 January 2011; Revised 18 May 2011; Accepted 25 May 2011

No conflicts of interest were declared.

Introduction

Haem oxygenases (HO) catalyse the first and rate-limiting step in haem catabolism towards biliverdin,carbon monoxide (CO), and free iron [1]. The stress-responsive HO-1 isoform, encoded by the Hmox1gene, is cytoprotective [2] and exerts anti-inflammatoryeffects [3,4] while modulating cell proliferation [5].HO-1 prevents tissue damage and regulates innateas well as adaptive immunity in a manner that sup-presses the pathogenesis of a broad range of immune-mediated inflammatory diseases [4,6,7]. The cytopro-tective and immunoregulatory effects of HO-1 areablated when its enzymatic activity is inhibited phar-macologically, being restored when CO is suppliedexogenously [3,8–10]. Consequently, CO mediates toa large extent the salutary effects of HO-1; however,

other products of haem catabolism by HO-1, ie ironand biliverdin, might act in a similar manner. Afew Hmox1 null (Hmox1 −/−) mice, obtained by mat-ing Hmox1 +/− mice, survive to adulthood. Surviv-ing Hmox1 −/− female mice are reported as infer-tile [11–13]. The placenta is a unique organ whichensures oxygen supply to the fetus via haemopro-teins [14]. Thus, haem metabolism, including itscatabolism by HO-1, should play an important role inmaintaining the homeostasis of haemoproteins, ensur-ing placental development and function. Expressionof HO-1 is highly induced during human, rat, andmouse pregnancy, namely in placental trophoblasts[14–17]. Reduced HO-1 levels are associated withhuman and murine miscarriages [16,18] and pre-eclampsia, the most severe pathological complica-tion of pregnancy [19]. HO-1 induction supports

Copyright 2011 Pathological Society of Great Britain and Ireland. J Pathol 2011; 225: 293–304Published by John Wiley & Sons, Ltd. www.pathsoc.org.uk www.thejournalofpathology.com

294 ML Zenclussen et al

pregnancy, diminishing the early onset of murinemiscarriages [17,20].

Here, we aimed to unravel the function of HO-1 inpregnancy and the molecular mechanism underlying itsaction. We provide irrefutable experimental evidencethat HO-1 expression is required to support placenta-tion and fetal development. The salutary effects of HO-1 rely on its ability to prevent the deleterious effects offree haem during pregnancy. This effect can be mim-icked by CO, an end-product of haem catabolism byHO-1 that binds to haemoglobin and prevents it fromreleasing its haem groups. Finally, we show that thisgasotransmitter can be used for therapeutic purposesin the treatment of pregnancy pathologies. Collectively,our results provide direct support for the HO-1/CO sys-tem as a pleiotropic coordinator of crucial reproductiveevents that together ensure species survival.

Materials and methods

Mice

Hmox1-competent or -deficient mice were kindly pro-vided by Dr Saw Feng-Yet [12]. CBA/J, DBA/2J,and BALB/c mice were obtained from Charles River(Sulzfeld, Germany) and maintained in our animalfacilities in Berlin and Magdeburg, Germany with a12 h light/dark cycle with water and food ad libitum.Experimental procedures were approved by the Ger-man authorities (LaGeSo Berlin 0062/03 and Saxony-Anhalt’s Ministry 2–868). For experiments performedat the Instituto Gulbenkian de Ciencia, Hmox1 orHmox1.SCID animals were bred and housed at theirpathogen-free facility, all experimental protocols beingapproved by the institutional animal care commit-tee. The progeny obtained by mating Hmox1 +/−females with Hmox1 +/− males in a BALB/c orBALB/c.SCID background was genotyped and docu-mented. Hmox1 +/+, Hmox1 +/− or Hmox1 −/− femaleswere mated with either Hmox1 +/+, Hmox1 +/− orHmox1 −/− BALB/c males. For free haem adminis-tration, hemin (Frontier Bioscience, Lancashire, UK)was diluted in a 0.2 M NaOH/HCl solution and20 mg/kg body weight was administered intraperi-toneally to Hmox1 +/+ or Hmox1 +/− mice at days 7, 9,and 11 of pregnancy. Hmox1 +/+ and Hmox1 +/− con-trols received vehicle solution. DBA/2J-mated CBA/Jfemales and Hmox1 +/− females mated with Hmox1 +/−males were treated with mixed air or CO in iso-lated cages. The presence of a vaginal plug indicatedday 0 of pregnancy. Implantation rate as well as thepercentage of fetal loss was analysed on day 14 ofpregnancy by calculating the percentage of haemor-rhagic, non-viable implantations to the total numberof implantations (viable + non-viable) multiplied by100. Placentas and fetuses of gas-treated animals wereweighed. Tissue samples were obtained for histopathol-ogy studies. Paraffin-embedded placenta samples were

stained with haematoxylin and eosin (H&E) for anal-ysis of their morphology. Total HO activity was anal-ysed in fresh liver samples of Hmox1 +/+, Hmox1 +/−or Hmox1 −/− animals as indicated in the Support-ing information, Supplementary materials and methods.For the in vitro implantation model, 3.5 days pregnantHmox1 +/+ or Hmox1 +/− females were used as blas-tocyst and uterine epithelial cell (UEC) donors.

CO treatments

Mice in their cages were placed in a 98-l Plexiglasanimal chamber (A-Chamber, BioSpherix, NY, USA)and exposed to CO (50 parts per million, ppm) eitherduring days 3–8 of pregnancy (Hmox1 +/− mice) orduring days 5–8 of pregnancy (CBA/J × DBA/2Jmodel). Control mice were maintained in a similarchamber with mixed air but without CO following thesame protocol. Time exposure and doses to be appliedwere previously tested. 50 ppm at the mentioned timeframes was found to be effective in preventing fetaldeath, while not provoking any toxic effects. Moredetailed information is available in the Supportinginformation, Supplementary materials and methods.

In vitro model of blastocyst attachment

For analysing the kinetics of Hmox1 +/+, Hmox1 +/−,and Hmox1 −/− embryo attachment to the uterine wallin vitro, blastocyts from 3.5 days pregnant femaleswere flushed from the uteruses, collected in medium,and transferred to a monolayer of autologous UECs24 h later and grown in extracellular matrix (ECM). Weblindly analysed blastocysts from either Hmox1 +/+ orHmox1 +/− females mated with Hmox1 +/+,Hmox1 +/− or Hmox1 −/− males, as shown in theSupporting information, Supplementary Figure 1, andcharacterized their phenotype at the end of the exper-iment by immunohistochemistry. Attachment of theblastocysts to the UEC monolayer was analysed micro-scopically and recorded every 12 h.

Rcho-1 cells culture and differentiation

Rcho-1 trophoblast cells represent a stem cell popu-lation capable of differentiation along the trophoblastgiant cell (GC) lineage [21]. They can be manipulatedto proliferate or differentiate under culture conditions[22]. For differentiation of Rcho-1 trophoblast stemcells into trophoblast GCs, fetal bovine serum (FBS)was replaced by 10% of horse serum for 7 days. Tostudy the effect of HO-1 modulation on Rcho-1 cellsurvival and differentiation to GCs, 50 and 100 µMof ZnPPIX or CoPPIX was added to the culture. In afurther experiment, 100 µM ZnPPIX was added to theculture while CO was applied at 500 ppm in cham-bers as indicated above. Protein isolation and westernblot details are available in the Supporting information,Supplementary materials and methods.


Essential role of HO-1 and CO in pregnancy 295

Measurement of fetuses and implantation areasWhole implantation sites containing ectoplacental cone,embryo, and decidua from Hmox1 +/− females matedwith Hmox1 +/− males at gestation days 8, 10, and12 were embedded in paraffin as described by Croyet al [23]. Areas containing the whole implantationsites were measured by using the ImageJ programand analysed blindly (http://rsb.info.nih.gov/ij/). Geno-typing revealed the nature of the tissue (Hmox1 +/+,Hmox1 +/− or Hmox1 −/−).

StatisticsSamples were analysed for the normality of theirdistribution. Unless otherwise indicated, data are notnormally distributed and non-parametric tests wereused: the Kruskal–Wallis test for comparison amongmore than two groups and the Mann–Whitney U -test for two particular groups. The chi square testwas employed to analyse the progeny of the Hmox1colony and the effect of CO on the living progeny.Our data were monitored by statistics advisors fromthe Universities in Magdeburg and Berlin, Germany.

Detailed information regarding genotyping HO-1activity, hormone measurement, measurement of freecirculating haem and immunohistochemistry may befound in the Supporting information, Supplementarymaterials and methods.

Results

Hmox1 deletion in both father and mother leads tofetal lossThe percentage of viable Hmox1 −/− mice fromBALB/c Hmox1 +/− breeding was significantly lower,ie 7.33%, than the expected Mendelian ratio, ie 25%(Table 1). We analysed the implantation rate as wellas the rate of fetal loss when mating Hmox1 +/+,Hmox1 +/− or Hmox1 −/− BALB/c females withHmox1 +/+, Hmox1 +/− or Hmox1 −/− BALB/c males.The total number of implantations (healthy plus dead)

Table 1. Percentages of F1 Hmox1+/+, Hmox1+/−, andHmox1−/− progeny when pairing Hmox1+/− females andHmox1+/− males of BALB/c and BALB/c.SCID backgroundMouse % % %strain n Hmox1+/+ Hmox1+/− Hmox1−/−

BALB/c 3683 29.81 62.86 7.33∗

BALB/c.SCID 865 30.97 58.15 10.98†

Hmox1+/− females were paired with Hmox1+/− males and born pups weregenotyped. The percentage of knockout progeny was much lower than theexpected 25% according to Mendelian rules. This was observed in BALB/c andBALB/c.SCID background, suggesting that the lower rate of knockout pups is notdue to immunological rejection of them.∗p = 0.0002 compared with the expected 25% and †p = 0.0004 comparedwith the expected 25%. The percentages of Hmox1−/− obtained in BALB/c andBALB/c.SCID background were comparable (p = 0.447). Data were analysed usingthe chi square test.

did not differ among the combinations (Support-ing information, Supplementary Figure 2). However,the percentage of fetal loss within the implantedembryos increased significantly when Hmox1 alle-les were deleted in both the father and the mother(Figures 1A–1C). Decreasing levels of HO-1 expres-sion and total HO activity (Figures 1H and 1I, respec-tively) led to higher fetal loss (Figures 1A–1C). Totalfetal loss at day 14 of pregnancy (100%) was mani-fested when both Hmox1 alleles were deleted in boththe father and the mother (Figures 1C and 1F). Non-viable fetuses were recognizable by their haemorrhagic,necrotic appearance and smaller size compared withnormally developing fetuses (Figures 1D–1G).

To assess whether the low percentage of Hmox1 −/−mice in the heterozygous combination is due to aderegulated immune response towards these fetuses,we analysed the percentage of viable Hmox1 −/− miceobtained when mating Hmox1 +/− SCID BALB/c micethat lack T and B cells. The result obtained was sim-ilar to that obtained when mating immunocompetentHmox1 +/− BALB/c mice (10.98% versus 7.33%; p =0.447, Table 1), which was also significantly lowerthan the expected Mendelian ratio, ie 25% (Table 1).In line with these observations, the relative numbersof effector T cells and regulatory T cells were compa-rable among Hmox1 +/+, Hmox1 +/−, and Hmox1 −/−mothers mated with either Hmox1 +/+, Hmox1 +/− orHmox1 −/− BALB/c mice on day 14 of pregnancy(data not shown). We did not observe differences incytokine secretion or their mRNA levels among anyof the groups (data not shown). Neither progesteronenor oestradiol differed between the Hmox1 genotypes(Supporting information, Supplementary Figure 3).

HO-1 expression favours on-time blastocystattachmentHmox1 +/+ blastocysts attached significantly fasterthan Hmox1 +/− or Hmox1 −/− blastocysts (Support-ing information, Supplementary Table 1). A consid-erable proportion of the blastocysts produced fromHmox1 +/− × Hmox1 +/− or Hmox1 +/− × Hmox1 −/−combinations failed to attach to UECs, which wasnot the case for blastocysts obtained from the controlHmox1 +/+ × Hmox1 +/+ combination, which had allattached at 72 h (Figure 2A). Blastocysts that failed toattach to UECs proved to be Hmox1 −/−.

HO-1 is crucial for placentation and fetal growthWe next assessed whether HO-1 regulates placenta-tion, a critical event to ensure the successful outcomeof implanted fetuses. In wild-type mice, invasive tro-phoblasts expressed high levels of HO-1 when com-pared with the surrounding epithelial cells (Figure 2B).Murine invasive trophoblasts present typical GC mor-phological features, namely large size polyploid cells(Figure 2B). GCs are the first cells from the ecto-placental clone to emerge and are critically involved inplacentation [21]. We tested whether HO-1 is required



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Figure 1. HO-1 deficiency negatively alters pregnancy outcome. (A–C) Fetal loss rates in % as calculated by [(number of deadfetuses/number of total fetuses) × 100] in Hmox1+/+, Hmox1+/− or Hmox1−/− females paired with either Hmox1+/+ (A), Hmox1+/−(B) or Hmox1−/− (C) males (+/+ × +/+: n = 6; +/+ × +/− = 7; +/+ × −/−: n = 5; +/− × +/+: n = 5; +/− × +/−: n = 9;+/− × −/−: n = 8; −/− × +/+: n = 4; −/− × +/−: n = 4; and −/− × −/−: n = 3). Data are shown as dot plots. ∗p < 0.05 and∗∗p < 0.01 as analysed by using the non-parametric Kruskal–Wallis test for analysing differences among all groups and the Mann–WhitneyU-test for differences between two particular groups. (D–F) Pictures of healthy and dead fetuses as documented on day 14 of pregnancyfrom Hmox1−/− females mated with Hmox1+/+ (D), Hmox1+/− (E) or Hmox1−/− (F) males. Dead fetuses (∗) are significantly smallerthan healthy ones (#) and present a haemorrhagic appearance as indicated in D and shown in G in more detail. Furthermore, in healthyimplantations, it is possible to distinguish between the fetus and its placenta, which is not the case for already dead fetuses (G). (H) ThePCR products for Hmox1+/+, Hmox1+/−, and Hmox1−/− tissues. Hmox1−/− fetuses show amplification only with the HO/E4-Neo1 primerset, which amplifies a 400 bp fragment of the mutated allele, whereas Hmox1+/+ fetuses show amplification only with the HO/E3-HO/I3Rset of primers, which amplifies a 456 bp fragment of the wild-type allele. Hmox1+/− fetuses show amplification with both sets of primers.More detailed information is provided in the Supporting information. The gel shown here is representative for all experiments carried outwith fetuses or placentas. The picture was obtained using a Benchtop UV Transilluminator BioDoc-It Imaging System. (I) Representationof the HO-1 activity in freshly isolated liver tissue from Hmox1+/+, Hmox1+/− or Hmox1−/− mice as measured by the release of bilirubinand expressed as pmol/60 min per mg.

for trophoblast proliferation or modulates trophoblas-tic stem cell differentiation into GCs in vitro. Inhibitionof HO-1 by zinc protoporphyrin IX (ZnPPIX; 50 µM)(Supporting information, Supplementary Figure 4A)reduced trophoblastic stem cell (Rcho-1 cell line)viability from 86.4% to 46.5% and to 17.2% whenZnPPIX was used at higher concentration (100 µM;Supporting information, Supplementary Figure 4B).This inhibitory effect was not observed using cobaltprotoporphyrin IX (CoPPIX), which does not inhibitHO-1 expression (Supporting information, Supple-mentary Figure 4A); eg 72.5% viability at 50 µMand 61.4% viability at 100 µM (Supporting informa-tion, Supplementary Figure 4B). While HO-1 proteinexpression remained stable during stem cell differen-tiation into GCs under normal conditions (Support-ing information, Supplementary Figure 4C), inhibition

of HO-1 activity by ZnPPIX suppressed trophoblas-tic stem cell differentiation into GCs (Figure 2C,lower panel), an effect not observed using CoP-PIX (Figure 2C, middle panel), which presented nodifferences to untreated controls (Figure 2C, upperpanel). This provides strong evidence that HO-1activity regulates both trophoblast survival and dif-ferentiation into a mature phenotype, hence enor-mously contributing to placentation. In keeping withthis notion, inhibition of HO-1 expression using siRNAinhibited the viability of human primary trophoblastsisolated from first-trimester pregnancies (Supportinginformation, Supplementary Figure 5).

To confirm the pivotal role of HO-1 in pla-centation, we next analysed the histopathology ofHmox1 +/+, Hmox1 +/−, and Hmox1 −/− placentasobtained from Hmox1 +/+ × Hmox1 +/+, Hmox1 +/− ×



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Figure 2. HO-1 expression favours on-time blastocyst attachment to the uterine wall and promotes stem cell differentiation into giantcells. (A) The percentage of in vitro attached blastocysts obtained from Hmox1+/+ × Hmox1+/+ (n = 6), Hmox1+/− × Hmox1+/−(n = 13), and Hmox1+/− × Hmox1−/− (n = 6) pairing combinations, which was analysed every 12 h using a light microscope. Thenumber of blastocysts was as follows: Hmox1+/+ n = 61; Hmox1+/− n = 48, and Hmox1−/− n = 10. At 72 h, 100% of the blastocystsfrom the Hmox1+/+ × Hmox1+/+ combination were attached, while blastocysts from the combinations Hmox1+/− × Hmox1+/− andHmox1−/− × Hmox1+/− never reached 100% of attachment. (B) A blastocyst positive for HO-1 as analysed by light microscopy afterimmunohistochemistry for HO-1. (C) The maturation process of Rcho-1 trophoblast stem cells into trophoblast giant cells (arrows indicateimmature stem cells, SC or mature giant cells, GC) after addition of a special medium containing horse serum. The addition of 50 µMCo-PPIX did not modify this process, while the addition of 50 µM ZnPPIX, which inhibited HO-1 protein expression (see the Supportinginformation), blocked the differentiation of stem cells into GCs as observed by the absence of multinucleated large cells after 7 days ofculture. The pictures are representative of three independent experiments, each performed in triplicate. ∗p < 0.05 as analysed by usingthe non-parametric Mann–Whitney U-test. Pictures were taken with a total magnification of 200× using light microscopy using theAxiovision Rel 4–6 program (Zeiss AX 10 microscope).

Hmox1 +/−, and Hmox1 +/− × Hmox1 −/− combi-nations after genotyping their respective embryos.Hmox1 +/+ placentas presented typical morphologywith definable areas containing GCs, spongiotro-phoblasts + glycogen cells (junctional zone), andlabyrinth cells (Figure 3A). The number of GCs inHmox1 +/− placentas (Figure 3B), and especially inHmox1 −/− placentas (Figure 3C), was significantlyreduced compared with wild-type (Hmox1 +/+) placen-tas (Figure 3A and Table 2). In addition, Hmox1 +/−and Hmox1 −/− placentas had enlarged labyrinth areasand a reduced or absent junctional area (consist-ing of spongiotrophoblasts and glycogen cells) withdetectable morphological abnormalities (Figures 3A–3C), revealing improper placentation. Hmox1 +/− andHmox1 −/− placentas presented areas of fibrosis(Table 2) and haemorrhage (Figures 3A–3C). Deletionof the Hmox1 allele therefore leads to abnormal pla-centation and likely to an insufficient nutrient and oxy-gen supply to the fetus. While at day 8 of pregnancyfetuses among all genotypes were comparable in size,

at day 10 of pregnancy Hmox1 +/− and Hmox1 −/−

fetuses and whole implantation sites, ie uterus, pla-centa, and fetuses, were already significantly smallerthan Hmox1 +/+ specimens (Figure 3D and Table 3).

When mating Hmox1 +/− with Hmox1 +/−, therewere no viable Hmox1 −/− fetuses detectable at day12, while Hmox1 +/− fetuses were significantly muchsmaller than Hmox1 +/+ fetuses (Table 3). At day 14of pregnancy, Hmox1 +/+, Hmox1 +/−, and Hmox1 −/−

fetuses from the Hmox1 +/− × Hmox1 +/− combi-nation did not differ significantly in weight, butHmox1 +/− and Hmox1 −/− placentas weighed signif-icantly less than Hmox1 +/+ placentas (Figure 3E).Fully resorbed, non-viable fetuses were smaller thanhealthy ones (15–25 g versus 110–130 g; Figure 3E,lower and upper panel, respectively). All fetuses fromthe Hmox1 −/− × Hmox1 −/− combination were non-viable at day 14 (Figure 1). These observations pro-vide conclusive evidence that deletion of the Hmox1allele has major pathological consequences during



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Figure 3. HO-1 absence leads to improper placentation and intrauterine fetal growth restriction. (A–C) The histopathology of representativeHmox1+/+ (A), Hmox1+/− (B), and Hmox1−/− (C) placentas from n = 7 Hmox1+/+, n = 12 Hmox1+/−, and n = 9 Hmox1−/− placentasanalysed by light microscopy of H&E-stained slides. Special attention should be paid to the area containing giant cells (GCs), which isdiminished in Hmox1+/− placentas and even absent in Hmox1−/− placentas compared with Hmox1+/+ placentas (Table 2). The junctionalzone (JZ) containing spongiotrophoblasts (spong) is much thinner in Hmox1+/− and Hmox1−/− placentas than in Hmox1+/+ placentas.Haemorrhage is indicated by arrows. (D) Photographs from Hmox1+/+ (upper panels), Hmox1+/− (middle panels), and Hmox−/− (lowerpanels) whole implantation sites containing fetus, placenta, and uterus on days 8 (left) and 10 (right) of gestation. The fetuses togetherwith the ectoplacental cone were measured using the ImageJ program and the area sizes are indicated in Table 3. (E) The weights ofHmox1+/+ fetuses (n = 8), placentas (n = 8), and resorptions (dead fetuses, n = 2); Hmox1+/− fetuses (n = 14), placentas (n = 14), andresorptions (n = 16), and Hmox1−/− fetuses (n = 3), placentas (n = 3), and resorptions (n = 7). Data are shown as dot plots and mediansand significance were analysed by the non-parametric Kruskal–Wallis test followed by the Mann–Whitney U-test between two groups.∗p < 0.05. Pictures were taken with a total magnification of 200× (A–C) or 20× using light microscopy using the Axiovision Rel 4–6program (Zeiss AX 10 microscope).

Table 2. Hmox1+/− and Hmox-1−/− placentas presentdiminished number of GCs as well as augmented fibrosis andcompared with Hmox1+/+ placentasGenotype of the Giant Haemorrhage Fibrosisplacenta cells

Hmox1+/+ (n = 7) 2 (2–3) 2 (0–3) 0 (0–1)Hmox1+/− (n = 12) 1 (0–2) 1 (0–3) 1 (0–2)∗

Hmox1−/− (n = 9) 0 (0–3)† 3 (2–3)† 3 (1–3)†

Scoring of histopathological findings in Hmox1+/+ , Hmox1+/− , and Hmox1−/−

placentas: Giant cells were quantified as follows: 0 = no GCs; 1 = 1–10 GCsper ten fields; 2 = 10–20 GCs per ten fields; and 3 = 20–30 GCs per ten fields,after analysis of H&E-stained slides. For haemorrhage, the following scores wereconsidered: 0 = no haemorrhage; 1 = light haemorrhage in single regions,2 = massive haemorrhage in single regions; and 3 = massive haemorrhagein more than four fields. Fibrosis was visualized using Azan-Mallory stainingand was scored as follows: 0 = no fibrosis, 1 = light fibrosis in single areas,2 = massive fibrosis in single areas; and 3 = massive fibrosis in more thanfour fields. For all parameters, fields were analysed using a 20× objective lens.Data are expressed as medians (min–max); significance was analysed using theKruskal–Wallis test followed by the Mann–Whitney U-test between two groups.Differences (∗p < 0.05 and †p < 0.01) are indicated regarding the wild-typeanimals.

Table 3. Feto-placental units from HO-1 partial or total deficientmice are significantly smaller than those of HO-1 competent miceon days 10 and 12 of gestation

Hmox1+/+ Hmox1+/− Hmox1−/−

Day 8 127 081 (n = 16) 135 543 (n = 15) 120 338 (n = 5)Day 10 205 861 (n = 15) 170 043∗ (n = 15) 145 074∗ (n = 2)Day 12 348 863 (n = 14) 284 722† (n = 15) None detectable

The total area of the whole feto-placental unit (pixel2) was measured usingthe ImageJ program. Data are expressed as medians (n = 5 per group per timepoint); significance was analysed using the Kruskal–Wallis test followed bythe Mann–Whitney U-test between two groups. Differences (∗p < 0.05 and†p < 0.001) are indicated regarding the Hmox1+/+ type animals. At day 12 ofpregnancy, no viable feto-placental units were detectable in this experiment.

pregnancy, leading to intrauterine growth restriction(IUGR) and eventually to fetal loss.

HO-1 protective effects can be mimicked byexogenous CO applicationWe next asked whether CO, an end-product of haemcatabolism by HO-1, could overcome Hmox1 allele



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Figure 4. HO-1 protective effects on placentation and fetal growth are mediated by CO. (A) The percentage of fetal loss of Hmox1+/−females mated with Hmox1+/− males being exposed to either mixed air (n = 6) or 50 ppm CO (n = 5) during days 3–8 of pregnancy.Data are expressed as dot plots. ∗∗p < 0.01 as analysed by the non-parametric Mann–Whitney U-test. (B–G) Representative pictures ofthe histopathology of Hmox1+/+ (B, E), Hmox1+/− (C, F), and Hmox1−/− (D, G) placentas resulting from the Hmox1+/− × Hmox1+/−matings treated with mixed air (B–D) or 50 ppm CO (E–G) during days 3–8 of pregnancy. Special attention should be paid to fact that COtreatment restored the GC area in Hmox1+/− and Hmox1−/− placentas. (H, I) The weight of fetuses and placentas from the Hmox1+/− ×Hmox1+/− combination treated with either mixed air or 50 ppm CO during days 3–8 of pregnancy (n = 30 fetuses and their placentasfrom six females treated with air and n = 45 fetuses and their placentas from five females treated with CO). Data are shown as mean ±SEM and statistical analysis was carried out using the unpaired t-test. #p < 0.1. Pictures B–G were taken with a total magnification of200× using light microscopy using the Axiovision Rel 4–6 program (Zeiss AX 10 microscope).

Table 4. CO treatment during implantation and early placentationcan restore the placental phenotype of Hmox1+/− and Hmox1−/−

placentasGenotype of the placenta Giant cells Haemorrhage Fibrosis

Hmox1+/+ + air (n = 5) 2 (2–3) 2 (0–3) 0 (0–1)Hmox1+/+ + CO (n = 5) 2 (2–3) 1 (0–2) 1 (0–2)Hmox1+/− + air (n = 8) 1 (0–2) 1 (0–3) 1 (0–2)Hmox1+/− + CO (n = 7) 3 (1–3)∗ 1 (0–2) 0 (0–2)∗

Hmox1−/− + air (n = 4) 0 (0–3) 3 (2–3) 3 (1–3)Hmox1−/− + CO (n = 3) 2 (1–3)∗ 2 (1–2)† 1 (0–1)†

Scoring of histopathological findings in Hmox1+/+ , Hmox1+/− , and Hmox1−/−

placentas from Hmox1+/− mothers treated either with mixed air or CO (50 ppm)in isolated boxes during days 5–8 of pregnancy (see the Materials and methodssection Supplementary Figure 10 for a more detailed description). Giant cellswere quantified as follows: 0 = no GCs; 1 = 1–10 GCs per ten fields; 2 = 10–20GCs per ten fields; and 3 = 20–30 GCs per ten fields, after H&E staining. Forhaemorrhage, the following scores were considered: 0 = no haemorrhage; 1 =light haemorrhage in single regions; 2 = massive haemorrhage in single regions;and 3 = massive haemorrhage in more than four fields. Fibrosis was visualizedusing Azan-Mallory staining and was scored as follows: 0 = no fibrosis, 1 = lightfibrosis in single areas; 2 = massive fibrosis in single areas; and 3 = massivefibrosis in more than four fields. For all parameters, fields were analysed usinga 20× objective lens. Data are expressed as medians (min–max); significancewas analysed using the Kruskal–Wallis test followed by the Mann–Whitney U-test between two groups. Differences (∗p < 0.05 and †p < 0.01) are indicatedregarding air-treated animals of the same genotype.

deletion and restore the beneficial effects of HO-1on reproductive outcome. When applied in vitro toRcho-1 cells, CO (500 ppm) prevented the deleteri-ous effect of ZnPPIX, restoring the viability of tro-phoblastic stem cells (Supporting information, Supple-mentary Figure 7). The CO salutary effect was notassociated with modulation of MAPK activation, asassessed for ERK1/ERK2, JNK1/2, and p38a. In a sim-ilar manner, CO did not impact on the activation ofthe transcription factor NFκB (data not shown). Whenapplied continuously via inhalation between days 3 and8 of pregnancy, CO (optimal dose 50 ppm) reducedthe percentage of fetal loss from Hmox1 +/− pair-ings to 28%, as opposed to 57% in air-treated con-trols (Figure 4A). CO increased significantly the rela-tive number of GCs (Table 4 and Figures 4B–4G) andreduced the extent of fibrosis (Table 5) in Hmox1 +/−and Hmox1 −/− placentas obtained from Hmox1 +/−mice. The size of the junctional area was also increasedafter CO treatment (Figures 4B–4G). We observedthat CO treatment diminished the rate of apoptosisbut did not affect lymphocyte infiltration in the pla-centa (data not shown). This clearly indicates thatHO-1/CO act at the trophoblast level, positively influ-encing its physiology, which has a profound impact



Table 5. Genotype of viable and dead fetuses from Hmox1+/− mothers treated with either mixed air or 50 ppm CO during days 5–8 ofpregnancy. Genotyping of fetuses (viable or dead) as found in n = 5 Hmox1+/− mothers treated with mixed air and in n = 5 Hmox1+/−mothers treated with 50 ppm CO. Statistical analysis was carried out by the chi square test

Hmox1+/+ Hmox1+/− Hmox1−/−

Viable Dead Viable Dead Viable Dead

Air (n = 50 embryos from 5 mothers) 6 2 14 24 1 350 ppm CO (n = 56 embryos from 5 mothers) 6 0∗ 35 9† 6 0∗

∗p < 0.05; †p < 0.01.

100

A B

* *

80

60

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20

0

% o

f fet

al d

eath

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le

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hemin

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Hmox1+/+

Hmox1+/-

Hmox1+/-

vehicle hemin hemin

#

#

##

*

*

*

*

Figure 5. Free haem provokes intrauterine fetal death. (A) The percentage of intrauterine fetal death observed in Hmox1+/+ (n = 6)and Hmox1+/− (n = 4) pregnant females treated with 20 mg hemin/kg body weight at days 7, 9, and 11 of gestation and sacrificed onday 14 of pregnancy. Hmox1+/+ (n = 4) and Hmox1+/− (n = 3) controls received vehicle containing NaOH. (B) Viable (#) and dead (∗)fetuses after vehicle (left panel) or haem application (middle and right panels) at day 14 of pregnancy. It should be mentioned that haemapplication led to either very high percentages of fetal death (middle panel) or to total fetal loss with unidentifiable fetuses/placentas andvery thick uteruses (right panel).

on placentation. CO increased the weight of fetuses(Figure 4H) and placentas (Figure 4I) in Hmox1 +/−pairings. CO did not affect the total number of implan-tations (healthy + dead) (Supporting information, Sup-plementary Figure 6), but greatly influenced the via-bility of fetuses. CO application resulted in a muchhigher percentage of viable Hmox1 −/− embryos com-pared with air-treated controls (Table 5). CO furtherreduced the intrauterine mortality from Hmox1 +/− andHmox1 +/+ mice (Table 5). This shows that CO medi-ates to a large extent the salutary effects of HO-1 onpregnancy outcome; its application seems to compen-sate for the partial absence of the enzyme in uterinetissue of Hmox1 +/− mothers.

Free haem mediates intrauterine fetal death

After observing that insufficient levels of HO-1 led toIUGR followed by fetal death, we wondered whetherthis may be due to inadequate catabolism of free haem,which is most likely generated from oxidized cell-free haemoglobin. Haem administration (20 mg/kg) towild-type (Hmox1 +/+) or heterozygote (Hmox1 +/−)mice during late placentation, at days 7, 9, and 11 ofpregnancy, increased the rate of fetal death (Figures 5Aand 5B) and led to placental haemorrhage (Supportinginformation, Supplementary Figure 8). Lower doses(10 and 15 mg/kg) did not provoke fetal death, while ahigher dose (30 mg/kg) was lethal for both the mother

and the progeny. These observations demonstrate thathigh levels of free haem mediate intrauterine fetaldeath and suggest that the haemorrhage observed inHmox1+/− and Hmox 1 −/1 placentas may be the causeof inadequate placentation followed by fetal death inthese animals.

CO acts therapeutically to suppress IUGR byreducing the levels of circulating free haem

To test whether CO can be used therapeutically to sup-press fetal loss in a clinically relevant situation, weapplied CO via inhalation to DBA/2J-mated CBA/Jmice. This model is a well-established experimen-tal model of fetal loss [24] that shares features withhuman recurrent miscarriage and IUGR [25]. We rea-soned that CO might compensate for the previouslyestablished reduced levels of HO-1 expression at thefetal–maternal interface of these mice [18]. Fetal deathwas abolished in mice receiving CO (50 ppm) fromdays 5–8 of pregnancy (Figure 6A), in contrast withair-treated controls, despite comparable total implan-tation rates (Supporting information, SupplementaryFigure 9). CO also significantly increased the weight ofboth fetuses and placentas (Figures 6B and 6C, respec-tively). The protective effect of CO was associated withdiminished levels of circulating free haem (Figure 6D),clearly indicating that the protective effects of CO relyto a large extent on the inhibition of haem release from



60A B C D

50

40

30

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%)

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eigh

t (m

g) 1.71.61.51.41.31.21.11.00.9

Fre

e ha

em

CO air CO air CO air CO

*** *** *** ***

Figure 6. CO prevents fetal death while diminishing the levels of free haem in a mouse IUGR model. (A) Fetal loss in percentages forDBA/2J-mated CBA/J females treated with either mixed air (n = 8) or 50 ppm CO (n = 8) during days 5–8 of pregnancy. Data are shownas dot plots. ∗∗∗p < 0.001 as analysed by the non-parametric Mann–Whitney U-test. (B, C) Weight of fetuses (B) and placentas (C) fromCBA/J × DBA/2J mating combinations treated with either mixed air or 50 ppm CO during days 5–8 of pregnancy (n = 44 fetuses and theirplacentas from eight females treated with mixed air and n = 56 fetuses and their placentas from eight females treated with CO). (D) Thelevels of circulating free haem in animals treated with either mixed air (n = 8) or 50 ppm CO (n = 8). Data are shown as mean ± SEM.∗∗∗p < 0.001 as analysed by unpaired t-test.

haemoprotein, leading to reduced levels of circulatingfree haem, as demonstrated for other pathologies [26].

Discussion

There are few examples of genes whose expressionis strictly required at multiple stages of pregnancyin mammals. Genes involved in uterine receptivityand implantation have been reviewed by Wang andDey [27]. The transcription factor P53 [28] and theleukaemia inhibitory factor (Lif ) [29] have both beenshown to support blastocyst implantation in the mouse.Less is known about genes involved in placentation[30,31].

The use of homozygous animals carrying a deletionfor a specific gene is a useful tool for understandinghow a given molecule affects reproduction. It hasbeen shown, for example, using knockout femalesfor the progesterone receptor that although developingnormally to adulthood, they display significant defectsin several reproductive tissues, being unable to ovulate[32]. Molecules whose expression is linked to thestress-responsive gene Hmox1, eg cox-2 [33,34], affectreproductive processes as well. Using animals carryinga deletion for the Hmox1 gene, we have now irrefutablyshown that HO-1 plays a central role in supportingpregnancy in mice. HO-1 expression is essential tosupport placentation, fetal development, and growth,thereby enabling pregnancy success that consequentlyensures species survival.

Contrary to what was previously suggested [11–13],Hmox1 −/− females are not infertile. They do getpregnant but their fetuses die in the uterus. Thisexplains the low yield of Hmox1 -deficient mice firstreported by Poss and Tonegawa [11] and confirmedthereafter by Yet et al [12], as well as the observationsby Kreiser et al reporting positive effects associatedwith Ad-HO-1 administration on pup weight in rats[35] and our observations that transduction with Ad-HO-1 promotes fetal survival [20].

HO-1 is expressed at the fetal–maternal interfaceas early as the stage of blastocyst implantation. While

not absolutely essential for implantation to occur, HO-1 supports timely blastocyst attachment to uterineepithelial cells in vitro. Delayed implantation is knownto have a profound impact on placentation and fetalgrowth [36]. We confirm that Hmox1-deficient embryospresent defective placentation. HO-1 is required togive rise to giant cells, important for placentation,and also to viable spongiotrophoblasts and glycogencells—the junctional zone—and a normal labyrinthstructure. Interestingly, haemorrhage is visualized inpathological placentas, which may be due to an excessof free haem as the exogenous application of haemprovokes placental haemorrhage. Increased fetal lossin mice with Hmox1 deletion in spite of normalimplantation numbers is then caused by defectiveplacentation, which results in suboptimal nourishmentof the fetuses. Fetuses and placentas partially or totallylacking Hmox1 show diminished size and weightcompared with wild-type embryos. These featuresclosely resemble intrauterine IUGR, which can lead tofetal intrauterine death. Together with the histologicalobservations made recently by Zhao et al in non-genotyped placentas from heterozygote mothers [37],our data unveil a key role for HO-1 in blastocystimplantation, placentation, and fetal growth. Notably,IUGR in humans has been associated with insufficientHO-1 levels [15,19]. Lack of HO-1 expression inHmox1 −/− blastocysts in Hmox1 +/− mothers maybe compensated to some extent by HO-1 expressionin the maternal uterus, which explains the ability ofHmox1 +/− mice to procreate, albeit not providing theexpected knockout yield.

The observation that free haem is sufficient perse to precipitate intrauterine fetal death in wild-typeHmox1 +/+ mice strongly supports the notion that,firstly, free haem in high doses can cause intrauterinefetal death and, secondly, expression of HO-1 is strictlyrequired to suppress the deleterious effects of free haemand to prevent intrauterine fetal death. As recentlyreported, free haem seems to have a dual effect, beingprotective at normal or slightly above normal concen-trations because it up-regulates the levels of HO-1,but highly pathogenic when present at higher levels



[38]. The effects of HO-1 seem to occur independentlyof the maternal adaptive immune system, suggestingthat the protective effect of HO-1 does not rely onits immunoregulatory effect [7]. The salutary effects ofHO-1 reported here are most probably mediated by oneof the end-products of haem catabolism, namely thegasotransmitter CO. This notion is strongly supportedby the observation that exogenous CO applied betweenpre-implantation and placentation prevents fetal loss inHmox1 +/− females mated with Hmox1 +/− males com-pared with air-treated controls, rescuing Hmox1 −/−fetuses. The protective effect of CO was associatedprimarily with the normalization of placenta morphol-ogy, as shown by normalized numbers of GCs andrestoration of the junctional zone, and increased fetaland placenta weights. In a clinically relevant modelof IUGR characterized, among others, by low HO-1 expression at the fetal–maternal interface [18], COdrastically improved fetal survival, while diminishingthe circulating levels of free haem. We conclude thatproduction of CO via haem catabolism by HO-1 pos-itively influences placenta formation, which leads to aproper oxygen and nutrient supply and results in ade-quate fetal growth. Maternal hypoxia during the earlystages of placentation activates the invasive endovascu-lar trophoblast cell lineage and promotes uterine vascu-larization, positively influencing placentation [39]. Thisstrongly supports our hypothesis on CO beneficiallyinfluencing placentation.

CO might be used therapeutically to suppress earlyand recurrent onset of fetus abortion, associated withhuman HMOX1 polymorphisms [40], or to preventIUGR in pre-eclampsia associated with low HO-1levels [15,16]. We found that CO can also be usedtherapeutically to fully suppress IUGR when appliedexogenously during late implantation and throughoutplacentation. This confirms the great potential of COfor pregnancy complications, as it is already known toprotect in a variety of pathologies [3,10,41,42]. Pre-eclampsia, known to be caused by shallow trophoblastinvasion [15,19], hence, abnormal placentation, notonly has been linked to HO-1 deficiency but itsincidence is also significantly lower in smokers [43,44].CO has been proposed as the main reason for this[45]. Our work is the first scientific proof that CO canin fact positively influence placentation and preventpregnancy pathology, most probably by diminishingelevated levels of circulating free haem.

Expression of HO-1 affords protection againstimmune-mediated inflammatory diseases [4,41,42]because of its cytoprotective action, which limits tis-sue damage in inflammatory conditions associated withhaemolysis [4,46,47]. Since haemolysis co-exists withpro-inflammatory cytokines during implantation andplacentation, and haem metabolism is required in theplacenta, it becomes apparent why the expression ofHO-1 is strictly required for viable pregnancies asdemonstrated here. The cytoprotective effect of HO-1 is mediated via the enzymatic degradation of free

haem, therefore limiting haem accumulation and avoid-ing tissue damage. This effect can be mediated viaCO that binds the Fe2+ in the haem group of ferroushaemoproteins such as haemoglobin [48], preventinghaemoglobin oxidation and haem release from oxidizedHb [42]. The combined effect of haem degradation perse and inhibition of haem release from haemoproteinsreduces the levels of circulating free haem, a potentpro-oxidant, thereby supporting proper placentation aswell as fetal development and growth.

In conclusion, we have demonstrated that HO-1plays a central role in reproduction, acting as anevolutionary conserved mechanism that ensures properplacentation and consequently normal fetal growthand development by preventing the accumulation offree haem. HO-1 emerges as a protective gene thatavoids cell and tissue damage and supports pregnancydevelopment through CO release. We propose thatthis enzymatic system can be used for therapeuticpurposes via the inhalation of minimal amounts ofCO to suppress several major pathological outcomesof pregnancy including IUGR, for example, associatedwith pre-eclampsia.

AcknowledgmentWe are very grateful to Dr SF Yet, currently atthe National Health Research Institutes, Taipei, Tai-wan, for providing the Hmox1 mice and to KatjaWoidacki, for her assistance with the histology pic-tures. We thank Markus Scharm and Silvia Cardosofor their excellent technical assistance, as well asRocio Soldati for her assistance with the HO-1 activ-ity assays. Very special acknowledgment goes to Pro-fessor Michael Soares, Department of Pathology andLaboratory Medicine, University of Kansas MedicalCenter, Kansas City, Kansas, USA, for his techni-cal advice and for critically reading the manuscript.This work was supported by grants from the DeutscheForschungsgemeinschaft (DFG ZE 526/5-1), the GEMIFund (GEMI 018/07) to ACZ, and the Walther-SchulzFoundation to ACZ and SF. Further support was pro-vided by the Boehringer Ingelheim Foundation (grantsto ACZ and MLZ). MLZ was a PhD fellow fromthe Charite, Berlin. This work was further supportedby Fundacao para a Ciencia e Tecnologia (Portugal)grants [POCTI/SAU-MNO/56066/2004, POCTI/BIA-BCM/56829/2004, PTDC/BIA-BCM/101311/2008,PTDC/SAU-FCF/100762/2008 (MPS)] as well as bythe European Community, 6th Framework Grant (LSH-2005-1.2.5-1 to MPS).

Author contribution statement

MLZ, PAC, and MPS designed and performed research,analysed data, and contributed to manuscript prepa-ration. TEM, SR, SL, and NL performed researchand analysed data. HDV and SF analysed data andcontributed to manuscript preparation. ACZ designed,



performed and supervised research, analysed data, andwrote the paper with the help of MPS.

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SUPPORTING INFORMATION ON THE INTERNET

The following supporting information may be found in the online version of this article.

Supplementary materials and methods.

Figure S1. In vitro model for implantation.

Figure S2. HO-1 expression does not influence implantation number.

Figure S3. HO-1 expression does not alter hormonal levels during pregnancy.

Figure S4. HO-1 down-regulation by ZnPPIX diminishes trophoblast cell viability.

Figure S5. HO-1 down-regulation by siRNA diminishes the viability of primary trophoblasts.

Figure S6. CO counteracts the negative effects of ZnPPIX on trophoblast viability.

Figure S7. CO application does not modify the implantation numbers of Hmox1+/− mice.

Figure S8. Application of free haem during pregnancy leads to haemorrhage in the placenta.

Figure S9. CO application does not modify the implantation numbers of DBA/2J-mated CBA/J females.

Figure S10. Gas chambers used for the in vivo application of either mixed air or CO.

Table S1. Time that Hmox1 +/+, Hmox1 +/− or Hmox1 −/− needed to attach to uterine epithelial cells in an in vitro implantation model.

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Haem oxygenase-1 dictates intrauterine fetal survival in mice via carbon monoxide