PPARγ Expression Is Diminished in Macrophages of Recurrent Miscarriage Placentas

PPARγ belongs to the group of nuclear receptors which is expressed in the trophoblast and together with other factors is responsible for the maintenance of pregnancy. Apart from that PPARγ is also a main factor for macrophage polarization. The aim of this study was to investigate the combined expression pattern and frequency of PPARγ under physiological circumstances and in spontaneous and recurrent miscarriages in the trophoblast and in maternal macrophages of the decidua. Human placental tissues of the first trimester (15 physiologic pregnancies, 15 spontaneous abortion and 16 recurrent miscarriage placentas) were analyzed for expression of the nuclear receptor PPARγ. Expression changes were evaluated by immunohistochemistry and real time PCR (RT-PCR) in trophoblast and in maternal macrophages of the decidua. Maternal macrophages were identified by double immunofluorescence using cluster of differentiation 68 (CD68) as marker for macrophages and further characterized regarding their M1/M2 polarization status. The intermediate villous trophoblast revealed a significantly lower PPARγ expression in spontaneous and recurrent abortion. Maternal macrophages express PPARγ. Their number is significantly enhanced in the decidua of spontaneous miscarriages whereas in recurrent miscarriages maternal macrophages seem to express PPARγ only in very few cases. PPARγ is associated with an M2 polarization state that is common for decidual macrophages. The lack of PPARγ in recurrent miscarriage decidual macrophages seems to be associated with a specific inflammatory response against the fetus.


Introduction
Miscarriage, which is defined as either spontaneous or recurrent, is a common disorder in pregnancy [1]. It affects 25-50% of all reproductive-aged women. Immunologic, endocrine and metabolic mechanisms are involved in the success of human pregnancy and disturbances in any of these processes can lead to fetal loss [2]. Established risk factors are fetal chromosomal or endocrine disorders for spontaneous miscarriages and the antiphospholipid syndrome, thrombophilia or maternal anatomical malformations, especially for recurrent pregnancy losses [3]. In nearly 50% of affected patients, however, the cause of miscarriage remains unknown [4].
Peroxisome proliferator-activated receptor γ (PPARγ) belongs to the family of nuclear receptors [5] that are key players in maintaining pregnancy [6,7]. PPARγ, together with its heterodimer binding partner retinoid X receptor alpha (RXRα), are involved in cell proliferation, cell differentiation, and organogenesis [8]. RXRα is upregulated in extravillous trophoblast in recurrent miscarriages in humans [9]. RXRα plays a pivotal role in the receptor family, due to its ability to form heterodimers with other nuclear receptors. Heterodimer partners include, e.g., peroxisome proliferator-activated receptor (PPAR), thyroid hormone receptor (TR), and liver X receptor (LXR) [2,[10][11][12]. Especially the expression of the isoform PPARγ is linked to trophoblast invasion [13] and downregulation of the isoform RXRα seems to protect from apoptosis in human trophoblasts [9].
Not only trophoblast cells express PPARγ, but also macrophages [14]. Macrophages play a key role in immune response and they can respond to environmental stimuli by acquiring specific phenotypes [15]. In response to external cues they will undergo classical M1 activation with high levels of inflammation and microbicide as well as anti-tumor activity. Alternatively, the M2 pathway contains mostly parasite containment, tissue remodeling and most importantly in this case immunomodulatory functions like pregnancy [16][17][18].
Our former studies showed that the number of decidual macrophages is increased at the feto-maternal interface of preeclampsia placentas [19] and also in spontaneous miscarriage cases. An additional finding was the FasL-positivity of these macrophages [20]. Therefore, the aim of this study was a phenotype characterization of macrophage populations in abortive placental tissue, its PPARγ expression and the characterization of PPARγ expressing trophoblast sub-types.

PPARγ-Expression in the Trophoblast
The expression of PPARγ in the nucleus and cytoplasm of trophoblast cells was analyzed in tissue from healthy pregnancies (    Immunohistochemical staining of decidual macrophages with CD68 as a marker for macrophage positivity. Decidual macrophages were increased in RM (16 cases) and SM samples (15 cases) compared to the control group (15 cases), (a). In recurrent miscarriage specimens, the decidual macrophages tended to be upregulated (p = 0.181) (b). In spontaneous miscarriage samples, the population of macrophages was significantly higher compared to the control (p = 0.013) (c). Summary of staining results of CD68 positive decidual macrophages (d). Scale is 200 μm. The insert picture is 100 μm scaled.

Identification of PPARγ-Expressing Cells in the Decidua Basalis
Decidua basalis tissue of regular first trimester pregnancies (15 cases), SM (15 cases) and RM (16 cases) was double stained using antibodies against PPARγ (green staining), and CD68 (red staining). Nuclear staining appeared in blue. PPARγ + CD68 double immunofluorescence staining was performed to investigate the macrophage expression of PPARγ in RM, SM and control groups. CD68 staining in the cytoplasm of normal decidual cells is shown in Figure 3a. Figure 3b

Identification of PPARγ-Expressing Cells in the Decidua Basalis
Decidua basalis tissue of regular first trimester pregnancies (15 cases), SM (15 cases) and RM (16 cases) was double stained using antibodies against PPARγ (green staining), and CD68 (red staining). Nuclear staining appeared in blue. PPARγ + CD68 double immunofluorescence staining was performed to investigate the macrophage expression of PPARγ in RM, SM and control groups. CD68 staining in the cytoplasm of normal decidual cells is shown in Figure 3a.  Figure 3i shows a strong co-expression of both markers. We identified CD68 positive macrophages also expressing PPARγ in the healthy and in the spontaneous miscarriage placenta. In the group of recurrent miscarriages only very few PPARγ-expressing macrophages (5-8% of the macrophages in RM are PPARγ-positive) were detected.

Characterization of the Macrophage Population in Recurrent Miscarriage Cases
Because macrophages in RM cases (16 cases) expressed PPARγ only in 5-8% of the total macrophage population compared to healthy control tissue (15 cases) and SM (15 cases), we further characterized these cells with a panel of M1/M2 markers and CD68. The M1 marker iNOS is expressed in 2-5% of the macrophages in healthy controls (CD 68 Figure 4a

Characterization of the Macrophage Population in Recurrent Miscarriage Cases
Because macrophages in RM cases (16 cases) expressed PPARγ only in 5-8% of the total macrophage population compared to healthy control tissue (15 cases) and SM (15 cases), we further characterized these cells with a panel of M1/M2 markers and CD68. The M1 marker iNOS is expressed in 2-5% of the macrophages in healthy controls (CD 68 Figure 4a

Evaluation of PPARγ Expression with Real-Time RT-PCR (TaqMan)
PPARγ mRNA (PPARG) expression was analyzed in placental tissue from SM, RM and healthy controls by quantitative RT-PCR. PPARγ was significantly downregulated in SM (15 cases, 1.8-fold; p = 0.010) and in RM (16 cases, 1.5-fold; p = 0.004) compared to the control group (15 cases, Figure 8).  (15 cases). This bar graph shows the mean of relative PPARγ expression; therefore, the presentation of error bars is not appropriate.

Discussion
Within this study we could show that PPARγ is downregulated in the intermediate villous trophoblast (IVT) in both spontaneous (SM) and recurrent miscarriage (RM) placentas. The downregulation of PPARγ was confirmed by RT-PCR in both miscarriage pregnancy cases.
In addition, we showed that in recurrent miscarriages, decidua basalis macrophages are nearly PPARγ-negative, whereas in normal controls and surprisingly also in SM decidua basalis macrophages are all PPARγ-positive. The additional characterization of the macrophage polarization status using the M1 polarization markers TLR2 and iNOS [22] and the M2 polarization markers CCL1 and CX3CR1 confirmed the loss of M2 polarized marcrophages [23] in recurrent miscarriages.
PPARγ as nuclear receptor is already known to be essential for the maturation of alternatively activated M2 macrophages [24]. M2 macrophages and decidual macrophages have mainly immune regulatory and homeostatic properties [25]. These macrophages have little in common with pro-inflammatory M1 macrophages, which is in line with the role for decidual macrophages in establishing and sustaining fetal tolerance [26].
In addition, rosiglitazone as a selective peroxisome PPARγ agonist has been shown to induce an M2 macrophage polarization via activating the PPARγ pathway [27,28]. The activation of PPARγ suppresses gene transcription by interfering with signal transduction pathways, such as the nuclear factor 'kappa-light-chain-enhancer' of activated B-cells (NF-κB), Signal transducer and activator of transcription (STAT), and Activator protein 1 (AP-1) pathways that are involved in pro-inflammatory immune responses [29][30][31]. It is a striking result of this study, that we could identify the loss of PPARγ in decidual macrophages of patients with recurrent miscarriages but not in patients with SM and of course in normal control placentas.
In spontaneous miscarriages, we identified a significant increase of decidual macrophages, although they were PPARγ positive. In former studies, we could show that these macrophages express FasL [20]. The expression of FasL on decidual macrophages had been already described before [32].  (15 cases). This bar graph shows the mean of relative PPARγ expression; therefore, the presentation of error bars is not appropriate.

Discussion
Within this study we could show that PPARγ is downregulated in the intermediate villous trophoblast (IVT) in both spontaneous (SM) and recurrent miscarriage (RM) placentas. The downregulation of PPARγ was confirmed by RT-PCR in both miscarriage pregnancy cases.
In addition, we showed that in recurrent miscarriages, decidua basalis macrophages are nearly PPARγ-negative, whereas in normal controls and surprisingly also in SM decidua basalis macrophages are all PPARγ-positive. The additional characterization of the macrophage polarization status using the M1 polarization markers TLR2 and iNOS [22] and the M2 polarization markers CCL1 and CX3CR1 confirmed the loss of M2 polarized marcrophages [23] in recurrent miscarriages.
PPARγ as nuclear receptor is already known to be essential for the maturation of alternatively activated M2 macrophages [24]. M2 macrophages and decidual macrophages have mainly immune regulatory and homeostatic properties [25]. These macrophages have little in common with pro-inflammatory M1 macrophages, which is in line with the role for decidual macrophages in establishing and sustaining fetal tolerance [26].
In addition, rosiglitazone as a selective peroxisome PPARγ agonist has been shown to induce an M2 macrophage polarization via activating the PPARγ pathway [27,28]. The activation of PPARγ suppresses gene transcription by interfering with signal transduction pathways, such as the nuclear factor 'kappa-light-chain-enhancer' of activated B-cells (NF-κB), Signal transducer and activator of transcription (STAT), and Activator protein 1 (AP-1) pathways that are involved in pro-inflammatory immune responses [29][30][31]. It is a striking result of this study, that we could identify the loss of PPARγ in decidual macrophages of patients with recurrent miscarriages but not in patients with SM and of course in normal control placentas.
In spontaneous miscarriages, we identified a significant increase of decidual macrophages, although they were PPARγ positive. In former studies, we could show that these macrophages express FasL [20]. The expression of FasL on decidual macrophages had been already described before [32].
The role of the Fas/FasL system in the conditions of spontaneous abortion and pregnancy had been described for T cell apoptosis by decidual and trophoblast cells earlier [33,34]. Our group was able to describe an increased expression of FasL in decidual macrophages of spontaneous miscarriages [20]. Therefore, we speculated that FasL expression by macrophages could be a part of an M2-like polarization. FasL-expressing macrophages could induce apoptosis to Fas-bearing activated T-cells reducing potentially harmful immune responses against the semi-allogenic embryo. We further assumed that macrophages might mediate the T-cell triggered trophoblast apoptosis highlighting an alternative way of inducing apoptosis in SM [15].
Placental growth is exponential in the first trimester of pregnancy and involves coordinated events in trophoblast and mesenchyme, one of these is the differentiation of progenitor cytotrophoblast cells into intermediate villous trophoblast cells (IVT). These IVT are programmed to either fuse with the syncytium, or are transferred to extravillous trophoblast cells [35]. We found a downregulation of PPARγ in the IVT compartment in both spontaneous and recurrent miscarriage on protein as well as on mRNA-level. Already decades ago, the natural binding partner of PPARγ, the RXRα was described in this trophoblast compartment: RAR and RXR, both types of receptors were present in the proliferative intermediate villous trophoblast [36]. Later, RXRα was found to play a crucial role in pregnancy and is a key regulator of apoptosis in trophoblasts of patients with recurrent miscarriages [9]. PPARγ, on the other hand, was also described to be dysregulated in different trophoblast compartments of the miscarriage placenta [37,38], although PPARγ expression in the IVT was never investigated before. Interestingly, Fournier et al. described that activation of PPARγ induces accumulation of lipids, villous trophoblast differentiation and inhibits trophoblast invasiveness [39]. In addition, the expression of PPARγ is downregulated by stimulation of trophoblast cells with either arachidonic acid or 15d-PGJ2 [40]. Because we identified a downregulation of PPARγ in the IVT of miscarriages and a missing PPARγ expression in macrophages of RM cases, we might speculate that PPARγ ligands (e.g., prostaglandins) are released to a higher extent under these pathological circumstances.

Patient Data
The Institutional Review Board of the Ludwig-Maximilian-University, Munich, (Number of approval: 337-06, 29 December 2006) approved this study. All women signed an informed consent allowing analysis of all clinical and laboratory data mentioned in this study. Placental tissue from spontaneous miscarriages (SM) (n = 15) and recurrent miscarriages (RM) (n = 16) at gestational weeks 4 to 13 was obtained at the Department of Obstetrics and Gynecology, LMU Munich. Placental tissue from legal terminations of healthy pregnancies (n = 15) served as control group. The tissue was collected at a private practice clinic in Munich, Germany. The control group specimens were confirmed as healthy by a blinded independent pathologist. All placental material was acquired by dilatation and curettage, without any prior pharmaceutical induction. In cases of SM and RM, the operation was performed within 24 h after diagnosis. Instantly, after the uterine curettage, the obtained tissue was either frozen or formalin fixed for further analysis. All patients included had an inconspicuous family and medical history, which was obtained systematically. Patients with common disorders, autoimmune diseases, thrombophilia and microbiological infections (Bacteria and Chlamydia trachomatis) were excluded. Chromosomal abnormalities were ruled out by karyotype analysis in all samples, as described recently [11,41]. Table 1 summarizes the number of samples used for immunohistochemical staining for each gestational week. Table 2 shows the demographic and clinical characteristics of the study population. 1.6 ± 0.9 (1-4) 3.1 ± 1.1 (2)(3)(4)(5) 0.001 Values are Mean ± S.D. * Mean, standard deviation, range.

Immunohistochemistry
Formalin-fixed tissue slides were embedded in paraffin wax for immunohistochemistry. Samples were deparaffinized in xylol for 20 min and rinsed in 100% ethanol. Methanol/H 2 O 2 incubation for 20 min was performed to inhibit endogenous peroxidase reaction. Afterwards, the specimens were rehydrated in deescalating alcohol gradients, starting with 100% ethanol and ending with distilled water. The samples were cooked in a pressure pot, containing a sodium citrate buffer (pH = 6.0), which consisted of 0.1 mM citric acid and 0.1 mM sodium citrate in distilled water. Subsequently, samples were washed in PBS twice and incubated with a blocking solution (reagent 1, ZytoChem Plus HRP Polymer System (Mouse/Rabbit), Zytomed, Berlin, Germany) for 5 min. Incubation with the primary antibody was performed with each section for 16 h at 4 • C. All antibodies used are listed in Table 3. Following every subsequent step, samples were washed twice in PBS (pH = 7.4). Blocking solutions, containing post block (reagent 2) for 20 min and HRP-Polymer (reagent 3) for 30 min, were applied. The chromogen-substrate staining was carried out using the Liquid DAB+ Substrate Chromogen System (Dako Scientific, Glostrup, Denmark), 1 min for CD68 and 2 min for PPARγ. The reaction was stopped by applying distilled water. Finally, tissue samples were counterstained with Hemalaun for 2 min and blued in tap water. Specimens were dehydrated in an ascending alcohol gradient and cover slipped with Eukitt ® quick hardening mounting medium (Sigma Aldrich, St. Louis, MO, USA). Positive control (human colon tissue) as well as negative control staining was carried out as described previously [10,40]. All slides were analyzed using the microscope Leitz Wetzlar (Wetzlar, Germany; Type 307-148.001 514686). The immunoreactive score (IRS) was used for evaluation of the intensity and distribution pattern of antigen expression. This semi-quantitative score is calculated as follows: the optical staining intensity (grades: 0 = none, 1 = weak, 2 = moderate, 3 = strong staining) is multiplied by the total percentage of positively stained cells (0 = none, 1 ≤ 10%, 2 = 11-50%, 3 = 51-80% and 4 ≥ 81% of the cells). This multiplication has a minimum of 0 and a maximum of 12. Analysis of all slides was performed independently by two experienced staff members. Total number of macrophages in a magnification field of 40× lens was calculated three times each in 3 different areas of the decidua basalis. The median number was calculated.

Evaluation of PPARγ-Expressing Cells as Macrophages
For the visualization of PPARγ-expressing cells in the trophoblast, tissue samples of SM, RM, both first-trimester abortion placentas, and healthy controls (first trimester) were used. The antibodies used are shown in Table 3. Double immunofluorescence staining for PPARγ and CD68 as a specific macrophage marker was performed to identify expression patterns in the nucleus and the cytoplasm.

Evaluation of M1/M2 Marker on Decidual Macrophages
In order to further characterize the macrophage polarization state, TLR2 and iNOS, as well as CCL1 and CX3CR1, were used for M1 and M2 polarization, respectively. Each specimen was incubated overnight at 4 • C with monoclonal anti-CD68 mouse IgG1 and one of the polyclonal IgG antibodies against PPARγ, TLR2, iNOS, CCL1, or CX3CR1. Polyclonal Cy-2-and polyclonal Cy-3-conjugated antibodies (Dianova, Hamburg, Germany) were used as secondary antibodies. Incubation was performed for 30 min at room temperature. Samples were fixed with Vectashield ® mounting medium with DAPI (Vector Laboratories; Burlingame, CA, USA) and analyzed with the Axioskop fluorescent photomicroscope (Zeiss; Oberkochen, Germany). Images were taken with the Axiocam camera system (Zeiss CF20DXC).

Reverse Transcription
According to the protocol reverse transcription (RT) was carried out with the High Capacity cDNA Reverse Transcription Kit (Applied Biosystems™, Fisher Scientific Company, Waltham, MA, USA) and placed in a mastercycler ® gradient (Eppendorf, Hamburg, Germany). RT conditions were as follows: 10 min at 25 • C, 2 h at 37 • C, 5 min at 85 • C and continued by a hold step at −20 • C.

Real-Time Reverse Transcription PCR
After conversion of RNA to cDNA, PCR was performed on all samples individually. Real-Time Reverse Transcription PCRs were covered with optical caps in optical 96-well (Applied Biosystems™, Fisher Scientific Company, Waltham, MA, USA) reaction microtiter plates. Each reaction was accomplished with a volume of 20 µL, including 1 µL cDNA, 8 µL H 2 O (DEPC treated DI water; Sigma, Taufkirchen, Germany) and 10 µL TaqMan ® Fast Universal PCR Master Mix 2× (Applied Biosystems, Nr. 4367846; 50 mL). The total contained 1 µL TaqMan ® Gene Expression Assay 20× (HS01115513_m1 for PPARγ, Applied Biosystems). The temperature protocol was as follows: 20 s at 95 • C, 40 cycles of amplification, denaturation for 3 s at 95 • C and denaturation plus annealing process for 30 s at 60 • C. Processing the PCR assays was performed using the 7500 Fast Real-Time PCR System (Applied Biosystems), and quantification was accomplished by the 2 −∆∆Ct method using β-actin as housekeeping gene (Applied Biosystems, Hs_99999903_m1).

Statistics
Analysis of the collection and statistical data was processed with the SPSS software version 24 (SPSS, Chicago, IL, USA) and Excel version 12.3.1 (Microsoft Windows 2016; Redmond, WA, USA). The Mann-Whitney U signed-rank test was used for the comparison of two independent groups. p-values < 0.05 were considered to be statistically significant.

Conclusions
Mouse knockout models showed that PPARγ is essential for placentation. PPARγ depletion leads to fetal loss in early pregnancy due to the missing PPARγ expression and extended placental defects [42]. In addition, decidual M1-like macrophage polarization events are associated with PPARγ modulation strategies [14]. Therefore, the PPARγ pathway is a new molecular target for future preventive strategies for the treatment of spontaneous and recurrent miscarriages.