Maternal Obesity Alters Placental Cell Cycle Regulators in the First Trimester of Human Pregnancy: New Insights for BRCA1

In the first trimester of pregnancy, placental development involves a wide range of cellular processes. These include trophoblast proliferation, fusion, and differentiation, which are dependent on tight cell cycle control. The intrauterine environment affects placental development, which also includes the trophoblast cell cycle. In this work, we focus on maternal obesity to assess whether an altered intrauterine milieu modulates expression and protein levels of placental cell cycle regulators in early human pregnancy. For this purpose, we use first trimester placental tissue from lean and obese women (gestational week 5+0–11+6, n = 58). Using a PCR panel, a cell cycle protein array, and STRING database analysis, we identify a network of cell cycle regulators increased by maternal obesity in which breast cancer 1 (BRCA1) is a central player. Immunostaining localizes BRCA1 predominantly to the villous and the extravillous cytotrophoblast. Obesity-driven BRCA1 upregulation is not able to be explained by DNA methylation (EPIC array) or by short-term treatment of chorionic villous explants at 2.5% oxygen with tumor necrosis factor α (TNF-α) (50 mg/mL), leptin (100 mg/mL), interleukin 6 (IL-6) (100 mg/mL), or high glucose (25 nM). Oxygen tension rises during the first trimester, but this change in vitro has no effect on BRCA1 (2.5% and 6.5% O2). We conclude that maternal obesity affects placental cell cycle regulation and speculate this may alter placental development.


Introduction
Adequate human placental development is crucial for embryonic and fetal growth. During the first trimester of pregnancy, placental development relies on several cellular processes, including trophoblast proliferation, differentiation, and fusion [1,2]. Within placental villi, the structural unit of the first trimester placenta, villous cytotrophoblasts (vCTs) proliferate and fuse to give rise to the

Results
In order to determine the effect of maternal obesity on placental cell cycle modulators, first trimester placental tissue from non-smoking women was divided into two groups, i.e., lean (n = 37, mean body mass index (BMI) = 22.2 kg/m 2 ) and obese (n = 21, mean BMI = 32.3 kg/m 2 ). Table 1 shows the sample characteristics for each experimental approach.

Maternal Obesity Affects Placental Cell Cycle Regulators Already in the First Trimester of Pregnancy
A PCR panel and a protein array were used to first test the effect of maternal obesity on gene expression and protein levels of several cell cycle regulators in placental tissue from lean and obese women (week 7). The results were analyzed using a multivariate linear regression (MVLR) model with BMI as the exposure variable adjusting for maternal age.

Maternal Obesity Affects Placental Cell Cycle Regulators Already in the First Trimester of Pregnancy
A PCR panel and a protein array were used to first test the effect of maternal obesity on gene expression and protein levels of several cell cycle regulators in placental tissue from lean and obese women (week 7). The results were analyzed using a multivariate linear regression (MVLR) model with BMI as the exposure variable adjusting for maternal age.

BRCA1 Is a Key Player in Cell Cycle Regulation in Early Pregnancy and Is Upregulated by Maternal Obesity
Among all the cell cycle regulators analyzed in the present study, only BRCA1 mRNA and protein were concordantly upregulated by maternal obesity (Figure 2A and Supplementary Figure  S1). Using the STRING database network analysis tool, we established a protein-protein association network to identify possible functional interactions between these obesity-upregulated cell cycle Figure 1. Induction of negative cell cycle regulators by maternal obesity in early pregnancy. Volcano plots show fold change for genes (A) and proteins (B) differentially expressed between lean (n = 7) and obese (n = 6) placental tissue (gestational week 7). PCR panel and protein array results were analyzed through multivariate linear regression using BMI as exposure, adjusting for maternal age. Differences were considered significant when p < 0.05 and the fold change threshold was set to 1.3. x axis = log2 fold change (lean versus obese), y axis = multivariate linear regression p value. Legend: BRCA1, breast cancer 1; ERRC5, excision repair 5; p (Ser1423) -BRCA1, phospho-BRCA1.

BRCA1 Is a Key Player in Cell Cycle Regulation in Early Pregnancy and Is Upregulated by Maternal Obesity
Among all the cell cycle regulators analyzed in the present study, only BRCA1 mRNA and protein were concordantly upregulated by maternal obesity (Figure 2A and Supplementary Figure S1). Using the STRING database network analysis tool, we established a protein-protein association network to identify possible functional interactions between these obesity-upregulated cell cycle modulators. Within this network, BRCA1 was located at a central position ( Figure 2B, arrow), interacting with proteins involved in different cell cycle events such as Chk1, Chk2, and Myc.  BRCA1 upregulation by maternal obesity was confirmed by Nanostring analysis and Western blotting. Nanostring data showed an increase in BRCA1 expression by 52.3% in placental tissue from obese versus lean women (p = 0.032, Figure 3A). Similarly, placental BRCA1 and p (Ser1423) -BRCA1 protein levels were also increased within the obese cohort (by 49.1%, p = 0.044 and 57.4%, p = 0.001, respectively) compared to the lean cohort ( Figure 3B-D). The differences observed were independent of fetal sex. Protein-protein interaction analysis of the upregulated proteins using the STRING database shows a central role of BRCA1 in the cell cycle regulation network (B, arrow). Line shape indicates the predicted mode of action, with nodes describing protein action effects (arrow: positive, dash: negative, circle: unspecified) and line color describing protein action types (blue: binding, black: reaction, green: activation, red: inhibition, pink: posttranslational modification, violet: catalysis). The minimum required interaction score was set to medium confidence (0.4), showing up to 10 interactions in the first shell. BRCA1 upregulation by maternal obesity was confirmed by Nanostring analysis and Western blotting. Nanostring data showed an increase in BRCA1 expression by 52.3% in placental tissue from obese versus lean women (p = 0.032, Figure 3A). Similarly, placental BRCA1 and p (Ser1423) -BRCA1 protein levels were also increased within the obese cohort (by 49.1%, p = 0.044 and 57.4%, p = 0.001, respectively) compared to the lean cohort ( Figure 3B-D). The differences observed were independent of fetal sex. Int. J. Mol. Sci. 2019, 20, x FOR PEER REVIEW 5 of 17 Figure 3. Placental BRCA1 is upregulated by maternal obesity. BRCA1 expression and protein levels were determined in first trimester placental tissue (gestational week 7) from lean (n = 6-7) and obese (n = 6) women by Nanostring (A) and Western blotting (B-D), respectively. Gene counts of Nanostring analysis were normalized to the mean of two different housekeeping (HK) genes (WD repeat domain 45B (WDR45L) and TATA box binding protein (TBP, A). Immunoblots for BRCA1 and p (Ser1423) -BRCA1 (B) were quantified by densitometric analysis (C and D). β-actin and α-tubulin were used for normalization as loading controls. Results are presented as mean ± SD. Statistical analysis was performed using the Mann Whitney test or t-test as appropriate.

BRCA1 DNA Methylation Is Not Altered by Maternal Obesity
Obesity influences placental gene expression through DNA methylation [26]. To test whether obesity-associated BRCA1 upregulation is the result of epigenetic changes in early pregnancy, placental BRCA1 methylation was assessed. We found no evidence that maternal obesity alters the DNA methylation profile of the 86 CpGs in the BRCA1 gene in the first trimester placenta ( Figure 4). Mean beta values for the sum of all CpG sites of obese total tissue DNA samples (0.319, gestational week 6 +0 -11 +6 , n = 15) were similar to those of the lean group (0.318, gestational week 5 +0 -11 +2 , n = 15). The BRCA1 methylation profile was also not affected by maternal age (data not shown). A list with all analyzed CpGs can be found in Supplementary Table S3. . Placental BRCA1 is upregulated by maternal obesity. BRCA1 expression and protein levels were determined in first trimester placental tissue (gestational week 7) from lean (n = 6-7) and obese (n = 6) women by Nanostring (A) and Western blotting (B-D), respectively. Gene counts of Nanostring analysis were normalized to the mean of two different housekeeping (HK) genes (WD repeat domain 45B (WDR45L) and TATA box binding protein (TBP, A). Immunoblots for BRCA1 and p (Ser1423) -BRCA1 (B) were quantified by densitometric analysis (C and D). β-actin and α-tubulin were used for normalization as loading controls. Results are presented as mean ± SD. Statistical analysis was performed using the Mann Whitney test or t-test as appropriate.

BRCA1 DNA Methylation Is Not Altered by Maternal Obesity
Obesity influences placental gene expression through DNA methylation [26]. To test whether obesity-associated BRCA1 upregulation is the result of epigenetic changes in early pregnancy, placental BRCA1 methylation was assessed. We found no evidence that maternal obesity alters the DNA methylation profile of the 86 CpGs in the BRCA1 gene in the first trimester placenta ( Figure 4). Mean beta values for the sum of all CpG sites of obese total tissue DNA samples (0.319, gestational week 6 +0 -11 +6 , n = 15) were similar to those of the lean group (0.318, gestational week 5 +0 -11 +2 , n = 15). The BRCA1 methylation profile was also not affected by maternal age (data not shown). A list with all analyzed CpGs can be found in Supplementary Table S3.

Placental BRCA1 Is Mainly Localized to vCTs and EVTs during the First Trimester of Pregnancy
BRCA1 location in early, mid, and late first trimester placental tissue (gestational week 5, 8, and 12, respectively) was detected by immunostaining. Both vCTs and EVTs showed strong BRCA1 staining which was localized to the nuclei and the cytosol ( Figure 5A, C, E). Stromal cells were also stained with anti-BRCA1. BRCA1 immunostaining in the ST was located in the nuclei ( Figure 5B, D, F, arrowheads). No differences in location were observed between early, mid, and late first trimester placental specimens. Negative (immunoglobulin G isotype) and positive (ovarian tumor section) controls showed no signal or strong nuclear and cytosolic BRCA1 staining, respectively (Supplementary Figure S2).
All the experiments were performed at 2.5% O2, which was considered as a physiological O2 tension for early first trimester placenta. Since oxygen tension in the intervillous space rises during the first trimester period covered by the explant experiments (weeks 5-10), we tested the potential effect of oxygen on BRCA1. To this end, chorionic villi (gestational week 5 +0 -9 +1 , n = 10-12) were also cultured under 6.5% O2 and the results compared to 2.5% O2. No differences in BRCA1 expression

Placental BRCA1 Is Mainly Localized to vCTs and EVTs during the First Trimester of Pregnancy
BRCA1 location in early, mid, and late first trimester placental tissue (gestational week 5, 8, and 12, respectively) was detected by immunostaining. Both vCTs and EVTs showed strong BRCA1 staining which was localized to the nuclei and the cytosol ( Figure 5A, C, E). Stromal cells were also stained with anti-BRCA1. BRCA1 immunostaining in the ST was located in the nuclei ( Figure 5B, D, F, arrowheads). No differences in location were observed between early, mid, and late first trimester placental specimens. Negative (immunoglobulin G isotype) and positive (ovarian tumor section) controls showed no signal or strong nuclear and cytosolic BRCA1 staining, respectively (Supplementary Figure S2).
The ratio between p (Ser1423) -BRCA1 and total BRCA1, reflecting activity of upstream ataxia telangiectasia mutated (ATM) kinase, was also not affected by any of the treatments described above (Supplementary Figure S3). In principle, the absence of a response to most of the treatments may have been due to low tissue viability. This can be ruled out since human chorionic gonadotropin, a major hormone produced by the trophoblast, was secreted into the culture medium, and its levels remained stable during the various treatments (Supplementary Figure S4). . BRCA1 is not regulated by short term exposure to obesity-associated inflammation, hyperglycemia, or oxygen tension in early pregnancy. First trimester placental chorionic villous explants from different placental tissues (n = 4-11, gestational week 5-11) were cultured at 2.5% O 2 with tumor necrosis factor α (TNF-α) (50 ng/mL), leptin (100 ng/mL), interleukin 6 (IL-6) (100 ng/mL), D-glucose (25 nM), and L-glucose (25 nM, osmotic control) for 48 h in triplicate. Explants (n = 10-12) were also cultured at 6.5% O 2 . BRCA1 gene expression was analyzed by RT-qPCR and normalized to the mean of hypoxanthine phosphoribosyltransferase 1 (HPRT1) and peptidylprolyl isomerase A (PPIA), which were used as housekeeping (HK) genes (A-C). BRCA1 and p (Ser1423) -BRCA1 protein levels were analyzed by Western blotting (D-F). Immunoblots were quantified by densitometric analysis (G-I for BRCA1 and J-L for p (Ser1423) -BRCA1), with β-actin used as a loading control. Data are shown as -∆Ct (A-C) or ratio to β-actin (G-L) and have been presented as mean ± SD from different placental tissues (n = 4-12). Statistical analysis included the Mann Whitney test or Friedman's test followed by Dunn's post hoc analysis.
All the experiments were performed at 2.5% O 2 , which was considered as a physiological O 2 tension for early first trimester placenta. Since oxygen tension in the intervillous space rises during the first trimester period covered by the explant experiments (weeks 5-10), we tested the potential effect of oxygen on BRCA1. To this end, chorionic villi (gestational week 5 +0 -9 +1 , n = 10-12) were also cultured under 6.5% O 2 and the results compared to 2.5% O 2 . No differences in BRCA1 expression ( Figure 6C) and BRCA1 and p (Ser1423) -BRCA1 protein levels ( Figure 6F, I, L) were detected between the two oxygen tensions.
The ratio between p (Ser1423) -BRCA1 and total BRCA1, reflecting activity of upstream ataxia telangiectasia mutated (ATM) kinase, was also not affected by any of the treatments described above (Supplementary Figure S3). In principle, the absence of a response to most of the treatments may have been due to low tissue viability. This can be ruled out since human chorionic gonadotropin, a major hormone produced by the trophoblast, was secreted into the culture medium, and its levels remained stable during the various treatments (Supplementary Figure S4).

Discussion
Adequate trophoblast proliferation, differentiation, fusion, and survival are required for successful placental development and function [22]. Although cell cycle regulation is crucial for these biological processes [27], placental cell cycle modulators and their potential regulation by the intrauterine environment have been scarcely investigated.
Obesity has been associated with low-grade sustained inflammation and oxidative stress, both classical triggers of cell cycle arrest [28]. In the present study, we demonstrated that maternal obesity increases the expression of several cell cycle regulators, i.e., 9 genes and 25 proteins, in the first trimester of pregnancy, suggesting that obesity affects placental cell cycle control already in early pregnancy. This might in turn compromise vCT proliferation and differentiation into EVTs, altering invasion and spiral artery remodeling, and may, thus, set the basis for obesity-associated pregnancy disorders such as preeclampsia [29]. Advanced maternal age is known to alter placental cell proliferation and apoptosis [30,31] and might also modify cell cycle regulation. Thus, this was accounted for in the present study by adjusting exposure-outcome relationships for maternal age. This should allow for the identification of obesity-mediated effects, although residual confounding cannot be excluded.
To subsequently characterize the effect of maternal obesity on specific cell cycle modulators, we selected BRCA1 as a potential candidate based on its consistent upregulation at the mRNA and the protein level. BRCA1 has been traditionally studied in the context of breast and ovarian cancer [32], where it plays a pivotal role in establishing an adequate DNA damage response [33,34]. It also plays an important role in cell cycle checkpoint regulation, inducing G1 cell arrest through p27 activation, blocking S phase entry through p53-dependent activation, and favoring G2/M arrest through p53-dependent 14-3-3 zeta/delta activation [35,36]. Interestingly, we found that BRCA1 was the only cell cycle modulator upregulated at both the gene and protein level by maternal obesity. Several BRCA1 protein binding partners were also upregulated by maternal obesity, including 14-3-3 zeta/delta, chk1, and chk2, and the latter is known to induce cell cycle arrest in a pathway involving cdc25 and p-BRCA1 [37,38]. We also observed that p (Ser1423) -BRCA1 was upregulated by maternal obesity. Several kinases are involved in BRCA1 phosphorylation, including chk2, Akt, and ATM, the latter being directly involved in BRCA1 phosphorylation at serine 1423 [39]. Phosphorylation of BRCA1 fine-tunes its function. The enhanced phosphorylation in obesity might reflect further BRCA1 interactions with other cell cycle regulators [40]. Indeed, our network analysis of functional protein associations using the STRING database identified BRCA1 as central in the pathways controlling placental cell cycle regulation in early pregnancy.
Only a few reports have investigated BRCA1 location in late first trimester and term human placental tissue [41,42]. Thus, a thorough characterization of BRCA1 location in early pregnancy was still required. We localized BRCA1 predominantly to vCTs and EVTs in early, mid, and late first trimester placental tissue. BRCA1 immunostaining within the ST was weaker and restricted to a few nuclei. A similar distribution pattern, i.e., absence or weak expression in ST versus high expression on vCTs and EVTs, has been described for several other cell cycle regulators including Ki67, cyclin A, p53, and p57 [24,43]. Considering that ST is characterized by the absence of an active cell cycle [44], BRCA1 positive nuclei within the ST might be the result of recent vCT-ST fusion.
It has been previously reported that subcellular BRCA1 location also determines its function [45]. We detected BRCA1 in the nuclei and cytosol of vCTs. Cytosolic BRCA1 location has been associated with highly proliferating breast cancer tumors [46]. Its presence in the cytosol of vCTs reflects the high proliferative potential, as can be expected from the trophoblast stem cell population. The essential role of BRCA1 for vCTs is also demonstrated by BRCA1 knock-down leading to increased apoptosis in the first trimester trophoblast cell line Swan71 [42], suggesting that BRCA1 promotes trophoblast survival. Likewise, its presence in EVTs might reflect the role of BRCA1 in cell cycle regulation during the gradual differentiation of vCTs into EVTs, which precedes invasion. Whether BRCA1 is directly involved in regulating cell proliferation and invasion in primary vCTs and EVTs needs to be further studied.
We then aimed to determine which obesity-associated molecular mechanisms could explain the differences observed in placental BRCA1 levels between lean and obese women. Among other mechanisms obesity has been shown to alter gene expression through DNA methylation [47], which may be a candidate mechanism to explain these differences. This hypothesis is also supported by a recent study demonstrating BRCA1 downregulation due to promoter hypermethylation in disorders characterized by trophoblast over-proliferation [48]. Despite the overall effects of obesity on placental DNA methylation [26], placental BRCA1 methylation profile was not affected by maternal obesity in early pregnancy.
To identify short-term drivers of BRCA1 changes related to obesity we chose those cytokines most prominently associated with the pro-inflammatory environment of obesity [49] and tested them in an in vitro villous explant culture model. TNF-α treatment altered BRCA1 gene expression without concomitant changes of BRCA1 protein. This lack of a short-term effect on BRCA1 protein levels does not preclude the possibility that chronic exposure to TNF-α in vivo can contribute to BRCA1 upregulation, as found in the obese cohort. Similarly, IL-6 and leptin did not regulate BRCA1 protein.
Although we carefully selected cytokine concentrations within the physiological range to mimic the in vivo environment in first trimester placenta [50], we did not investigate a potential interplay between these cytokines, which might also fine-tune placental BRCA1 regulation. Moreover, treatment of placental chorionic villous explants from lean women with pro-inflammatory cytokines only allows testing for their short-term effects (up to 48 h), which may not be long enough to induce changes seen as the result of long-term tissue exposure to the obesity environment.
Obesity-associated hyperglycemia has been shown to alter placental development and function [51]. Interestingly, hyperglycemia is able to promote cell cycle arrest via cyclin D1 and p21 upregulation [52]. Here, high glucose treatment did not alter human placental BRCA1 gene expression or protein levels. Intriguingly, women with BRCA1 mutations are more prone to developing diabetes, a risk that also increases in women with a high BMI [53]. In this regard, hyperinsulinemia is also a common feature of both obesity and diabetes, and high insulin levels have been linked to cell cycle arrest in mouse keratinocytes [54]. Thus, further studies assessing the potential role of obesity-associated hyperinsulinemia on BRCA1 levels are warranted.
Oxygen tension is one of the major regulators of vCT proliferation and EVT differentiation. Hence, we determined whether oxygen might directly regulate first trimester placental BRCA1 levels. Physiological oxygen concentrations rise from 2.5% to 6.5% O 2 in early pregnancy [55]. Although we have shown that BRCA1 and p (Ser1423) -BRCA1 levels remained stable under both physiological oxygen concentrations, BRCA1-associated RING domain protein 1 (BARD1) is increased in first trimester trophoblast under physiological low oxygen tension (6.5% O 2 ) [56]. The BARD1-BRCA1 dimer results in BRCA1 stabilization [35]. Thus, BRCA1 activity might indirectly be regulated by oxygen tension.
To the best of our knowledge, this is the first study assessing the influence of maternal obesity on placental cell cycle regulators during the first trimester of pregnancy. The results using placental tissue, which preserves the spatial arrangement of cells and the extracellular matrix, in which they are embedded, clearly demonstrated an effect of maternal obesity on several cell cycle modulators. Among these we could identify BRCA1 as a central node in the network of cell cycle regulator proteins. It localizes to vCTs and EVTs independently of maternal BMI. Key components of the obesity-associated inflammatory and metabolic environment are unlikely to contribute to these changes. The physiological increase in oxygen tension and DNA methylation do also not appear to drive BRCA1 upregulation in obesity.
In the present study all the experiments were conducted on human first trimester placental tissue in a physiological, i.e., low, oxygen environment. This is a major strength and avoids potential hyperoxic effects of ambient oxygen (21% O 2 ). The main limitation of this study was the lack of an in-depth analysis of the cellular consequences of obesity-associated BRCA1 changes. Moreover, concentration and time-dependent effects of pro-inflammatory cytokines and the hyperglycemic environment on BRCA1 regulation cannot be excluded. Likewise, other maternal metabolic factors that could explain the obesity associated long-term effects on BRCA1 levels, e.g., hyperinsulinemia and insulin resistance, were not investigated in the present study and their contribution cannot be ruled out.
Collectively, our results suggest that BRCA1 might play a role in first trimester trophoblast biology. Thus, its upregulation by maternal obesity might alter placental development with potentially ensuing adverse consequences for maternal and fetal health.

Study Subjects
The study was approved by the institutional review board and the ethical committee of the Medical University of Graz (29-095 ex16/17, 23 December 2016). Women with a singleton pregnancy scheduled for legal elective pregnancy termination were recruited upon signing written informed consent. Since smoking has major effects on metabolism and inflammation, we carefully excluded smoking women, who were identified by serum cotinine levels ≥0.03 nmol/L [57]. Women with other comorbidities and those under current medication were also excluded from the study.
Upon inclusion, clinical data was collected (Table 1). Maternal BMI was calculated using the formula BMI = weight (kg)/height (m) 2 . Gestational age was calculated based on the patient's last menstrual period and verified by measurement of the fetal crown-rump length.

Human Placental Tissue Collection
First trimester placental tissue (gestational week 5 +0 -11 +6 ) was obtained after surgical pregnancy termination, washed with phosphate-buffered saline (PBS) and cryopreserved at −80 • C until further use, or was fixed in 4% paraformaldehyde (PFA) and paraffin-embedded. Fetal sex was assessed by gene expression analysis (DDX3Y and XIST, see 4.6). Based on the maternal pre-pregnancy BMI, gestational age matched samples were subsequently divided into two groups, i.e., lean (mean BMI = 22.2 kg/m 2 ) and obese (mean BMI = 32.3 kg/m 2 ).

DNA/RNA Isolation and Reverse Transcription
First trimester placental tissue was homogenized in RLT Plus Buffer (Qiagen, Venlo, Netherlands) with 1% β-mercaptoethanol (v/v, Merck) using a tissue lyser (MagNa Lyser, Roche, Basel, Switzerland). DNA and total RNA was isolated with the AllPrep DNA/RNA/miRNA Universal Kit (Qiagen) according to the manufacturer's guidelines. After a quality check (Bioanalyzer, RNA integrity number (RIN) > 3.0), mRNA reverse transcription was performed using the SuperScript II Reverse Transcriptase kit (Life Technologies, Carlsbad, CA, USA) as per the manufacturer's protocol.

PrimePCR Panel
Differential expression of 187 genes associated with DNA damage-repair and cell cycle regulation was analyzed using a PrimePCR Collection panel (DNA damage H384 Predesigned 384-well, BioRad Laboratories, Munich, Germany) according to the manufacturer´s guidelines. A complete list of the genes and controls can be found on the manufacturer's website (https://www.bio-rad.com/de-at/primepcr-assays/pathway/dna-damage-collection-panel). For each PCR reaction 10 ng of cDNA were used. Real-time PCR was then conducted using the CFX384 PCR detection system (BioRad Laboratories) and Ct values were generated by the associated software. Results were analyzed using the 2 −∆∆Ct method. Hypoxanthine phosphoribosyltransferase 1 (HPRT1) and TATA box binding protein (TBP) were selected as housekeeping genes since their expression was unaffected by maternal obesity.

Real Time PCR
BRCA1 expression was determined by quantitative real-time PCR using FAM-labeled TaqMan gene expression assays (Life Technologies, BRCA1: Hs01556193_m1). Fetal sex was determined in a multiplex PCR setup using FAM-labelled DDX3Y and VIC-labelled XIST expression assays (Life Technologies, DDX3Y: Hs00965254_gH, XIST: Hs01079824_m1) as described elsewhere [58]. RT-qPCR was performed using TaqMan Universal PCR Master Mix (Life Technologies) using the CFX96 Thermocycler (BioRad Laboratories). A calibrator sample was added onto each plate to control for inter-run variations. Ct values were generated by the BioRad CFX Manager 3.1 software and relative gene expression was calculated by the 2 −∆∆Ct method, with HPRT1 (Life Technologies, HPRT1: Hs02800695_m1) and peptidylprolyl isomerase A (Life Technologies, PPIA: Hs04194521_s1) used as housekeeping genes.

Nanostring
PrimePCR Panel validation was performed using the NanoString nCounter system (Nanostring Technologies, Seattle, WA, USA), which is based on direct digital detection of mRNA molecules using target-specific, color-coded probe pairs that hybridize directly to target molecules. Gene expression was measured by counting the barcode for each specific molecule, which is detected by a digital analyzer.
Positive normalization to the geo-mean of the top three positive controls and codeset normalization on the reference genes WD repeat domain 45B (WDR45L) and TBP was performed using nSolver software (Nanostring Technologies). Results have been expressed as gene counts of mRNA molecules in 100 ng/µL RNA.

Protein Isolation and Quantification
Placental tissue was lysed in RIPA buffer (Sigma Aldrich) containing complete protease inhibitors (Roche) using a tissue lyser (MagNa Lyser, Roche). Protein concentration was determined using the bicinchoninic acid assay (BCA, Thermo Fisher Scientific) as per the manufacturer's guidelines.