South Korea has one of the lowest fertility rates in the world, reaching a record low of 0.98 in 2018. Understanding socioeconomic differentials in fertility in South Korea has become an important social and policy issue.This study examines socioeconomic differentials in first and second childbirths among married women using various indicators of socioeconomic status at the individual and household level.Using the Korean Labor and Income Panel Study (1998–2017), discrete-time hazard models are used to evaluate the relationships between multiple indicators of socioeconomic status and the transition to first and second births.Higher socioeconomic status  is conducive to a transition to parenthood and second births. However, the wife’s employment – standard employment in particular – is negatively associated with both first and second childbirth. Among the indicators of socioeconomic resources, stable housing arrangements and the husband’s employment security appear to be the most important factors for a married couple’s fertility decisions.Socioeconomically disadvantaged married couples tend to delay their transition to parenthood. In addition, those with high SES are more likely than their counterparts with low SES to have second births. If these patterns persist, they have important implications for the demographic process and social stratification.The findings of this study contribute to a comprehensive understanding of socioeconomic differentials in fertility in South Korea and therefore have important policy implications. These findings will also prove useful to other societies with very low fertility rates. Specifically, this study evaluates how the socioeconomic conditions of both wife and husband  and of household  are associated with the transition to first and second births. The findings of this study will shed light on the socioeconomic determinants of fertility in Korea by providing a more comprehensive picture of socioeconomic differentials in parity-specific fertility, which have important policy implications. These findings also have implications for other societies that are experiencing low fertility and growing socioeconomic differences in family behaviors . According to second demographic transition theory, changes in values and attitudes, such as individualization and secularism, are the main drivers of low fertility and related family changes in Europe and other Western societies e.g., . While tOther scholars have emphasized the importance of economic factors for understanding low fertility. With the increase in female education and labor force participation, the opportunity costs of childbearing also increase e.g., . AccordiEconomic explanations of low fertility have gained importance in East Asian countries, especially after the Asian financial crisis in the late 1990s. Korea and other East Asian societies have since undergone substantial changes in economic structure, resulting in the growth of nonstandard jobs  and widening income inequality. Moreover, rising economic inequality and employment insecurity have created feelings of instability and apprehension, leading to greater investment in education and careers among young adults e.g., . ConsequWhile these theoretical explanations provide a generalized framework for understanding the socioeconomic determinants of family formation, it is also important to note that decisions about childbearing are made in a broad socioeconomic and cultural context . For ins2.22The fertility decline in Korea is largely driven by the trend toward delaying and even foregoing marriage, as childbearing occurs almost exclusively within marital union. As is well documented, the level of nonmarital fertility has been very low in Korea. In 2014 the percentage of nonmarital births was 1.9, the lowest among OECD countries, followed by Japan (2.3%) and Turkey (2.8%) . The OECReflecting the trend toward fewer and later marriages in Korea, the percentage of ever-married women aged 25–29 decreased from 83% in 1985 to 41% in 2005 . SimilarJeong-gyu-jig) and nonstandard workers (Bi-jeong-gyu-jig) has become salient in Korea. According to recent data, about one in three waged workers is classified as holding a nonstandard job, and the share of self-employed workers is over 20% , and Opo sedae is a generation giving up five things (the first three plus employment and homeownership)  and educational achievements . Nevertheless, it is clear that Korean young adults are keenly aware of economic inequality and insecurity as major barriers to family formation.With rising inequality in the labor market the perception of economic insecurity and inequality has spread, in particular among young people. For example, p) e.g., . Accordip) e.g., . The skeIt is also important to consider distinctive gender contexts in order to understand fertility behaviors in Korea. Korea and other East Asian societies are still influenced by traditional family values and gender norms in spite of the rapid expansion in female education and labor force participation . In East2.3One important trend documented in recent decades is the widening socioeconomic inequality in family outcomes e.g., . For insWhether a similar pattern can be observed in Korea and other East Asian countries is an interesting and important question. In particular, given the very low level of fertility in this region, whether individuals’ fertility decisions differ across socioeconomic strata has very important societal and policy implications. However, evidence from prior research on socioeconomic differentials in fertility is still limited. Existing literature also brings mixed results in that the relationship between socioeconomic status and fertility often varies by gender as well as by a specific indicator examined. For men, evidence suggests that higher socioeconomic status, such as income, education, or employment status (while differences in employment type are often ignored) is positively associated with parenthood, reflecting a strong expectation for men to assume the role of breadwinner e.g., . AnotherIn most developed countries, women’s education is closely linked to labor force participation . HoweverAt the same time, it is important to recognize that the educational differences in attitudes toward and behaviors surrounding childbirth found in Korea often contradict those found in other Western countries. Studies show that among unmarried Korean women, the highly educated hold more favorable views of marriage than those with lower education e.g., . This fiJeonse. Jeonse is a real estate contract unique to Korea in which a renter makes a lump-sum deposit to a landlord, called ‘key money,’ when a lease is signed. The amount of ‘key money’ usually ranges between 50% and 80% of the property’s value. The Jeonse contract is usually made for a 2-year term and, once signed, the tenant stays at the property without paying any additional monthly payments as the landlord collects interest earned on the deposit during the contract period. The amount of key money has been increasing following rising house prices in recent years, and according to a report from the early 2010s the average key money to rent an apartment with Jeonse in Seoul has reached around 300,000 USD  becomes a common housing arrangement, in particular among households headed by young adults   .While limited, evidence from prior research arrives at two scenarios regarding the relationship between socioeconomic status and fertility in the South Korean socioeconomic context. If having a child is a normative pathway after marriage, but higher-order births are perceived to be optional since the married couple have met normative and individual expectations, then socioeconomic differentials might be more pronounced for second births than for first births. However, it is also plausible that few socioeconomic differences are found by birth order if young people in Korea delay (or give up) childbearing for economic reasons, in particular factors beyond individual control . In addition, there might be gender differences in the role of socioeconomic status in childbirths depending on a specific indicator examined in light of distinctive gender contexts in family and at work. In particular, women’s employment would be negatively associated with childbirths due to the difficulty of combining work and family responsibilities. On the contrary, husbands having a better labor market status  would be conducive to the transition to childbirths, while having an insecure labor market status, especially a nonstandard job, would deter the transition, given the rigid labor market segmentation and the persistent male-breadwinner norm.3.3.1Data come from the Korean Labor and Income Panel Survey (KLIPS). The KLIPS is an annual longitudinal study based on a representative sample of 5,000 households in urban areas at the baseline (1998). Through direct face-to-face interviews, the household reference person is asked to provide information on all household members. If face-to-face interviews are not available, telephone interviews and self-administered questionnaires are used. The analytic sample includes data from wave 1 (1998) to wave 20 (2017), the most recent survey available at the time of conducting the current analyses. The KLIPS well suits the purpose of this study since it covers the period from after the financial crisis of 1997 to the present, during which time economic inequality has increased in Korea.In light of very low nonmarital birth rates in South Korea, only marital fertility is considered by restricting analyses to those in first marital union. This study examines (1) transitions to first births among childless married women and (2) transitions to second births among those with one child. Due to very low fertility and the possibility of selection , any higher order births  are excluded from the analyses  having increased in recent years in Korea . In addi3.2The first birth is coded as 1 if women in their first marriage who were childless at survey year (t) had given birth by survey year (t+1), and 0 for women who were still childless at year (t+1). The second birth is coded as 1 if women in their first marriage with one child at survey year (t) had another childbirth by survey year (t+1), and 0 for women who still had only one child at year (t+1). This means that only childless married women at year (t) are included in the models estimating the associations between socioeconomic status and the first birth, since they can only have the risk of a first childbirth. Similarly, in models for the second birth, only those who had one child are included as they are exposed to the risk of a second birth. Married women are censored after the event of interest  occurs and those who do not make the transition  are censored at last interview or observation.Various indicators of socioeconomic status at the individual and household level are considered in this study. First, educational attainment and employment type for both wife and husband are included: the level of education is a categorical variable, which identifies less than high school education, high school education (reference), some college or junior college degree, and university (or postgraduate) degree. The wife’s and husband’s employment type, based on information on labor force participation  and employment status , is categorized as standard employment (reference), nonstandard employment , self-employment (including family business), and non-employment. Following the classification of the Korean Statistics Bureau see  and consJeonse (key money deposit), Weolse (rent with a monthly payment), and other types .Annual household income for the past calendar year (logged) is included as a proxy for family economic conditions. Household income  includes wages (from employment), capital gains, social insurance, income from real estate property, and income from all other sources. Homeownership is a categorical variable consisting of four housing arrangements: homeowner (reference), All models control for demographic characteristics that might confound the relationship between socioeconomic status and fertility, including age of husband and wife and place of residence (Seoul (reference), metropolitan cities other than Seoul, and other areas). In addition, time since first marriage in the first-birth sample and time since first birth in the second-birth sample are included as a baseline hazard see .3.35 All variables included in the analyses, except for the sex of the first child, are time varying and are lagged by a year so that socioeconomic and other demographic characteristics precede the event of birth , logistic regression models were used. Results are presented with coefficients and 95% confidence intervals to show the uncertainty of results (7 results . In addiResults from Model 1 in Turning to relationships between wife’s employment type and the likelihood of first birth (Model 3), wives who are out of the labor force are more likely to have first births than employed women (in both standard and nonstandard employment). Those in self-employment also have a lower risk of first birth than non-employed women, but the 95% CI includes zero (−0.869 to 0.001). It is likely that women who plan to have a child may have left the labor force for that specific purpose, especially those whose husbands have better economic standing. In fact, women tend to be out of the labor force if their husbands hold standard employment . It is also worth noting that wives’ employment type makes little difference to the likelihood of them having a first birth. Even those with careers in standard employment who may benefit from recent policy changes  do not differ from wives in unstable nonstandard jobs in terms of the likelihood of having a first birth. In addition, husbands’ non-employment deters the transition to parenthood, according to results from Model 4. This finding is not surprising, in light of the normative expectations for men to be breadwinners in Korean society. Both standard work and self-employment are positively associated with first birth, but the 95% confidence interval for nonstandard employment includes zero (−0.026 to 1.504).Weolse (monthly rent) are less likely to have a first birth than homeowners, suggesting the importance of a stable housing arrangement to the timing of family formation  do not differ from zero.The next two models show results for household-level socioeconomic resources. According to Model 5, additional household income  is not associated with first birth. However, the likelihood of first birth is associated with type of housing arrangement (Model 6): those with on e.g., . HoweverWeolse (monthly rent) and first birth is attenuated: the 95% confidence interval now includes zero (−0.853 to 0.015).Model 7 includes all posited SES indicators in order to evaluate whether the associations observed in Models 1–6 change with the inclusion of all indicators. First, the strong positive association between tertiary education of the wife and the risk of first birth observed in Model 1 is attenuated. However, the disadvantage of lowest-educated women (relative to high school graduates) is still observed. In addition, the husband’s educational level is no longer associated with first birth when the wife’s education is considered . On the contrary, the relationship between employment type of both wife and husband and first birth (Models 3 and 4) remains the same: the wife’s employment delays the transition to first birth while the husband’s employment facilitates the transition. In addition, the coefficient for the husband’s nonstandard employment is now different from zero (95% CI are 0.124 to 1.730). Annual household income is not associated with the likelihood of first birth, as was found in Model 5. At the same time, the negative association between 5.2Next, Weolse (monthly rent) have the lowest likelihood of having a second birth, while no differences are found among other groups.Results from Model 1 also show that wife’s education is not associated with the transition to second birth. Lowest-educated women (no high school degree) have a lower likelihood of a second birth than high school graduates, but the 95 CI includes zero (−1.088 to 0.023). The results for husband’s education (Model 2) are similar for first births , as womeIn Model 7, which includes all indicators of socioeconomic status, most indicators are not associated with second childbirths. One exception is that the negative association between women’s standard employment and second births (Model 3) becomes stronger: women in standard employment are least likely to make the transition to a second birth among women of various employment statuses.6.As one of the countries with the lowest-low fertility and fastest-aging population, the issue of fertility decline has become a subject of serious concern in Korea. The government’s efforts to reverse the falling fertility trend include subsidizing childcare leave, child allowances, and expansion of nurseries, but with little success. Given the level of societal attention and the governmental resources provided to address this issue, it is surprising that we still have a limited understanding of fertility behaviors across socioeconomic status. To fill this gap in the literature, the present study examines the socioeconomic characteristics of both wives and husbands and household economic conditions, and their relation to transitions to first and second childbirth.Jeonse lease have a higher likelihood of having first and second births than those who rent by the month (Weolse).8 At the same time, it is important to note that the association between socioeconomic status and fertility often varies across indicators and gender. In general, higher socioeconomic status seems to be conducive to the transition to parenthood and second birth; however, employed wives are less likely than housewives to have a first child. This reflects the difficulty of combining work and motherhood due to gender norms emphasizing women’s domestic responsibilities and a family-unfriendly work culture   are positively associated with having a first and second childbirth. Also, those who can secure enough money to purchase a house or to deposit a lump-sum for a herings) . While wherings) . Indeed,herings) . A recenherings) .In addition, fertility differentials by husbands’ employment status are worth taking into consideration. Women have a lower likelihood of giving birth if their husbands hold nonstandard jobs compared to those whose husbands work in standard employment or self-employment. As discussed above, employment status is not only an indicator of one’s current labor market status but also of one’s future career trajectory in a rigidly segmented Korean labor market. In this context, married couples may postpone having their first child or subsequent children if the husband is not in secure standard employment (or does not run his own business), reflecting the persistent male-breadwinner norm. These findings are consistent with the finding that deteriorating employment prospects for young men are linked to declining marriage and fertility rates . By consWeolse (monthly rent) are least likely to make the transition to parenthood.9 With skyrocketing home prices and the lack of affordable housing, securing ‘key money’ becomes a challenge and is causing rising household debt in Korea  in fertility so as to design effective policy measures to reduce socioeconomic differentials in fertility and increase fertility rates. Also, gender division of labor might affect the transition to parenthood in light of the difficulty of combining work and family responsibilities that Korean women experience , which may be related to their socioeconomic status, can be selected into parenthood or into remaining childless. Employing analytic designs to explicitly deal with the issue of selection would be a useful extension in order to establish a causal relationship between socioeconomic status and fertility decisions. Second, building upon the findings of the current study, future research will need to examine socioeconomic differentials in fertility in more depth. For example, it is important to understand the role of housing debt and the source of money for housing purchases or perience . The preDespite these limitations, this study contributes to a comprehensive understanding of socioeconomic differentials in fertility behaviors in Korea by documenting the relationships between multiple indicators of socioeconomic status and parity-specific fertility. One of the main findings from this study is that men’s employment security and affordable housing are conducive to the transition to parenthood and subsequent births. Socioeconomic status driven by structural issues such as the real estate market, mortgage law, and employment relations cannot be easily addressed at the individual or household level. Therefore, large-scale policies with long-term investments  may be more fruitful to reverse or at least halt the trend of falling fertility rates in Korea. This study’s findings on socioeconomic differentials in fertility have further important implications beyond Korean society, given that many other developed countries also face growing inequality – in particular employment precarity and rising housing prices, as well as fertility decline.


We also found two small control-dominated clusters. We replicated these data on an independent, large birth cohort. Separation testing showed significant similarity scores between the clusters from the two independent cohorts of patients. Canonical pathway analysis of the unique genes defining these clusters demonstrated enrichment in inflammatory signaling pathways, the glucocorticoid receptor, the insulin receptor, EGF and B-cell signaling, These results support a genetic architecture defined by subgroups of patients that share variants in genes in specific networks and pathways which are sufficient to give rise to the disease phenotype.The likely genetic architecture of complex diseases is that subgroups of patients share variants in genes in specific networks sufficient to express a shared phenotype. We combined high throughput sequencing with advanced bioinformatic approaches to identify such subgroups of patients with variants in shared networks. We performed targeted sequencing of patients with 2 or 3 generations of preterm birth on genes, gene sets and haplotype blocks that were highly associated with preterm birth. We analyzed the data using a multi-sample, protein–protein interaction (PPI) tool to identify significant clusters of patients associated with preterm birth. We identified shared protein interaction networks among preterm cases in two statistically significant clusters,  However, GWAS have failed to demonstrate the “missing heritability” in many common diseases8. The computational approaches underlying GWAS reflect the “common disease-common variant hypothesis,” that complex disease architecture is due to additive genetic effects of variants in individual genes. However, the genetics of complex diseases suggests that is unlikely. The more likely genetic architecture is that subgroups of patients share variants in genes in specific networks and pathways which are sufficient to give rise to a shared phenotype. It is also likely that variants in genes in different networks and pathways express similar phenotypes and define different subgroups of patients.Genome-wide association studies (GWAS) are a contemporary approach to the investigation of complex diseases that have made possible discovery of insights not previously recognized11. We built the Database for Preterm Birth, containing a validated collection of genes with an a priori connection to preterm birth9. This was the result of a semantic data mining and curation of published literature and publicly available, high-throughput databases. We used this resource to analyze a large genome wide association study to identify the biological networks and pathways associated with preterm birth10. In this report, we present the results of targeted sequence of the genes, flanking sites and haplotype blocks identified by gene set enrichment of that GWAS10. Further, in order to leverage the likelihood of genetic discovery, we exploited an “extreme phenotype” of preterm birth by concentrating our enrollment on patients with a family history of preterm birth. We compared variants identified in women with 2–3 generations of preterm birth with term controls without history of preterm birth. We then used Proteinarium, a multi-sample, protein–protein interaction analysis (PPI) tool, to identify clusters of patients with shared PPI networks associated with preterm birth11.Preterm birth is an important, complex genetic disorder affecting up to 10% of pregnant women14. We therefore concentrated our efforts on identification of maternal genetic variants. Women & Infants Hospital of Rhode Island is the sole provider of high-risk perinatal services in Rhode Island, northeastern Connecticut and southeastern Massachusetts. We used this population-based service to enroll patients with a prior history of preterm birth. The study was approved by the Institutional Review Board of the Women & Infants Hospital of RI, 08-0117. All methods were performed in accordance with the relevant guidelines and regulations. An informatically driven retrieval from our electronic medical record gave us a daily report on all preterm births. A clinical research assistant, formally trained in genetic interviews, reviewed the records of all patients delivering ≥ 24 weeks and < 34 weeks. Following informed consent, women underwent an interview focused on family history of preterm birth. We asked explicit questions about preterm birth in mother, grandmother, her first order relatives and also paternal relatives. Careful clinical history with an emphasis on additional risk factors for prematurity including medical illnesses, drug use, psychiatric disorders and employment history was recorded on all patients. We excluded patients delivered prematurely for considerations related to preeclampsia, drug use, diabetes or multiple gestation. Controls were patients who delivered ≥ 37 weeks gestation in whom the same, formal genetic history revealed no history of preterm birth on either maternal or paternal side of the pedigree. All of the patients’ identifying data was coded and redacted for the purposes of data analysis. 190 patients were enrolled for targeted sequencing. Samples were taken from 122 women with multiple generations of preterm birth, and 68 race, ethnicity matched control women at term. Residual maternal whole blood was obtained for extraction of genomic DNA. The samples were stored continuously at − 80 °C until processing.Large epidemiological studies drawn from population based analyses support a predominantly maternal origin for the genetic contribution(s) to risk of preterm birth, with little contribution by paternal or fetal genetic factors10. Genomic DNA from maternal whole blood was extracted using QIAamp DSP DNA blood mini kit from Qiagen following the manufacture’s protocol. Samples were quantified using Qubit technology  and sequencing libraries were constructed from 2 μg each of case/control DNA. Library preparation was performed using Illumina TruSeq DNA LT Sample prep Kit , with enzymatic fragmentation using dsDNA Fragmentase (NEB), followed by indexing and clean-up. DNA capture was performed using custom capture probes from SeqCap EZ choice kit (Roche NimbleGen).We targeted the 329 genes and 132 haplotype blocks for sequencing that are highly associated with preterm birth15, sorted and indexed with SAMtools16.The library was sequenced on an Illumina HiSeq 2500 using 100 bp paired-end protocols. Following sequencing, the multiplex indices were used to bin the samples for each patient and QC sequence data was recorded. High quality sequence data from well-balanced pools was observed. There was an average of 25 million reads from each patient, with an average Q30 of 91%. Reads were then mapped to the to the human reference sequence (Hg19) with BWA17. Duplicate reads were marked and removed using Picard Tools version 1.77. Haplotype caller was applied for variant detection18. Twenty-five base pairs upstream and downstream of each exon were included in the design of capture probes and in variant detection. Variants were annotated using ANNOVAR for pathogenicity prediction scores. We used Eigenstrat to control for population stratification during genotype testing of differential abundance of variants in cases and controls19. To investigate the frequency of potentially relevant single variants, we extracted variants with the following filters: coverage ≥ 10×, a Polyphen 2 HDIV prediction if a change is damaging (≥ 0.957), a SIFT score (< 0.05), a CADD score > 10, and minor allele frequency (MAF) < 0.05 from the Exome Aggregation Consortium (ExAC)20, and significant difference by genotype testing22.Variants were flagged as low quality and filtered using established metrics: if three or more variants were detected within 10 bp; if four or more alignments mapped to different locations equally well; if coverage was less than ten reads; if quality score < 30; if low quality for a particular sequence depth (variant confidence/unfiltered depth < 1.5); and if strand bias was observed . A variant identified by any one of these filters was labeled “low quality” and not considered for further analysis. For variant discovery we used the Gene Analysis Tool Kit (GATK) version 3.2 to analyze the sequence readsProteinarium11. This is a tool for analysis of protein–protein interactions that uses the String interactome database, Dijkstra’s algorithm and the Jaccard index to build a network similarity matrix of protein–protein interactions (PPI) between samples11. The top 30 genes, based on the most significant variants  for each patient, were used as the seed genes for input into Proteinarium. We selected Proteinarium’s user defined output minimum path length of 2, which includes pathways in which seed proteins are connected directly to each other and/or via a single intermediary protein. We refer to these intermediary connecting proteins as imputed proteins. The output of Proteinarium is a UPGMA generated dendrogram that shows clusters of patients with shared PPI networks, gene lists forming the networks and the group assignment for each patient. Statistical significance for each branch under the dendrogram is calculated by Fisher exact test comparing the probability of observing a cluster of that size relative to the total number of samples and group assignment.In order to identify patients with shared networks and pathways associated with preterm birth, we used 21. This network-based approach compares the shortest distances between proteins within each disease or network to the shortest distances between the disease networks. This approach has been applied to other complex disease phenotypes22. We computed the separation between our clusters using the seed genes identified for each of the patients. Using the union of the seed genes for patients within each of the clusters, we identified genes unique to each cluster to use as input for the separation analysis21. A negative score indicates an overlap between networks within the interactome. The more negative the score the greater the similarity/overlap between two networks.Computational methods have been developed to identify disease-disease similarity by comparison of individual networks from the protein–protein interactomeProteinarium, we used a Bayesian generalized linear model implemented via the Arm package in R and the optimal model was determined using stepwise model selection with the MASS package (https://CRAN.R-project.org/).In order to identify significant differences in the clinical, phenotypic characteristics between the clusters identified by The clinical characteristics of the patients and their distribution by race/ethnicity are shown in Table 10. We restricted our subsequent analyses to variants in regions with greater than tenfold coverage which resulted in 39,472 variants. There were also almost 7000 exonic variants and several splice variants. After application of the initial filters for coverage and variant pathogenicity, there were a total of 264 variants . INOVA provided the variant data for the genes and haplotype block intervals described above. With similar functional filters, among the 264 variants identified from our cohort, 165 (63%) were also identified in the INOVA sequence data.For replication of these univariate data, we compared our results to a cohort of patients recruited at the INOVA Translational Medicine Institute, Falls Church VA Proteinarium. The resulting dendrogram is shown in Fig. Among our cases with a family history of preterm birth, we found that each patient had an average of 163 variants that passed the coverage and Eigenstrat genotype testing above. In order to identify clusters of patients with shared networks associated with preterm birth, the top 30 genes based on the most significant variants  for each patient were used as the seed genes for input into p < 0.001. Out of the 190 patients sequenced, a total of 66 subjects were assigned to statistically significant clusters. The two largest significant clusters (A and B) had significantly more cases than controls, encompassing 45 of the 122 cases. There were also two small control-dominated clusters. The layered networks for the case-dominated clusters A and B are shown in Fig. For ease of visualization the dendrogram has been circularized and the significant clusters have been highlighted in colors. The inset in Fig. Proteinarium. There were four significant case-dominated clusters identified encompassing 40 of the 60 cases. The layered networks for these four clusters are shown in Fig. For replication of this network analysis, a similar approach using the filters described above to identify variants in individual patients was applied to the INOVA cohort. The top 30 seed genes for each subject were used for input into Proteinarium. The two case dominated clusters from our preterm birth cohort (clusters A and B) showed overlap with each other within the interactome. This was reflected by negative separation values (separation = −0.285) when compared to the control dominated clusters that showed higher separation values (ranging from −0.037 to 0.11).We used separation testing to compare the networks identified by p values ranging from 106 to 108. The pathway similarity is illustrated in the heatmap shown in Fig. We also compared the separation of the case dominated clusters from our preterm birth cohort to the case dominated clusters in the INOVA replication cohort. The results are shown in Table Proteinarium. The clinical phenotype of the case patients in Cluster A, were compared to the characteristics of the remaining patients not included in Cluster A or Cluster B. An analogous analysis was performed for Cluster B. In addition, the characteristics of patients in Clusters A and B were compared to each other. The distribution of clinical characteristics for these comparisons is shown in Table We also sought to determine if there were underlying phenotypic differences between the patients in the different clusters we identified using Proteinarium, a multi-sample, protein–protein interaction tool, to identify clusters of patients with shared protein–protein interaction networks associated with preterm birth11. Using seed genes from each patient, Proteinarium mapped the input genes onto the STRING PPI interactome to build individual networks. The similarities between all subjects’ PPI Networks were used as the distance metric for clustering samples. We identified two significant clusters with a predominance of preterm birth patients encompassing 45 out of the 122 women with a multi-generation history of preterm birth. We also found two small control-dominated clusters. For replication, we compared our data to a large birth cohort collected at INOVA Health 24. Sequence data analyzed from INOVA’s 60 cases and 321 controls identified 40 preterm cases in four significant clusters. Separation analysis of the layered PPI networks of the significant clusters from our preterm birth cohort and the replication cohort demonstrated overlap of these clusters within the interactome. Canonical pathway analysis of the unique genes defining these clusters demonstrated enrichment in inflammatory signaling by IL-6, IL-7, IL-15, IL-8, JAK/Cytokine signaling, toll-like receptors, the glucocorticoid receptor, the insulin receptor, EGF and B-cell signaling, p ~ 10–6 to 10–8.We performed targeted sequencing of gene sets and haplotype blocks that are highly associated with preterm birth on carefully phenotyped patients. We enrolled women with idiopathic, singleton births < 34 weeks gestation, the majority of whom had multiple generations of preterm birth. We compared them to term controls with no family history of preterm birth. We used CXCR4 is among the gene sets and pathways upregulated in whole blood from women with spontaneous preterm birth when compared to patients delivering at term25. Moreover, CXCR4 is located in genomic regions with large ROH that we have shown to be in greater abundance in women delivering preterm than full term26. CXCR4 has also been evolutionarily linked to preterm birth27. CXCL8 (IL-8) is a monocyte macrophage chemoattractant. It has been widely studied in labor and shown to be expressed in multiple gestational tissues including myometrium, cervix and decidua28. CXCL8 is upregulated in chorio-decidua samples collected from preterm labor patients when compared to patients at term and in labor28. This is consistent with the increase in inflammatory cells in the decidua during labor. The gene FOXO3 codes for the forkhead box 03 transcription factor that regulates inflammation in non-gestational tissues. It has also been shown to be expressed in human myometrial tissue29. Further, higher FOXO3 gene and protein expression have been demonstrated in myometrium from women in labor compared to non-laboring samples. In isolated cells, FOXO3 silencing was associated with a significant decrease in IL-1 induced I-L6 and IL-8 expression and cyclooxygenase 2 production. Overexpression of FOXO3, increased cytokine expression, prostaglandin production and MMP9 expression are all observed in myometrial cells following administration of IL-1B. Thus, FOXO3 may be implicated in the pathways regulating labor and it may be a potential target for prevention of preterm birth29. The STAT5A gene is present in a network of cell-mediated immune responses, cellular movement and hematologic system development that was identified in a genome-wide association study looking at single nucleotide polymorphisms (SNP) in peripheral blood of patients who are in preterm labor compared to full term labor30. Most importantly, serial sampling and transcriptional profiling of circulating immune cells during pregnancy has been carried out to identify patterns of gene signatures across pregnancy that are associated with gestational age and thus define a “gestational clock” 31. These studies revealed an important role for IL-2 dependent STAT5A signaling in modulating T-cell function during pregnancy. Baseline gene expression was compared with expression following activation with receptor-specific ligands including interferon and a cocktail of interleukins. The endogenous STAT5A signal in naïve cells and the phospho-STAT5A response to interleukins and neutrophils were among the strongest features correlated to immunological adaptations to pregnancy and association with gestational age31. The Comparative Toxicogenomics Database (CTD) contains more than 5,000 curated and inferred gene–disease associations (including preterm birth) extracted from the published literature by formal curation32. STAT5A is also among the CTD gene disease phenotypic associations with preterm birth. MAPK8 is also among the CTD gene disease associations associated with preterm birth32. Lastly, several genes in Cluster A are involved in cellular migration and invasion, which may be important in the onset of labor. CTTN is the cortactin gene. It functions as a key regulator of actin cytoskeleton and has roles in actin-based cellular processes including cell migration and invasion. Polymorphisms in the VEGFA gene in a discrete haplotype block have been shown to be associated with preterm birth30. This may play an important role in angiogenesis during placentation. Nonetheless, the latter study was of modest size and the findings just reached nominal significance.The individual, unique genes from the two case-dominated preterm birth clusters are shown in Table EGF and the EGF receptor in human amnion cells regulates their proliferation and increases calcium mobilization and PGE2 production which may also have significant effects on uterine contractility33. PAK1, one of the genes identified in the Database for Preterm Birth9, encodes a member of the serine threonine P21 activating kinases. PAK1 is only present in pregnant myometrial tissue. PAKs have shown to regulate uterine contractility34. We have also previously reported that PAK1 is located in a genomic region with runs of homozygosity (ROH) which are significantly more abundant in mothers delivering preterm than term26. Activation of the CHRM1 receptor by acetylcholine increases uterine contractility35. Of functional significance, CHRM1 has been shown to be down-regulated in preterm human myometrium compared to patients at term and not-in-labor36. WASF1 is an A-kinase anchoring protein. It has been implicated in preterm labor by the Ontario Birth Study where it was shown to be differentially expressed in patients undergoing preterm labor37. It was further shown to be responsive to glucocorticoids in another study of peripheral blood mononuclear cells from patients delivering preterm37.In support of the network analysis, many of the genes are involved in uterine contractility, signal transduction and cell–cell signaling. Moreover, there is substantial literature based evidence that 4 out of the 6 unique genes in Cluster B are involved in uterine contractility. Signaling via 41. This has included case controlled studies of single nucleotide polymorphisms (SNP) in the protein coding regions, regulatory and intronic sequences of specific genes have been described. Because of the prominent inferences of inflammatory reactivity and alterations in uterine contractility, candidate have largely been investigated53. Modest associations in some SNPs and alterations in the expression of genes regulating inflammatory mechanisms have been identified. Nonetheless, the results do not explain the cause of prematurity without evidence of inflammation or infection. Additionally, while treatments directed at infection and inflammation in animal models have been successful, they have not demonstrated benefit in the treatment of preterm birth or prolongation of gestation in humans49.Previous investigations have been undertaken to identify social, environmental and genetic associations with preterm birth54. Using a combination of techniques, including ATAC-seq, Hi-C and ChIP-seq, they identified the chromatin landscapes in decidua-derived stromal cells. These were then compared to the heritability of annotations in the GWAS of pregnancy-related traits. They showed the heritability of gestational-duration was enriched for functional annotations in decidual stromal cells54. Volozanoka recently carried out targeted sequencing of genes shown to be related to cervical insufficiency following a systematic literature analysis similar to that underlying this study55. They identified 12 genes that were normally linked to cervical insufficiency. However, this was a modest study involving only 21 patients and there were no overlaps with our gene set. Zhou et al. recently analyzed publically available gene expression data sets derived from maternal blood in the second and third trimesters of women with spontaneous preterm birth and term birth56. Expression of a single gene, EBF1, was associated with preterm birth57. In a large genome-wide association study of over 1300 cases of spontaneous preterm birth in comparison to 12,000 ancestry-matched controls, they identified only two intergenic loci associated with preterm birth. The authors concluded that the genetic contributions to preterm birth are unlikely due to single common genetic variants but could be explained by interactions of multiple variants or environmental influences57. Meta-analysis of maternal and fetal transcriptomic data from genomic databases for studies related to preterm birth identified genes differentially expressed in spontaneous preterm birth relative to term. Ontogeny analysis demonstrated that maternal changes were enriched in immune-related pathways, upregulation of innate immunity and downregulation of adaptive immunity58. By comparison, analysis of the transcription profile in cord blood showed a downregulation of innate immune findings. Nonetheless, the results demonstrate a significant relationship of immune functions in the pathogenesis of preterm birth. Multi-omic analysis of preterm birth from the parent study of our INOVA replication samples has been reported24. It was a large study integrating whole genome sequencing, RNA sequencing and DNA methylation data for 270 preterm births and 520 control families. In their univariate analysis there were no variants that reached genome-wide significance. However, there were groups of genes associated with preterm birth in subsets of patients that were identified in secondary analyses24. Integration of the three data sources  identified a set of 72 candidate biomarker genes for very early preterm birth (VEPTB) and genes associated with PTB. RAB31 and RBPJ were identified by all three data types in preterm birth patients. Additionally, pathways associated with VEPTB included the EGFR and prolactin signaling pathways, inflammation- and immunity-related pathways, chemokine signaling, IFN-γ signaling, and Notch1 signaling24.More recently, investigations using high throughput and multi-omic techniques have been undertaken. Sakabe et al. compared transcriptome and regulatory maps of decidua-derived stromal cells to a genome-wide association study of gestational durationOur study has many strengths which contributed to successful identification of genetic variants in genes in networks associated with preterm birth. First, we used a very carefully phenotyped cohort of women with a strong family history of preterm birth. Our control cases were as carefully ascertained to have no family history of preterm birth. Second, we carried out targeted sequencing on genes with a demonstrated role in preterm birth. Third, we employed a novel analysis of protein–protein interactions to identify clusters of patients with shared PPI networks associated with preterm birth. Our study also has limitations and areas that deserve consideration. A power calculation was not carried out. This was an opportunistic, discovery sample. The significant clusters of preterm birth cases included unique genes that were both in our targeted sequencing as well as imputed via network analysis. Even though not included in our original sequencing, the fact that the unique imputed genes had a strong association with preterm birth, uterine contractility and immune responses is noteworthy. This was a modest sample size. We identified significant clusters of patients with networks and pathways associated with preterm birth but those findings were restricted to 45 out of the 122 cases. We did not anticipate being able to assign each of the cases to a significant cluster. We believe that the targeted sequencing contributed to our successful discovery even though all cases were not assigned to significant clusters. Nonetheless, the fact that we were able to identify significant case dominated clusters in our preterm birth cohort at all and that we were able to demonstrate similarity to clusters of patients with shared networks in the replication cohort lends validity to our hypothesis on the genetic architecture of this complex disease and the genetic leverage provided by the family history of preterm birth. The fact that this was not a whole exome study likely contributed to the number of cases we were able to assign to significant clusters. Futures studies employing similar techniques but with whole exome sequencing are likely to expand the number of case clusters that we will be able to identify using this approach. It is beyond the scope of this report to thoroughly discuss the control dominated clusters. Nonetheless, several elements deserve mention. We interpret the networks and pathways that are shared between control patients to represent protective genes or genes that confer resiliency against preterm birth. It is notable that we were able to identify significant clusters in the controls from our preterm birth cohort but none were identified in the replication cohort. We attribute this to the careful phenotyping that was used to enroll patients in the primary study.share variants in genes in specific networks and pathways which are sufficient to give rise to the disease phenotype.In summary, we used a novel multi-sample, protein–protein interaction tool, to identify clusters of patients with shared protein–protein interaction networks associated with preterm birth. We showed similarity between these networks and results from an independent replication cohort. Our results provide insights into the genetics of PTB and support a genetic architecture defined by subgroups of patients that Supplementary Tables.


To assess the efficacy of vitamin E oral spray in pregnancy.This was a retrospective study aimed to evaluate efficacy of vitamin E oral spray (vitamin E acetate in a medium chain tryglicerides vehicle – patented formulation) starting from the first trimester of pregnancy, with a control group.A total of 100 women were included in the study and were compared with a matched control group. Only 25/200 women reported to have at least one teeth cleaning during pregnancy. Women who received the oral spray had a significantly lower risk of preterm birth compared with the control group, and lower risk of periodontal diseases.Use of oil-based vitamin E oral spray in pregnancy is associated with a decreased risk of periodontal diseases and therefore preterm birth. Periodontitis, an inflammatory disease caused primarily by gram-negative bacteria that destroy tooth-supporting connective tissue and bone, is associated with an increased risk of preterm birth, as well as low birth weight and pre-eclampsia. Use of oil-based vitamin E oral spray in pregnancy is associated with a decreased risk of periodontal diseases and therefore preterm birth. Preterm birth, defined as delivery before 37 weeks of gestation, is the leading cause of perinatal morbidity and mortality . Differeet al. preterm labor should be seen as one syndrome with many causes associated with intra-amniotic infection, decidual senescence and breakdown of maternal–fetal tolerance [The etiology of preterm birth and preterm labor is not completely understood . Accordiolerance . More tholerance . Micro-oolerance . TherefoRecently, several studies have reported a significant association between oral hygiene and preterm birth by using image analysis tools to quantify dental plaque biofilm . PeriodoThe aim of the study was to assess the efficacy of oil-based vitamin E oral spray for oral health in pregnancy in reducing the risk of preterm delivery.This was a retrospective cohort study using data of women at risk of preterm birth or prior preterm birth, or history of prior cervical surgery. Women with multiple gestations were excluded. In this study, we retrospectively analyzed data on oral health and use of fluoride toothpaste, mouthwash and flossing. We planned to compare women who used oil-based vitamin E oral spray (vitamin E acetate in a medium chain tryglicerides vehicle – patented formulation) with a control group who did not.Inclusion criteria were singleton gestations and at least one risk factor for preterm birth including prior preterm birth, short mid-trimester transvaginal ultrasound cervical length, obesity or prior cervical surgery.Exclusion criteria were multiple gestations, and women with fetal abnormalities. Women with placenta previa/accreta were also excluded .Women in the intervention group used vitamin E acetate (in a caprylic/capric triglycerides vehicle) spray. Women used the therapy at least daily during the pregnancy. Women in the control group did not receive the oil-based vitamin E oral spray.The primary outcome was preterm birth. The secondary outcomes were pre-eclampsia, perinatal death, admission to neonatal intensive care unit and birth weight.Preterm birth was defined as delivery before 37 weeks. Pre-eclampsia was defined as a blood pressure elevation (≥140/90 on two occasions 4 h apart or ≥160/110 once), after 20 weeks of gestation, with proteinuria (≥300 mg on 24 h protein or >0.3 protein/creatinine ratio) or any of the following if proteinuria not present: platelets <100,000; creatinine >1.1 ; doubling of aspartate aminotransferase (AST) or alanine aminotransferase (ALT). Perinatal death was defined as either stillbirth  or neonatal death .-test to test group means by assuming equal within-group variances. Normality assumption check of continuous variables was performed using the Kolmogorov–Smirnov test.Statistical analysis was performed using Statistical Package for Social Sciences (SPSS) v.19.0 . Data were shown as means ± standard deviation or as a number (percentage). Categorical variables were compared using the chi-square test with continuity correction. Within-group comparison was undertaken using tA p-value < 0.05 was considered statistically significant.A total of 100 women who received the oral spray were included in the study and were compared with a matched control group (n = 100) who did not. The two groups were similar in terms of maternal demographics (the available maternal demographics are reported in This study aimed to assess the efficacy of oil-based vitamin E oral spray for oral health in pregnancy. We found in women at risk of preterm birth that use of oil-based vitamin E oral spray during pregnancy was associated with a decreased risk of preterm birth. The decreased preterm birth rate may be associated with the decreased risk of periodontal diseases in the intervention group.The main limitations of the study were the small sample size, and the nonrandomized approach. Given the retrospective study design, we cannot evaluate the compliance with the therapy. Moreover, we did not evaluate the presence of gingivitis or periodontal disease in the participants. The information about oral health was self-reported by the women leading to possible reporting bias and recall bias, potentially affecting our study findings. The major limitation of this study was the absence of randomization. Another limitation was the asimmetric gestational ages at assumption of the intervention.Preterm birth is defined as delivery before 37 week of gestations . Common Although intervention trials reported contradictory results ,37, oralVitamin E is a potent antioxidant with anti-inflammatory properties ,44. VitaIn our study we investigated the role of oil-based vitamin E oral spray for oral health in pregnancy. We found that in a small cohort of pregnant women, with risk factors for preterm birth and with a low compliance for professional teeth cleaning, use of vitamin E oral spray is associated with a reduction in the rate of preterm delivery.In summary, daily use of oil-based vitamin E oral spray in pregnancy may be associated with a decreased risk of preterm birth.Periodontitis is associated with an increased risk of preterm birth.Use of oil-based vitamin E oral spray in pregnancy is associated with a decreased risk of periodontal diseases and, therefore, preterm birth.We suggest to offer to women at risk of preterm birth or periodontal diseases the use of daily oil-based vitamin E oral spray.


Preterm birth is one of the main health problems encountered in the neonatal period, especially because it is also the first cause of death in the critical 1st month of life and the second in children under 5 years of age. Not only preterm birth entails short term health risks due to low weight and underdeveloped organs, but also increases the risk of suffering from non-transmissible diseases in the long term. To date, it is known that medical conditions and lifestyle factors could increase the risk of preterm birth, but the molecular mechanisms that control this process remain unclear. Luteolysis, increased inflammation or oxidative stress have been described as possible triggers for preterm birth and, in some cases, the cause of dysbiosis in preterm neonates. Several murine models have been developed to shed light into the mechanistic of preterm birth but, for the most part, are inflammation-based labor induction models and the offspring health readouts are mainly limited to survival and weight. Using a set of SWISS-CD1 mice born prematurely we analyzed inflammation and gut permeability parameters compared with term pups at weaning age. Overall, preterm mice presented higher systemic inflammation and gastrointestinal tract permeability. In this perspective article, we discuss the recent discoveries on preterm birth and the necessity of non-inflammatory murine models to really understand these phenotypes and be able to design strategies to prevent the sequels of this traumatic event in neonates. Preterm birth is defined by the World Health Organization as any birth before 37 completed weeks of gestation. It can be further divided based on gestational age (GA) as: extremely preterm (<28 weeks); very preterm (28 – < 32 weeks); moderate or late preterm (32 – < 37 completed weeks of gestation) . Around Early birth entails a huge range of short- and long-term health complications and disabilities in survivors. Preterm babies born with low weight, underdeveloped organs and they are more likely to have long-term neurodevelopmental and body function deficits, such as impairments on infant growth and weight gain, cerebral palsy, sensorial and motor disabilities, learning and behavioral disorders, cardiorespiratory problems, even an increased risk for accelerated brain aging was observed in young adults after premature birth. Moreover, they undergo an increased risk of falling in non-communicable diseases such as diabetes, obesity, asthma or mental disorders .Premature delivery can be considered as a syndrome with many causes and factors . MedicalBifidobacterium or Bacteroides genera, and predominance of facultative anaerobes like enterobacteria (Escherichia or Klebsiella), enterococci, some clostridia or lactobacilli at the 1st months of life in preterm babies , can impair the inflammatory process and diminish the rate of neonatal death  alarmin, which was also accompanied by higher neonatal death . The samIt is important to note that in most of these models, only the survival of neonates or inflammatory features of the fetus were analyzed, while other features already associated with preterm birth, like changes in the microbiota have not been assessed. This is surprising since, as commented previously, gut microbiota changes are present in preterm neonates. In this respect, in vivo T-cell activation through the intraperitoneal injection of α-CD3, progesterone improved the rate of preterm birth . These mice were analysed at weaning (3 weeks of age for this murine strain). We measured a very sensitive general marker of systemic inflammation in serum  and the general permeability by using FITC-dextran marker . Our resDelivery is a huge revolution for the newborn. Even in normal conditions, the baby passes from the comfort of the intrauterine environment to the unfriendly external world. Human babies are considered as unmatured. There is a specific developmental sequence that allows the correct growth and development of the newborn. It means that, even in term babies, both physical and emotional development is required to progress toward maturity. This complex process is even more complicated in preterm babies . AlthougIn the case of preterm birth, the immaturity of the immune and physiological systems seems to be a major driver of the alterations found, being inflammation and the consequent production of reactive oxygen species an elicitor of microbiota dysbiosis and gut hyperpermeability which in turn increase inflammation . NeverthThe raw data supporting conclusions of this article will be made available by the authors, without undue reservation.The animal study was reviewed and approved by local ethical committee for animal experiment (COMETHEA), INRAE Jouy en Josas, France.SA, DR-C, MG, and RM designed the perspective and wrote the manuscript. RM and PL designed the experiments. RM and FM performed the experiments. SA, DR-C, PL, MG, and RM corrected the manuscript. All authors approved the last version of the manuscript and contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Cognitive dysfunction in schizophrenia (SZ) is thought to arise from neurodevelopmental abnormalities that include interneuron hypomyelination in the prefrontal cortex (PFC). Here we report that RNA-sequencing of the medial (m)PFC of the APO-SUS rat model with SZ-relevant cognitive inflexibility revealed antioxidant metabolism as the most-enriched differentially expressed pathway. Antioxidant-related gene expression was altered throughout postnatal development and preceded hypomyelination. Furthermore, reduced glutathione levels and increased mitochondria numbers were observed in the mPFC. Strikingly, chronic treatment with the glutathione precursor N-acetylcysteine (NAC) from postnatal days 5–90 restored not only antioxidant-related mRNA expression and mitochondria numbers, but also myelin-related mRNA expression and mPFC-dependent cognitive dysfunction, while blood glutathione levels remained unaffected. The promyelinating effect of NAC was at least partly due to a positive effect on oligodendrocyte lineage progression. Together, our findings highlight that oxidative stress may contribute to cognitive symptoms in the APO-SUS rat model of SZ and encourage antioxidant therapy in early phases of SZ. Schizophrenia (SZ) is a severe neurodevelopmental disorder that includes cognitive symptoms arising from the prefrontal cortex (PFC). The neurobiological basis of the cognitive symptoms of SZ remains elusive. Oxidative stress, defined as an imbalance between the production of reactive oxygen species (ROS) and the clearance of ROS by antioxidants, has been suggested to play a key pathological role . In SZ pTo explore this question, we used a well-characterized, idiopathic rat model with SZ-relevant features, namely the apomorphine-susceptible (APO-SUS) rat along with its phenotypic counterpart, the apomorphine-unsusceptible (APO-UNSUS) rat. APO-SUS rats do not require genetic or pharmacological manipulation to display SZ-relevant behavioral characteristics, such as reduced prepulse inhibition, increased exploratory behavior, dopamine-induced stereotypic behavior –10, reduHere, we report that RNA-sequencing (RNA-seq) analysis of the mPFC of APO-SUS and APO-UNSUS rats revealed a central role for glutathione antioxidant metabolism. Intriguingly, treatment with the glutathione precursor N-acetylcysteine (NAC) restored not only glutathione metabolism, but also improved mPFC hypomyelination and cognitive functioning of APO-SUS rats.Generation of the APO-SUS and APO-UNSUS rat lines has been described in detail elsewhere , 17. BriFor micropunching, naive postnatal day (P)21 (±1 day), P60 (±1 day), P90 (±1 day), P120 (±1 day) and P365 (±14 days) APO-SUS and APO-UNSUS rats were sacrificed by direct decapitation and brains were isolated, frozen on dry ice and stored at −80 °C. Micropunching was performed in a cryostat (Leica) at −15 °C and the Paxinos and Watson rat brain atlas was taken as a reference to aid the dissection. mPFC was collected with a 1.20 or 2.00-mm punch needle (Harris) from 300 µm coronal sections at Bregma 4.00–2.20. Dissected tissues were immediately frozen on dry ice and stored at −80 °C until further analysis.2O. RNA concentration and purity were measured using a DS-11 spectrophotometer (Denovix). RNA was kept at −80 °C until further analysis.RNA for RNA-seq analysis was extracted from P365 APO-SUS and APO-UNSUS mPFC with a Nucleospin RNA II kit following the instructions of the manufacturer . For all other RNA isolations, tissues were homogenized using Trizol reagent (Sigma) and stainless-steel beads in a Tissue Lyser (Qiagen), followed by chloroform extraction and RNA precipitation with isopropanol in the presence of 20 μg of glycogen (Fermentas). Pellets were washed twice with 75% ice-cold ethanol and dissolved in MilliQ Hn = 4, pooled) and APO-UNSUS  mPFC were analyzed by RNA-seq at the Hudson Alpha Institute for Biotechnology . RNA-seq libraries were formed from ~850 ng total RNA of each sample. RNA-seq was performed using paired end sequencing on Illumina HiSeq (Illumina), at 100 base pairs, generating over 30 million paired reads. RNAseq FASTQ files were analyzed using GeneSifter software (VizX Labs). Transcript abundance was calculated by estimating the reads per kilobase of exon per million mapped reads (RPKM) and normalization to the number of mapped reads was used for comparison. The mRNAs differentially expressed in the APO-SUS versus APO-UNSUS mPFC and meeting our two predefined criteria  were used as input data for analysis with the Ingenuity Pathway Analysis (IPA) software package (Qiagen) to identify overrepresented biological pathways with a focus on ‘Canonical Pathways’.For RNA-seq analysis, total RNA quality of the samples was assayed using Agilent 2100 Bioanalyser (Applied Biosystems). Total RNA concentration was estimated by Qubit Fluorometer (Invitrogen). RNA samples  prepared from P365 APO-SUS  and contained 2.0 μL diluted cDNA, 0.8 μL 5 μM forward primer, 0.8 μL 5 μM reverse primer, 5 μL SybrGreen mix (Roche) and 1.8 μL MilliQ H2O. qPCR was performed with a Rotor Gene 6000 Series (Corbett Life Sciences) using a 3-step paradigm with a fixed gain of 8. Fifty cycling steps of 95, 60, and 72 °C were applied, and fluorescence was acquired after each cycling step. Primers were designed with NCBI Primer-Blast or Primer Express 2.0 and synthesized by Sigma  were used to determine the normalization factor with GeNorm  = 2.264, p = 0.142, main effects rat line F = 8.072, p = 0.007 treatment F = 0.571, p = 0.455). However, in the mPFC NAC treatment significantly changed APO-SUS mRNA expression levels of a number of glutathione-related genes to APO-UNSUS levels , including genes involved in the production and breakdown of glutathione, as well as the binding of glutathione to target oxidative molecules . We conclude that NAC treatment during postnatal development alleviates oxidative stress in the APO-SUS mPFC.We next wondered whether treatment with the glutathione precursor NAC from P5 onwards could restore the APO-SUS mPFC alterations in glutathione metabolism and mitochondria numbers Fig. . NAC treles Fig. . In APO-les Fig. . MoreoveF[6] = 3.050, p = 0.031, main effects rat line Wilks’ Lambda F[6] = 1.813, p = 0.153 treatment Wilks’ Lambda F[6] = 2.553, p = 0.057). Our ultrastructural analysis did not reveal a significant effect of NAC treatment on the number of myelinated axons in APO-SUS IL , likely due to the low sample sizes commonly used in electron microscopy experiments. Myelin thickness as measured by the G-ratio remained unaffected . In addition, there was a trend towards an increased percentage of OPCs, and a decreased percentage of preOLs in APO-SUS and APO-UNSUS OPC cultures exposed to CoCl2 . We therefore conclude that oxidative stress hinders the maturation of APO-SUS and APO-UNSUS OPCs in vitro. In line with these in vitro data, we found in vivo a reduced number of OLIG2 + OL lineage cells and mature OLIG2 + CC1 + OLs in the mPFC of oxidative-stress-exposed APO-SUS rats as compared to the mPFC of APO-UNSUS rats. Following NAC treatment, the numbers of OPCs and mature OLs in the APO-SUS mPFC were significantly increased , indicating that the promyelinating effects of the antioxidant are, at least partially, caused by a positive effect on OL lineage progression.Interneuron hypomyelination in APO-SUS mPFC is caused by impaired OL lineage progression . OLs andent Fig. . HoweverF = 0.003, p = 0.956 main effects rat line F = 0.221, p = 0.640 treatment F = 0.835, p = 0.044; Shift 2 Two-way ANOVA interaction F = 5.774, p = 0.019 main effects rat line F = 11.69, p = 0.001 treatment F = 3.39, p = 0.070, post-hoc pairwise comparisons APO-SUS versus APO-UNSUS t[74] = −4.17, p < 0.001 APO-UNSUS versus APO-UNSUS + NAC t[74] = −3.003, p = 0.003; Shift 3 Two-way ANOVA interaction F = 2.129, p = 0.149 main effects rat line F = 4.479, p = 0.038 treatment F = 0.011, p = 0.917). However, error analysis revealed that untreated APO-SUS rats made significantly more perseverative errors than untreated APO-UNSUS rats during all three shifts combined, indicating that APO-SUS rats kept following the initial rule and showed cognitive inflexibility. NAC treatment rescued the number of perseverative errors in APO-SUS rats, while not affecting perseverative error numbers of APO-UNSUS rats . NAC treatment also increased the number of regressive errors in APO-SUS and APO-UNSUS rats, but these rats make five times more perseverative errors than regressive errors and as such the effect of NAC treatment is twice as large on perseverative errors than on regressive errors.We next wondered whether alleviating oxidative stress and restoring myelination in the APO-SUS mPFC would lead to improved cognitive behavior. We therefore treated APO-SUS and APO-UNSUS rats with and without NAC, and performed the extra-dimensional set-shifting task that is modeled after the Wisconsin card sorting test used to examine cognitive symptoms in SZ patients , 27. DurF = 1.394, p = 0.242 main effects rat line F = 3.634, p = 0.061 treatment F = 0.001, p = 0.983; Reversal 2 Two-way ANOVA interaction F = 0.874, p = 0.353 main effects rat line F = 1.213, p = 0.274 treatment F = 0.562, p = 0.456; Reversal 3 Two-way ANOVA interaction F = 0.004, p = 0.948 main effects rat line F = 2.392, p = 0.126 treatment F = 0.487, p = 0.488). However, during error analysis we found that NAC treatment causes a trend towards a lower number of perseverative errors as well as a significant decrease in the number of regressive errors in APO-SUS and APO-UNSUS rats . Thus, NAC treatment improved cognitive flexibility in APO-SUS rats in both extra-dimensional set-shifting and reversal learning cognitive tasks.To further investigate the effects of NAC on cognitive inflexibility, we performed reversal learning. Rats were trained to press the lever on one side of the cage regardless of cue light illumination and during the reversal sessions, rats were required to reverse their behavior and press the lever on the other side of the cage. We quantified the number of errors the rats made until they reached the criterion in three subsequent reversal sessions . We found a trend towards an increased number of errors to criterion in APO-SUS rats during reversal 1, but no further differences in the performance of APO-SUS and APO-UNSUS rats during reversals 2 and 3  the positive, negative, and cognitive symptoms of SZ are instrumental to understand the molecular and cellular mechanisms underlying these symptoms. In this study, we used the APO-SUS rat model displaying SZ-relevant cognitive inflexibility to explore the elusive neurobiological basis of PFC-related cognitive symptoms in SZ. Unbiased transcriptomic analysis revealed that genes related to glutathione metabolism were enriched among the genes differentially expressed in mPFC of APO-SUS versus APO-UNSUS rats. The observed reduced levels of glutathione in APO-SUS mPFC were accompanied by an increase in the number of mitochondria. Oxidative stress and mitochondrial dysfunction have been hypothesized to play a role in SZ etiology . In indiReduced PFC glutathione levels have been associated with decreased PFC white-matter integrity that is thought to arise from defective myelination . ExpressTo investigate whether oxidative stress causes hypomyelination in the APO-SUS mPFC, we chronically treated postnatal APO-SUS rats with the antioxidant glutathione precursor NAC. We found normalized mPFC glutathione metabolism-related mRNA expression levels in adult NAC-treated APO-SUS rats as well as a reduced number of mPFC mitochondria in NAC-treated APO-SUS and APO-UNSUS rats. Remarkably, besides its antioxidative properties, NAC also showed promyelinating effects in that the mRNA expression of all major myelin components was significantly increased in the mPFC of APO-SUS rats treated with the antioxidant. Thus, a proper glutathione metabolism appears to be essential for developmental myelination in SZ. The notion that oxidative stress underlies PFC hypomyelination in SZ is corroborated by the results of our analysis of the developmental time course of mPFC mRNA expression, which showed that the dysregulation of glutathione-related genes preceded the reduced expression of myelin-related genes in APO-SUS rats.Since OLs, and in particular premyelinating OLs, are extremely vulnerable to oxidative stress , 25, we APO-SUS rats show mPFC-dependent cognitive inflexibility in the extra-dimensional set-shifting task . This coCollectively, the findings increase our understanding of the neurobiological mechanisms leading to cognitive symptoms in SZ and encourage the use of chronic NAC treatment as a preventive measure for individuals at high risk for developing SZ and early-phase SZ patients.The authors declare that they have no conflict of interest. This study was supported by grants from the programs ‘Top talent’ Donders Centre for Medical Neuroscience (to DAM), Van Gogh travel grant , Investissements d’Avenir ANR-10-IAIHU-06 (IHU-A-ICM) and ANR-11-INBS-0011 (NeurATRIS) (to BNO) and Dutch Top Institute Pharma T5-209 (to VDE and GJMM).Supplemental MaterialSupplementary Dataset


Cacna1h gene. Impaired, function of the low-voltage-activated T-Type calcium channel CaV3.2 is involved in genetic/mutational as well as acquired/transcriptional channelopathies that emerge with epileptic seizures. We show CRISPR-induced activation and inhibition of the Cacna1h locus in NS20Y cells and primary cortical neurons, as well as activation in mouse organotypic slice cultures. In future applications, the system offers the intriguing perspective to study functional effects of gain-of-function or loss-of-function variations in the Cacna1h gene in more detail. A better understanding of CaV3.2 channelopathies might result in a major advancement in the pharmacotherapy of CaV3.2 channelopathy diseases.Precise genome editing in combination with viral delivery systems provides a valuable tool for neuroscience research. Traditionally, the role of genes in neuronal circuits has been addressed by overexpression or knock-out/knock-down systems. However, those techniques do not manipulate the endogenous loci and therefore have limitations. Those constraints include that many genes exhibit extensive alternative splicing, which can be regulated by neuronal activity. This complexity cannot be easily reproduced by overexpression of one protein variant. The CRISPR activation and interference/inhibition systems (CRISPRa/i) directed to promoter sequences can modulate the expression of selected target genes in a highly specific manner. This strategy could be particularly useful for the overexpression of large proteins and for alternatively spliced genes, e.g., for studying large ion channels known to be affected in ion channelopathies in a variety of neurological diseases. Here, we demonstrate the feasibility of a newly developed CRISPRa/i toolbox to manipulate the promoter activity of the  Ion chaactivation (CRISPRa) and CRISPR interference/inhibition (CRISPRi) technology. CRISPRa uses a catalytically dead Cas9 (dCas9) enzyme fused with a highly efficient transcriptional activator complex consisting of the tripartite transcriptional activator VP64-p65-Rta (VPR), shown to increase target gene expression even up to 320-fold  system could be a powerful approach for the manipulation of large proteins that cannot be done by conventional techniques. Recently, substantial progress has been made in genomic editing by using specific applications of the CRISPR technique, including the CRISPR 320-fold . CRISPRi320-fold . Using tCacna1h, encoding the low-voltage-activated T-Type calcium channel CaV3.2. Ion channelopathies for Cacna1h have been described particularly for epilepsy variants  in a total volume of 10 μL, the reaction was performed by 30 cycles at 37°C for 5 min and 23°C for 5 min.All primer sequences used for cloning are shown in sI sites . BrieflyAfeI/KpnI-digested pLenti-Ef1a-dCas9-VPR. In a second step, the Cacna1h and LacZ sgRNA sequences from the pAAV-U6-sgRNA plasmids (see above) were cloned by IFC into the PacI site of pLenti-syn-dCas9-VPR, resulting in pLenti-U6-sgRNA(Cacna1h/LacZ)-Syn-dCas9-VPR.The all-in-one CRISPRa lentiviral system was generated by replacing the promoter of pLenti-Ef1a-dCas9-VPR  or CMV-# 63798, ] 200 ng,4 transfection of HEK293-AAV cells  as described previously  4 in 24 well plates containing 70,000 cells/well with a multiplicity of infection (MOI) of approximately 14. Cells were collected on DIV15 in lysis/binding buffer  and stored at −80°C until mRNA extraction or at DIV20 for Western blot.AAV1/2 viruses were produced by large-scale triple CaPOeviously . Lentivieviously . Dissocieviously . All aniRNA isolation and cDNA production was performed using Dynabeads mRNA Direct Micro Kit  and RevertAid H Minus First strand cDNA Synthesis Kit  following the manufacturer’s instructions. Quantitative PCR was performed in a Thermal Cycler . The reaction was performed using 1X Maxima SYBR Green/Rox qPCR Master Mix , 0.3 μM of each primer . Following washing steps in PBS-T (0.1% Tween) the membrane was incubated with IRDye800 Goat anti-Rabbit  for 45 min, washed and imaged. The membrane was subsequently incubated with anti-Tubulin antibody  for 2 h at RT, washed and incubated with IRDye680 Goat anti-Rat . Bands were detected with infrared Odyssey system (LI-COR Biosciences) and quantified using the software Image Studio Lite.Transduced cortical neurons  were lysed in RIPA buffer , loaded on 8% SDS polyacrylamide gels and transferred onto nitrocellulose membranes. Blots were blocked in 5% milk for 1 h and incubated overnight with a primary antibody against Ca2, without CaCl2), 25% horse serum, 0.65% D(+)-glucose, 0.01 M Hepes, 2 mM L-glutamine and 0.5X B27). Cultured slices were kept at 37°C, 5% CO2.Organotypic hippocampal slices were prepared from mice (C57Bl6/N) at postnatal day 3–6 as described before . In brieImages were obtained using an inverted phase contrast fluorescent microscope  and processed using Fiji. The cell surface area, set by the GFP fluorescence, was determined using the Weka trainable segmentation plugging. The integrated density, determined by the mRuby fluorescence was normalized to the cell surface area to obtain the reported values of integrated density/cell surface (IntDE/cell surfase).t-test, Student t-test, and two way ANOVA followed by multiple comparison tests were used to compare significance of the results. Values were considered significant a p < 0.05. For all graphs data are displayed as mean ± SEM.Statistical analyses were performed using GaphPad software. One sample Cacna1h gene, we first designed sgRNAs sequences targeting the mouse and rat Cacna1h gene. Using the Benchling computational tool, we selected two sgRNAs binding 66 (sgRNA1) and 131 (sgRNA2) base pairs (bp) upstream of the start-ATG of the Cacna1h gene  a minimal reporter unit expressing mRuby under control of the rat Cacna1h promoter, (ii) a ubiquitous promoter expressing eGFP, and (iii) either a CRISPRa construct [dCas9-VPR, Cacna1h promoter was observed after co-transfection with the CRISPRa construct and an inhibition after co-transfection with the CRISPRi construct , and reached similar levels as observed for the combination of the two sgRNAs (p = 0.015). Also for the CRISPRi system, comparable modulatory effects were observed for sgRNA1 , sgRNA2  and the combination of the two sgRNAs  and a decrease for the CRISPRi system . Also here, no significant differences were observed between the two sgRNAs or the combination of the two sgRNAs.To confirm and precisely quantify the Cacna1h mRNA expression levels after manipulation with the CRISPRa and CRISPRi systems using quantitative real-time RT-PCR. Since both sgRNAs showed similar effects on the Cacna1h reporter constructs . No changes in Cacna1h expression were observed for the CRISPRa and CRISPRi controls  and at the protein level (p = 0.0046). In addition, also inhibition of the system using CRISPRi in neuronal cultures resulted in a reduced Cacna1h expression at the mRNA (p = 0.032) as well as at the protein level (p = 0.042), indicating that sgRNA2 can efficiently modulate Cacna1h promoter activity in primary neurons. Although we observed additional non-specific bands in our Western blot experiments  after Cacna1h-CRISPRa/i targeting using quantitative real-time RT-PCR. No alterations were observed for any of the other calcium channels tested  and lentiviruses encoding our Cacna1h-CRISPRa toolbox and a fluorescent reporter. After co-transduction with AAV-sgRNA, lenti-EF1a-dCas9-VPR , and a decrease in Cacna1h expression after TET-ON-CRISPRi-Cacna1h transduction and doxycycline treatment (p = 0.027). No effect on Cacna1h expression was observed when using a control sgRNA targeting LacZ /genetic gene ablation (knock-out), respectively. Although very valuable, the applicability of these techniques has limitations  can be easily augmented using rAAV and lentiviruses, which can be produced in most molecular biology labs. Another advantage of the Cacna1h-CRISPRa system over conventional gain-of-function approaches is the endogenous modulation of the Cacna1h genomic locus, providing the opportunity to study the effects of abundant alternative splicing variants of Cacna1h. For Cacna1h, at least 12–14 alternative splicing sites have been described, resulting in the possibility to generate more than 4,000 alternative Cacna1h transcripts  used in our system does not cleave the DNA, the possible off-target effects are more likely to be less deleterious than using the conventional Cas9 to induce a genetic knock-out  a mutation in Cacna1h has been described, which augments the expression of Cav3.2 at the cell surface and increases calcium influx (Cacna1h-CRISPRa could be used as a gain-of-function approach to mimic the enhanced expression seen in GEARS. In addition, a transient inhibition of Cacna1h via the Tet-on inducible cacna1h-CRISPRi system in the GAERS rats at different stages during development could also reveal potentially interesting time windows for Cacna1h-associated channelopathies.Also in the context of mutational/genetic ion channelopathies, the potential of inducible m influx . Here, tCacna1h-CRISPRa/i modular approach could thus be used to model transient gain-of-function or loss-of-function effects in the Cacna1h gene and to study CaV3.2 channelopathies in more detail in vivo.Taken together, our here described The original contributions presented in the study are included in the article/The animal study was reviewed and approved by the LANUV Recklinghausen, Leibnizstr. 10, 45659 Recklinghausen.DT, SS, AB, and KL conceived and planned the study. DT and AT carried out the molecular and cellular experiments. HS, TO, and SS contributed to the study design and analysis. DT wrote the initial draft. AB, SS, and KL contributed to the review and editing. All authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.