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Unravelling the genetic landscape of cervical insufficiency: Insights into connective tissue dysfunction and hormonal pathways
Genetics of cervical insufficiency
https://orcid.org/0000-0003-0413-1481
Voložonoka Ludmila Conceptualization Data curation Formal analysis Funding acquisition Methodology Validation Visualization Writing – original draft 1 2 *
Bārdiņa Līvija Data curation Formal analysis Methodology Software Writing – original draft 1 2
Kornete Anna Investigation Methodology 1 3
Krūmiņa Zita Data curation Investigation Methodology Writing – review & editing 1
Rots Dmitrijs Conceptualization Methodology Software Writing – review & editing 1 2
Minkauskienė Meilė Investigation Supervision Writing – review & editing 4
Rota Adele Formal analysis Investigation 1 3
Strelcoviene Zita Investigation Writing – review & editing 4
Vilne Baiba Conceptualization Formal analysis Methodology Resources Software Supervision 1
Kempa Inga Conceptualization Data curation Formal analysis Funding acquisition Project administration Resources Supervision Writing – review & editing 1
Miskova Anna Conceptualization Funding acquisition Investigation Project administration Resources Supervision 1 3
Gailīte Linda Conceptualization Funding acquisition Project administration Resources Supervision Writing – review & editing 1
Rezeberga Dace Funding acquisition Project administration Resources Supervision Writing – review & editing 1 3 5
1 Riga Stradins University, Riga, Latvia
2 Children’s University Hospital, Riga, Latvia
3 Riga Maternity Hospital, Riga, Latvia
4 Lithuanian University of Health Sciences, Kaunas, Lithuania
5 Riga East Clinical University Hospital, Riga, Latvia
Bayraktar Burak Editor
Ankara Etlik City Hospital, TÜRKIYE
Competing Interests: NO authors have competing interests.

* E-mail: ludmilavolozonoka@gmail.com
19 9 2024
2024
19 9 e031071829 2 2024
5 9 2024
© 2024 Voložonoka et al
2024
Voložonoka et al
https://creativecommons.org/licenses/by/4.0/ This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

Background

The intricate molecular pathways and genetic factors that underlie the pathophysiology of cervical insufficiency (CI) remain largely unknown and understudied.

Methods

We sequenced exomes from 114 patients in Latvia and Lithuania, diagnosed with a short cervix, CI, or a history of CI in previous pregnancies. To probe the well-known link between CI and connective tissue dysfunction, we introduced a connective tissue dysfunction assessment questionnaire, incorporating Beighton and Brighton scores. The phenotypic data obtained from the questionnaire was correlated with the number of rare damaging variants identified in genes associated with connective tissue disorders (in silico NGS panel). SKAT, SKAT-O, and burden tests were performed to identify genes associated with CI without a priori hypotheses. Pathway enrichment analysis was conducted using both targeted and genome-wide approaches.

Results

No patient could be assigned monogenic connective tissue disorder neither genetically, neither clinically upon clinical geneticist evaluation. Expanding our exploration to a genome-wide perspective, pathway enrichment analysis replicated the significance of extracellular matrix-related pathways as important contributors to CI’s development. A genome-wide burden analysis unveiled a statistically significant prevalence of rare damaging variants in genes and pathways associated with steroids (p-adj = 5.37E-06). Rare damaging variants, absent in controls (internal database, n = 588), in the progesterone receptor (PGR) (six patients) and glucocorticoid receptor (NR3C1) (two patients) genes were identified within key functional domains, potentially disrupting the receptors’ affinity for DNA or ligands.

Conclusion

Cervical insufficiency in non-syndromic patients is not attributed to a single connective tissue gene variant in a Mendelian fashion but rather to the cumulative effect of multiple inherited gene variants highlighting the significance of the connective tissue pathway in the multifactorial nature of CI. PGR or NR3C1 variants may contribute to the pathophysiology of CI and/or preterm birth through the impaired progesterone action pathways, opening new perspectives for targeted interventions and enhanced clinical management strategies of this condition.

Fundamental and Applied Research Projects grant of The Latvian Council of Science Nr. 2020/1-0042 Rezeberga Dace The study was funded by the Fundamental and Applied Research Projects grant of The Latvian Council of Science. Project ‘Elucidating comprehensive etiology of cervical insufficiency to foster timely diagnosis of preterm delivery and prevent adverse outcomes in obstetrics’, Nr. 2020/1-0042 (to DR). The funders had no role in the study design, data collection or analysis, decision to publish, or preparation of the manuscript. Data AvailabilityAll relevant data are within the manuscript and its Supporting Information files.
Data Availability

All relevant data are within the manuscript and its Supporting Information files.
==== Body
pmcIntroduction

Preterm birth (PTB) is a significant yet poorly understood outcome of pregnancy, representing a major public health concern [1]. The prevalence of PTB is estimated to range from 5% to 18% globally. PTB accounts for approximately 75% of all neonatal deaths [2,3]. Prolonged stays in PTB neonatal-intensive care units for PTB infants, recurrent hospital admissions, and specialized PTB follow-up needs adds of significant financial strain [1,4].

Preterm birth: One outcome—Multiple aetiologies and distinct molecular pathways

The aetiology of PTB is multifaceted and complex. Various factors, including uterine anomalies, placental abruption, preterm premature rupture of membranes (PPROM), inflammatory conditions (such as intraamniotic inflammation and/or infection), and cervical insufficiency (CI), either individually or in combination, contribute to the occurrence of PTB [5]. Furthermore, genetic predisposition has been recognized as a potential influence on PTB [6,7]. Studies have suggested heritability estimates for PTB ranging from 15% to as high as 30–40% [8].

Findings from the literature collectively support the prevailing hypothesis that PTB is driven through two main pathways: 1) the inflammatory pathway–suggesting that PTB, at least in part, has an inflammatory aetiology. Inflammatory pathway can be induced by infection, or may result from sterile inflammation due to intracellular processes yet to be identified [6]; 2) connective tissue dysfunction pathway–biomechanical properties of the connective tissues can be disrupted by e.g. uterine overdistension, cervical or pelvis tissue laxity leading to preterm cervical dilatation, rendering it impossible to sustain an otherwise healthy pregnancy to full term. Nevertheless, lesser-known pathways and candidate genes within these pathways continue to emerge as significant in relation to prematurity, including, but not limited to: hemopoietic pathway, haemostasis, coagulation, focal adhesion, and cell communication pathways [9,10].

Nonetheless, determining the genetic factors contributing to PTB has been challenging. Despite advancing genetic studies in almost all clinical disciplines, our understanding of the genetic aetiologies and molecular pathways of PTB and PTB-associated phenotypes remains limited. Consequently, our ability to develop interventions to prevent and/or treat PTB by specifically targeting the underlying phenotypes–e.g., CI, PPROM, maternal-foetal immune rejection, etc.–is hindered. Currently, the existing treatment of PTB in clinical practice predominantly rely on observation and symptomatic/prophylactic management (e.g. progesterone, cervical cerclage and pessary) rather than targeting the specific underlying aetiology [11,12].

To date, the majority of studies have predominantly focused on idiopathic PTB. However, perceiving PTB as a single, homogeneous condition is overly simplistic and hinders our ability to recognize distinct classes of pathologies, each with its own unique or overlapping genetic or acquired origins, all ultimately culminating in the same outcome–PTB [13]. A comprehensive evaluation of the specific clinical characteristics of PTB and subgrouping patients based on their clinical presentations before embarking on any genetic study can strengthen the analytical approach, facilitating the identification of distinct causes and genetic markers for subtypes of PTB.

Deciphering the skewed underlying molecular pathways and understanding pathophysiological mechanisms resulting in PTB would eventually provide an opportunity to develop rationale and efficacious targeted intervention strategies to treat PTB etiologically and improving clinical outcomes tailored to the unique phenotypes observed [14–17].

Cervical shortening—A distinct phenotype in prematurity

Short cervix is defined as a cervical length of ≤25mm before 24 weeks of gestation on transvaginal sonographic examination [18]. Cervical insufficiency is characterized by rapid, painless shortening and opening of the cervix, often accompanied by prolapse of the foetal membranes into the cervical canal, or PPROM, occurring in the second or early third trimester of pregnancy, leading to pregnancy loss or PTB. Isolated (i.e. sporadic) CI occurs in approximately 1–3% of all pregnancies [19], but stands out for its correlation with poor pregnancy outcomes, including PTB rates reaching 40–50%, accompanied by significant perinatal and neonatal mortality rates [20].

Causes of CI are also complex and among others include intra-amniotic infection/inflammation, decidual haemorrhage, uterine overdistension, disruption of maternal-foetal tolerance. There is also evidence of genetics as a contributing factor to CI development [21]. A systematic literature review identified 12 genes linked to CI, majority of which are associated with connective tissue disorders [22]. Indeed, heterogeneous collagenopathies like Ehlers-Danlos (EDS), osteogenesis imperfecta (OI), and Marfan syndromes are clinically recognized risk factors for PTB, PPROM and CI, resulting in a syndromic form of the condition [23], however the direct evidence from genetic studies validating this connection has been notably scarce [24]. This data is further supported by our pilot study in which we conducted clinical exome sequencing in a limited number of patients with CI. Through target gene panel variant analysis and pathway enrichment analysis, we were able to demonstrate that the increased susceptibility to the development of CI is likely influenced by rare damaging variants in genes involved in extracellular matrix (ECM)/collagen synthesis. Therefore, our preliminary study suggests the hypothesis of CI as a subtle form of connective tissue disorder in apparently non-syndromic patients [22].

Considering the aforementioned, the primary objective of the present study is to identify genes and molecular pathways contributing to CI–a clinically distinct group of patients with particularly high risk of PTB. Specifically, our focus is on exploring the hypothesis that CI may stem from rare damaging variants in genes associated with connective tissue and related disorders. To pursue this investigation, we have curated a substantial cohort of females (n = 114) affected by idiopathic CI, capturing a wide range of clinical data (including Beighton [25] and Brighton criteria [26] used to clinically diagnose joint hypermobility and certain connective tissue disorders) that could influence CI development. The exomic data, acquired through next-generation sequencing (NGS) of all patients, has undergone an unbiased and comprehensive characterization. As a result, our study has taken a step toward unravelling the intricate genetics of CI, shedding light on genes not conventionally associated with this condition.

Methods

Ethical principles

The research conducted in this study adhered to the ethical guidelines outlined in the Declaration of Helsinki. Approval for the study protocol was obtained from Latvia’s Central Medical Ethics Committee under reference number 2/18-03-21 and Lithuanian Kaunas Regional Medical Ethics Committee under reference number 2021-10-14/BE-2-20. All patients were informed of the nature of the study and gave informed consent before enrolment to the study and collection of blood samples.

Patient inclusion criteria and data collection

Seventy-five patients from Riga Maternity Hospital in Riga, Latvia were included in the prospective longitudinal cohort study. Women with a history of second-trimester pregnancy loss, extreme PTB (22–27 weeks), or those undergoing routine antenatal care were examined for a short cervix or CI and recruited when treatment for cervical shortening was required. The inclusion criteria for these participants were a singleton pregnancy and the presence of a short cervix or CI. Short cervix was defined as a cervical length of ≤25mm before 24 weeks of gestation on transvaginal sonographic examination [18]. Exclusion criteria included multiple pregnancies, uterine anomalies, any genetic disorder other than known connective tissue disorder, age younger than 18 years, and patients in active labour. Active labour was defined as spontaneous preterm labour, characterized by the presence of regular uterine contractions occurring at a frequency of at least four contractions every 20 minutes before 37 weeks of pregnancy. In addition, 32 cases based on the same inclusion/exclusion criteria were recruited from the Lithuanian cohort. Patients were recruited from March 21st 2018 until December 31st 2023. During the same period detailed medical history was extracted from medical records; authors had access to information that could identify individual participants during and after data collection. Additionally, participants also completed a connective tissue dysfunction assessment questionnaire developed for this study (S3 Table). The questionnaire included Beighton criteria for the evaluation of joint hypermobility [25], Brighton criteria for the evaluation of EDS hypermobility type [26], as well as additional questions regarding family history of PTB and connective tissue functionality not covered by the Beighton/Brighton assessment. The Beighton and Brighton criteria are standard tests used to aid in the diagnosis of joint hypermobility syndrome, which may be associated with heritable connective tissue disorders such as Ehlers-EDS, Marfan syndrome, and OI. The Beighton and Brighton criteria were assessed according to the methods described in the original publications [25]. Each question in the connective tissue dysfunction assessment questionnaire was scored as follows: one point was assigned for a positive response (positive phenotype), while the absence of the phenotype was scored as zero points. The questionnaire had a maximum possible score of 25 points, with nine points allocated for the Beighton joint hypermobility criteria and eight points for the Brighton criteria (which also includes Beighton criteria, see S3 Table). Scores for the Beighton and Brighton criteria, as well as total scores, were calculated for each patient individually, as well as for the cohort on average.

During the enrolment process into the study, a clinical assessment of patients was conducted by an obstetrician-gynaecologist. Patients who met the Brighton criteria and those found to have P/LP variants in the connective tissue gene panel were offered a clinical geneticist consultation to evaluate the potential clinical diagnosis of a connective tissue disorder.

Controls for this study were chosen from an in-house WES/whole genome sequencing database (n = 588; 34.8% females and 65.2% males) comprising individuals who underwent genetic testing for various disorders, primarily neurologic or cardiological conditions. The presence of CI in the controls was not assessed. However, given the anticipated rarity of CI in the population, it is not expected to markedly influence genetic association analysis.

DNA sample processing and bioinformatics pipeline

The exomes of all CI patients were analysed using the Twist Human Core Exome Kit (Twist Bioscience, San Francisco, CA, USA). Sequencing was performed on the NextSeq 500 platform (Illumina) with 75 paired-end (PE) reads. This sequencing service was provided by CeGaT GmbH in Tübingen, Germany.

Variant calling and Quality Control (QC)

Bioinformatics analysis was conducted, and all project samples and associated data were securely stored on the DNAnexus cloud platform. Utilizing an in-house pipeline implemented within the DNAnexus cloud platform, WES data from samples related to patient (CI) and control samples, provided in FASTQ format, were aligned to the GRCh38 reference genome using BWA-MEM [27]. Duplicate reads were systematically removed from the analysis. Variant calling for each sample was performed using DeepVariant (v1.2.0) [28], resulting in variant calls provided in both VCF and GVCF formats. To ensure data quality, reference metrics for quality control (QC) were calculated based on an in-house WES database, and these metrics were subsequently applied as QC criteria to all CI sample data. All CI WES sample data successfully met these QC criteria and were considered for further analysis. For joint variant calling, GVCF format files from both CI samples and controls were integrated using GLnexus (v1.4.1) to generate the final variant calling output in pVCF format [3].

Removal of relatives

Kinship analysis was conducted using the PLINK 1.9 software suite with a relationship-based pruning approach (—rel-cutoff 0.1) (Purcell, Neale et al. 2007). This analysis aimed to identify and remove individuals with relationships up to the 3rd degree. In cases where related pairs were detected, one individual from each pair was systematically excluded from subsequent analysis. As a result, no cases and 16 control individuals were excluded from the final dataset.

Variant filtering and prioritization

Variants were subjected to annotation using Ensembl VEP (release 107) [29]. Subsequent variant exclusion was carried out through the application of bcftools (v.1.14) [30] and vcftools (v0.1.16) [31], adhering to the following filtering criteria:

Variants with a read depth (DP) of less than 10 were excluded.

Heterozygous variants with a variant allele frequency (VAF) of less than 0.3 were removed.

Variants failing the Hardy-Weinberg equilibrium (HWE) test with a p-value less than 1.5e-15 were excluded.

Variants with call rates in less than 99.9% of all individuals were eliminated.

Variants with an allele count exceeding 5% of the entire cohort (both cases and controls) were removed.

Variants with an allele frequency (AF) in the gnomAD Non-Finnish European population (NFE) or gnomADg NFE exceeding 0.01 were also excluded.

The filtering pipeline also incorporated an individual-level variant missingness filter using vcftools (v0.1.16) (—missing-indv) to exclude individuals with excessive amount of missing genotype data. No individuals had a missingness level exceeding 10%.

Loss-of-function (LOF) variants were identified utilizing the Ensembl VEP LOFTEE [32] plugin. Specifically, variants with VEP consequences such as stop gained, frameshift variant, splice acceptor variant, and splice donor variant were included. Variants marked with a LOFTEE filter "LC," indicating low-confidence LOF variants that failed at least one LOFTEE filter, were excluded from the analysis. Additionally, variants with VEP consequences of splice region and synonymous, accompanied by a SpliceAI [33] score exceeding 0.5, were incorporated into the LOF variant set.

Missense variants were selected based on a PHRED-scaled CADD [34] score greater than 25 and a REVEL [35] score exceeding 0.65. Only variants with VEP consequences such as missense variant, in frame deletion, in frame insertion, and start lost were included in the missense variant set.

Genome wide rare variant burden test

We conducted three gene-based exome-wide variant association tests: SKAT, SKAT-O, and burden test using the default settings of the SKAT R package v.2.2.4 [36]. This package allows us to treat genes as the fundamental units for statistical testing, enhancing our ability to identify disease-associated genes. The SKAT test is powerful when a small fraction of variants is causal and effects are mixed. Burden tests are sensitive when a significant fraction of variants is causal with consistent effects. The SKAT-O test combines strategies from both to maximize power. All three tests were applied to three variant sets: LOF variants, missense variants, and a combination of LOF and missense variants. CI samples were matched against 527 controls (including 62 whole genome sequencing samples). We converted the filtered variant file from VCF to plink format and used the SKAT R package for analysis.

Individual variant analysis in SeqR platform

Simultaneously with the joint analysis, individual variant analysis of genes clinically associated with connective tissue disorders were conducted for each case using a locally installed SeqR platform [37]. For each individual case, mitochondrial variants were identified using Strelka2 [38] with the—callContinuousVf chrM option. The VCF variant calls from Strelka2 were merged with VCF DeepVariant, resulting in the final VCF variant call file. This file was subsequently annotated using Ensembl VEP, and only variants with a ’PASS’ annotation were uploaded to the SeqR platform for further analysis.

Following variants were retained in the SeqR platform: nonsense, essential splice site (applying SpliceAI cut off of 0.2), missense and in-frame indels. An allele frequency (AF) threshold of 0.001 was applied for variants occurring in gnomAD exomes, gnomAD genomes, or the TOP-Med population database. Custom filters were overridden if a variant was found in ClinVar as P/LP or in the HGMD database as (likely) disease-causing.

We compiled a connective disorder gene panel by accessing data from the PanelApp database’s homepage (https://panelapp.genomicsengland.co.uk/), encompassing genes from EDS, OI, and Stickler syndrome panels (n = 102, S4 Table). Specifically, we included genes classified under green (established) and amber (putative) evidence categories, omitting those marked as red. We focused particularly on these genes as particular disorders are clinically associated with CI, and this is also in accordance with our hypothesis that CI is a subtle form of collagenopathy. Although due to allelic heterogeneity and pleiotropy analysis of this gene panel could reveal non-typical connective tissue disorders. Current gene panel also covered all genes previously linked to CI as identified in our pilot study [22]. Subsequently, to confirm any existing connective tissue disorder at the molecular level, the variants returned from this panel underwent classification according to ACGS guidelines [39] by two certified molecular geneticists.

Next, we correlated the variants identified in connective tissue gene panel among our patients with their Beighton/Brighton scores and responses to the connective tissue dysfunction assessment questionnaire (S3 Table). Given that the phenotype of isolated (non-syndromic) CI lacks a clear-cut gene-disease association in a Mendelian fashion, no SNV can be evaluated using ACGS criteria as it was done in the previous step. To overcome this, we implemented a cut-off of AF<0.001 in population databases and considered variants with a CADD score >10, designating these variants of interest as ‘rare damaging SNVs’.

Gene expression data

For the gene expression data, we sourced information from the consensus dataset on proteinatlas.org, which combines the HPA and GTEx transcriptomics datasets, and is presented in normalized expression levels (TPM).

Pathway-based burden test of ECM-related pathways

Pathway-based burden test was performed for the ECM pathways. Specific ECM pathways were taken from our pilot study [22] and encompassed following pathways: collagen formation, ECM-receptor interaction-Homo Sapiens (human), ECM organization, collagen biosynthesis and modifying enzymes, type I hemidesmosome assembly, assembly of collagen fibrils and other multimeric structures, laminin interactions, collagen chain trimerization, integrin, non-integrin membrane-ECM interactions, ECM proteoglycans, beta 1 integrin cell surface interactions, focal adhesion-Homo Sapiens (human), alpha6 beta4 integrin-ligand interactions. Additionally, we assessed ‘HSP90 chaperone cycle for steroid hormone receptors (SHR) in the presence of ligand’ pathway. The test was conducted against 527 (including 62 whole genome sequencing samples) controls in a manner identical to the gene-based rare variant burden test, but based on rare damaging variants in a pathway. Each test (Burden, SKAT, SKAT-O) was performed separately for LOF and missense variants, as well as jointly for LOF and missense variants.

Pathway analysis

In order to perform genome wide pathway enrichment of genes having rare damaging variants as well as to obtain insight about genes shown to be enriched for rare damaging variants, we conducted pathway enrichment analysis using ConsensusPathDB (available at http://cpdb.molgen.mpg.de/CPDB). We exploited interaction database gene set analysis function ‘over-representation analysis’ and looked for ‘Pathway-based sets’ in all built-in pathway databases with a p-value cut-off of 0.01. Genome-wide pathway enrichment analysis was conducted using two distinct modes: initially, genes were selected based on SNVs present exclusively in the CI group (genes shared between cases and controls). The second pathway analysis mode involved selecting genes devoid of rare damaging SNVs in controls (with no gene overlap between cases and controls).

Results

We conducted whole exome sequencing (WES) analysis on 114 patients diagnosed with a short cervix, CI or a history of CI in previous pregnancies. Patients were recruited from Latvia (n = 82) and Lithuania (n = 32)–genetically related populations of the Baltic region [40,41]. Average age of our patients was 33 ± 5.4 years; 43 patients were primiparas and 71 multiparas. On average the short cervix was diagnosed on 20 ± 4 week of gestation with average cervical length at the time of diagnosis being 17 mm. The phenotype data obtained from the connective tissue dysfunction assessment questionnaire (S3 Table), developed by our group for this study and incorporating both the Beighton and Brighton criteria [25,42], was subjected to correlation analysis with the genetic findings. The WES data from all patients fulfilled the requisite quality criteria, ensuring its suitability for subsequent analysis. An overview of the study design is depicted in the Fig 1. As controls, we utilized data from 588 individuals from our internal in-house WES database.

10.1371/journal.pone.0310718.g001 Fig 1 Overview of the study design.

CI–cervical insufficiency. WES–whole exome sequencing. WGS–whole genome sequencing. ACGS–The association for clinical genomic science. P/LP–pathogenic/likely pathogenic variant.

Analysis of variants in genes implicated in connective tissue disorders

Out of 166 filtered single nucleotide variants (SNVs) within genes known to cause connective tissue disorders in CI group (n = 114), four variants were classified as P/LP (Table 1) according to ACGS guidelines [39]. From the control samples (n = 588) 763 variants remained after filtering step, and 39 variants were classified as P/LP (S1 Table), including FKBP14 variant NM_017946.3:c.362dup which was also found in CI group. Thus, frequency of P/LP variants in connective tissue gene panel was 0.035 in patient and 0.067 in control group (p = 0.193). Clinical geneticist evaluations of patients carrying P/LP variants did not reveal any underlying Mendelian disorder during consultation (Table 1).

10.1371/journal.pone.0310718.t001 Table 1 Pathogenic / Likely pathogenic variants in connective tissue and related disorders gene panel and anamnesis of patients with cervical insufficiency.

Age	Beighton/Brighton/Total score	Gene
Associated Disease	Coding sequence
Protein alteration
ClinVar ID	ACGS Criteria	RNA expression within cervix (TPM)	Medical history	Clinical geneticist conclusion	CI diagnosis week, cervical length	Pregnancy outcome	
38	3 / 1 / 7	COL1A1
AD Ehlers-Danlos syndrome, arthrochalasia type, AD osteogenesis imperfecta	NM_000088.3:c.1663C>T
p.(Pro555Ser)
ClinVar ID: 1702168	LP (PM1, PP3, PP2, PM5 [43])	1284.0	Urinary incontinence, scoliosis, umbilical hernia. One PTB at 23 weeks of gestation (intrauterine foetal death), four pregnancy losses, two term deliveries	The patient could not attend clinical geneticist consultation	16th week of gestation,17 mm	Term delivery	
33	0 / 1 / 1	COL6A1
AD/AR Bethlem myopathy, AD/AR Ullrich congenital muscular dystrophy	NM_001848.2:c.244C>T
p.(Arg82Ter)
ClinVar ID: 1375454
	LP (PVS1, PM2)	483.6	Excessively thin /elastic skin, abnormal scarring. One term delivery, one PTB (23rd week of gestation)	Does not confirm COL6A1-associated disease. Due to signs of muscular hypotonia, the patient has been sent for a more in-depth neurological examination	20th week of gestation,0 mm	Extremely preterm (22–27 weeks of gestation) delivery	
34	0 / 0 / 0	FKBP14
AR Ehlers-Danlos syndrome, kyphoscoliotic type, 2	NM_017946.3:c.362dup
p.(Glu122ArgfsTer7)
ClinVar ID: 279809	LP (PVS1, PM3)	9.5	Three early pregnancy losses, one PTB (weeks 22–27)	Does not confirm FKBP14-associated disease	22nd week of gestation,0 mm	Extremely preterm (22–27 weeks of gestation) delivery	
39	4 / 1 / 6	ALPL
AD/AR odonto-hypophosphatasia	NM_000478.6:c.571G>A
p.(Glu191Lys)
ClinVar ID: 13670	P (PS3, PS4, PM1, PP3)	3.3	Five pregnancy losses, one PTB (31 week of gestation), family history positive for PTB	At the age of 20, six teeth were lost. Although there are no persuasive features of hypophosphatasia, additional analyses, including alkaline phosphatase, Ca, P in blood, and osteodensitometry have been requested	15th week of gestation,11 mm	Very preterm (28–31 weeks of gestation) delivery	
LP–likely pathogenic; P–pathogenic. ACGS criteria according to [39]. Beighton criteria according to [25]. Brighton criteria according to [26]. RNA expression obtained in GTEx transcriptomics dataset.

Next, we correlated the variants identified in connective tissue gene panel among our patients with their Beighton/Brighton scores and responses to the connective tissue dysfunction assessment questionnaire (S3 Table). The analysis revealed a total of 132 rare damaging SNVs in 64 genes known to be clinically associated with connective tissue disorders in 77 patients (67.5%). All genes with rare damaging SNVs showed at least minimal expression within cervical tissues (Fig 2A). The average number of rare damaging SNVs per patient with a variant was 1.71, compared to 1.79 identified in controls (p = 0.474).

10.1371/journal.pone.0310718.g002 Fig 2 Exploring genetics of cervical insufficiency: Connective tissue perspective.

A) Expression (Y axis) of connective tissue disorder genes (blue) in relation to all genes showing at least some expression within cervix (TPM>0) (light blue) (X axis). B) Beighton (pink) / Brighton (blue) / Total score (green) (Y axis) obtained by each study patient (X axis) in the connective tissue disfunction assessment questionnaire. C) Patients age (X-axis) correlation with Beighton hypermobility score (Y-axis). A larger circle size indicates a greater number of patients with the same age and Beighton score. D) Total score obtained in connective tissue dysfunction assessment questionnaire (X-axis) correlation with number of rare damaging gene variants in connective tissue gene panel (Y-axis). A larger circle size indicates a greater number of patients with the same score and number of rare variants.

Overall 27 (23.7%) patients had joint hypermobility according to Beighton score and 12 (10.5%) were positive for Brighton criteria (Fig 2B). Average age of patients with joint hypermobility comparing to those not showing signs of hypermobility according to Beighton was 31.8 ± 5.3 and 32.8 ± 5.1 (p = 0.4) (Fig 2C).

Among the 77 patients who had at least one rare damaging variant, the scores of Beighton/Brighton/connective tissue dysfunction assessment questionnaire were 1.98/1.21/4.27, respectively. On the other hand, the 37 patients with no SNVs identified had corresponding scores of 2.0/1.19/4.29 (p > 0.05). Conversely, patients with joint hypermobility according to the Beighton criteria (≥ 4 points) or a positive Brighton score had, 1.24/1.45 rare damaging SNVs accordingly vs 1.11/1.16 rare damaging SNVs of those without joint hypermobility according to Beighton (< 4 points; 87 patients) or a negative Brighton score (102 patients) (p > 0.05).

Individual number of rare damaging SNVs showed only a trend towards a positive correlation with the total score obtained in connective tissue dysfunction assessment questionnaire without reaching a statistical significance (Fig 2D).

Genome-wide rare variant burden test results

To identify genes associated with CI without a priori hypothesis (i.e. genome wide approach), we conducted three rare damaging SNV burden tests. Each test identified a number of genes enriched with rare damaging SNVs (nominal p-value < 0.01): LOF (loss of function) burden test n = 27 genes; LOF SKAT-O n = 33; LOF SKAT n = 15; missense burden n = 112; missense SKAT-O n = 123; missense SKAT n = 54; LOF/missense burden n = 122; LOF/missense SKAT-O n = 136; LOF/missense SKAT = 64 genes. In total 179 unique genes were found in at least one of the tested models (S2 Table). To gain insights into the relevance of these enriched genes in the context of CI, we conducted an evaluation that encompassed pathway enrichment analysis (Table 2), gene expression data as well as clinical and functional association (S2 Table).

10.1371/journal.pone.0310718.t002 Table 2 Top-20 pathway enrichment results of genes having burden of rare damaging SNVs in patients with cervical insufficiency.

Pathway name	p-value	Pathway name	p-value	
Prednisolone Action/Metabolism Pathway	8.73E-05	Signalling by activated point mutants of FGFR1	0.00454	
HSP90AA1
	0.000817	PI3K-Akt signalling pathway	0.00463	
Corticosteroids and cardio protection	0.002	Nuclear receptors	0.00535	
Recycling of eIF2:GDP	0.00236	FGFR1c ligand binding and activation	0.00542	
VEGFR1 specific signals	0.00246	Tetrahydrobiopterin (BH4) synthesis, recycling, salvage and regulation	0.00542	
Transport of inorganic cations/anions and amino acids/oligopeptides	0.0027	Extracellular matrix organization	0.00563	
Tyrosine metabolism	0.00346	PI3K-Akt signalling pathway—Homo sapiens (human)	0.006	
Collagen biosynthesis and modifying enzymes	0.00359	Ras signalling pathway—Homo sapiens (human)	0.00622	
Organic anion transporters	0.00374	FGFR2c ligand binding and activation	0.00636	
ROS and RNS production in phagocytes	0.00424	eNOS activation	0.00636	

Thus, out of 179 genes enriched for rare damaging SNVs, 159 had at least some expression within cervical tissues (TPM ≥ 0.1); 121 genes did not show any clinically associated phenotype (OMIM, PanelApp). Fifty-eight genes had known association with certain Mendelian diseases, although vast majority of these are unrelated to the phenotype of interest (i.e. connective tissue and related disorders).

A closer examination of genes exhibiting an increased variant burden and pathway enrichment results has sparked particular interest in the PGR (progesterone receptor), HSP90AA1 (heat shock protein HSP 90-alpha) and NR3C1 (glucocorticoid receptor) genes.

Progesterone as a naturally occurring steroid is necessary for the maintenance of pregnancy and plays a key role in maintaining cervical integrity prior to labour induction [44]. PGR’s expression within cervical tissue is 61.6 TPM. Six out of 114 patients with CI had rare SNVs in PGR (Fig 3) without having any additional rare damaging SNVs in the connective tissue disorder gene panel. Three variants were localized in (highly) missense variation intolerant regions (assessed through Metadome) [45] within known functional progesterone receptor domains–DNA binding domain and ligand binding domain. Across 588 control samples we identified four PGR variants in six patients, two SNVs were present both in patients and controls. Thus, frequency of PGR SNVs in patients was 0.052 vs 0.011 in controls (p = 0.0034). 3D analysis of the identified variants demonstrated that position Arg615 is localized proximally to the DNA binding site and amino acid change potentially could affect receptor’s and DNA interaction affinity. In turn, Arg788 localizes in the progesterone binding site and its change to Trp is predicted to affect ligand binding, potentially leading to the protein loss of function. Three remaining SNVs were predicted as benign by in silico tools, two of which are resided in an N-terminal (modulatory) domain and one SNV–C-terminally out of any domain.

10.1371/journal.pone.0310718.g003 Fig 3 PGR (progesterone receptor) variants identified in patients with cervical insufficiency and PGR amino acid positions tolerance map to missense variation.

PGR functional domains: 1) an N-terminal (modulatory) domain. 2) a DNA binding domain. 3) a hormone/ligand binding domain. GnomAD–variant frequency in a GnomAD V4 population database. CADD–combined annotation dependent depletion score, variant effect in silico prediction tool. Revel score–variant effect in silico prediction tool.

Next, across our patients we identified two rare damaging HSP90AA1 variants: NM_001017963.2:c.1843T>C p.(Tyr615His) and c.1724C>G p.(Ser575Cys) localized in a highly conserved and missense constraint positions. In controls we identified two HSP90AA1 variants, including c.2536_2538del located C-terminally out of domain and c.942A>C located in a missense intolerant region (p = 0.0001). HSP90AA1 has particularly high expression within cervix– 272.1 TPM.

Two of our patients had two NR3C1 SNVs: NM_001018077.1:c.1084G>A p.(Glu362Lys) and c.1475A>G p.(Lys492Arg). A more in-depth analysis of variants demonstrates that the position Lys492 plays a role in DNA binding and undergoes modification to N6-acetyl-lysine. Substituting the position with arginine could potentially impact the glucocorticoid receptor’s DNA binding capability. In turn, Glu362 is located within the disordered region. Across 588 controls we encountered three NR3C1 variants, including c.1094A>G in one sample located in a missense tolerated region and c.1639G>T located in missense intolerant region out of domain in two samples (p = 0.0007).

Pathway enrichment analysis results

Next, we performed targeted pathway-based burden tests (Burden, SKAT, SKAT-O) for the pathways (n = 14) demonstrating enrichment in our pilot study [22] (Methods). Additionally, we analysed burden of rare damaging variants in ‘HSP90 chaperone cycle for steroid hormone receptors in the presence of ligand’ pathway which includes PGR, HSP90AA1 and NR3C1 genes. Result revealed rare deleterious variant enrichment (nominal p-value, p < 0.05) in a few ECM-related pathways by some of the enrichment tests used: missense SKAT test revealed enrichment within ‘focal adhesion’ pathway; LOF SKAT-O and burden tests identified enrichment in ‘collagen formation’, ‘collagen chain trimerization’, and ‘collagen chain trimerization pathway’.

All tests used demonstrated statistically significant rare damaging variant enrichment in steroid hormone receptors pathway containing PGR, HSP90AA1 and NR3C1 genes, even if corrected for Benjamini-Hochberg method, e.g., LOF/missense burden test, p-adj = 5.37E-06.

In turn, genome-wide pathway enrichment analysis uncovered predominant enrichment in ECM pathways as well as highlighted several distinct pathways depending on the analysis mode (Fig 4A and 4B).

10.1371/journal.pone.0310718.g004 Fig 4 Genome wide pathway enrichment analysis results.

A. Genome-wide pathway enrichment conducted by selecting genes with single nucleotide variants present exclusively in the cervical insufficiency group (genes shared between cases and controls). B. Genome-wide pathway enrichment conducted by selecting genes devoid of rare damaging single nucleotide variants in controls (with no gene overlap between cases and controls).

Discussion

Our overarching objective was to delve into the genetic aetiology of CI. The impetus for our study emerged from a critical clinical imperative, given the current limitations in timely prediction and prevention of CI consequences in clinical settings [46]. To accomplish this, we performed WES on a meticulously phenotyped cohort comprising individuals with preterm cervical shortening or CI. Employing a comprehensive analytical approach, we ensured a thorough exploration of the WES data. Our patient cohort, consisting of 114 females from Latvia and Lithuania, represents the most extensive group of CI patients subjected to WES to date. This underscores the novelty and significance of our study in shedding light on the genetic landscape of CI.

Rethinking the role of connective tissue gene variants

Several connective tissue disorders are well-established in their clinical links to adverse pregnancy outcomes such as PTB, PPROM, and CI, [23,47]. Supporting this, compelling studies shed light on the intricate connection between CI and predisposition to pelvic organ prolapse and uterine rupture attributing to impaired collagen functioning [48]. Despite the long-standing implication of collagen in the development of CI, direct evidence from genetic studies validating this connection has been notably scarce [24]. Findings in our pilot study on the genetics of CI [22], prompted the formulation of a hypothesis that CI is a subtle manifestation of connective tissue disorder. To rigorously explore and substantiate our hypothesis, our current investigative approach comprised three key components. Firstly, we assessed the connective tissue functionality of individuals with CI by employing recognized Beighton and Brighton scores clinically used to diagnose joint hypermobility and some connective tissue disorders complemented with targeted inquiries concerning personal and family histories of PTB, CI, and general connective tissue functionality (S3 Table). Secondly, we conducted a comprehensive analysis of variants within a connective tissue gene panel. To contextualize the frequency of P/LP variants in collagen genes, we juxtaposed our findings with an analysis of our internal WES database, representative of our population (n = 588). Lastly, clinical geneticist consultations were extended to individuals exhibiting positive Brighton criteria and those harbouring P/LP variants in the analysed gene panel to assess whether a clinical diagnosis of connective tissue disorder can be established.

Analysis of genes causing connective tissue disorders according to ACGS guidelines [39] revealed four P/LP variants in COL1A1, COL6A1, FKBP14, and ALPL. COL1A1 is associated with several conditions, including arthrochalasia-type EDS and OI. It is considered that the penetrance in individuals heterozygous for a COL1A1 pathogenic variants is 100%, although disease expression may vary considerably, even within the same family [49]. Furthermore, COL1A1 has already been implicated in the development of CI from the data of case-control studies [50,51]. Regrettably, the patient harbouring the LP variant in COL1A1 in our cohort proved unreachable for discussion regarding her genetic testing outcomes. Absent a clinical geneticist’s evaluation, asserting whether patient clinically manifests a diagnosis related to COL1A1-associated conditions remains challenging. There are no reports linking COL6A1, FKBP14, and ALPL associated disorders with CI. Patients carrying P/LP variants in these genes did not demonstrate clinical features associated with the particular gene disorders.

Thus, applying ACGS guidelines to assess P/LP variants of connective tissue gene panel, indicated their rarity in patients with CI. No variants could be associated with monogenic connective tissue disorder clinically associated with CI (except the case with COL1A1 that could not be resolved). This suggests that CI in non-syndromic patients is not attributed to a single gene in a Mendelian fashion.

To gain further insights into the potential relationship among the three studied entities–genetics, CI, and connective tissue disorders–we correlated rare SNVs of connective tissue genes with the phenotypic data of patients with CI. We developed connective tissue dysfunction assessment questionnaire (S3 Table), aiming to encompass a comprehensive range of phenotypic information related to connective tissue functionality. For instance, we expanded the validated Beighton/Brighton questionnaire by incorporating details about muscle pain, a common symptom of connective tissue disorders [52]. Similarly, questions pertaining to scoliosis and the occurrence of frequent or atypical bone fractures were included to capture subtler phenotypical expressions in seemingly unaffected individuals. Recent studies indicate that women with a history of CI experienced a higher rate of pelvic organ prolapse and urinary symptoms [53], therefore, this information was also incorporated into the questionnaire.

Analysing connective tissue disorder related phenotypes revealed that in our cohort, 23.7% of patients exhibited joint hypermobility based on the Beighton criteria; while 10.5% met the clinical criteria outlined in the Brighton scoring system, theoretically indicative of hypermobility spectrum disorder. Correlation analysis between number of rare damaging SNVs and connective tissue phenotypes demonstrated a positive trend but lacked a clear correlation, hinting at the multifactorial nature of CI development. The clinical geneticist’ conclusion, following the evaluation of these patients, could only confirm benign joint hypermobility spectrum. Additional features, as assessed using the Brighton criteria, were considered as separate phenotypic units, not confirming any disease attributable to classic connective tissue disorder. As the literature suggests, joint hypermobility, including hypermobility type EDS–the most prevalent hypermobility spectrum disorder, currently lacks an identified genetic cause [42]. Notably, none of the patients positive for the Brighton criteria in our cohort had a P/LP variant in the connective tissue disorder gene panel either. Hence, the question of whether CI lies on a spectrum of joint hypermobility attributable to sub-clinically reduced connective tissue function remains open and requires further exploration.

Despite the negative results of the rare variant analysis of connective tissue genes, targeted and genome-wide examination of molecular pathways revealed a significant enrichment of variants in genes associated with collagen pathways. Interestingly, ECM-associated pathways were predominantly enriched when analysing genes with excluded overlapping variants in controls. The disparity in pathway outputs between the two analysis modes suggests that genotype-phenotype interactions may be influenced by diverse variant effects, potentially involving variants that either protect against or contribute to the CI phenotype. Pathway analysis findings not only replicate several of ECM-associated pathways identified as enriched in our pilot study [22], but also further strengthen the indication of potential cumulative involvement of multiple inherited connective tissue gene variants in the development of CI as a multifactorial disorder.

Rare variant burden analysis identifies PGR as a promising gene for CI

In order to continue a comprehensive search of a genetic markers with the potential role in the development of CI, we performed a genome wide burden analysis of rare damaging variants. As a result, 179 genes occurred to be significantly enriched as demonstrated three different tests used. Our attention captured involvement of genes associated with steroid pathways, specifically PGR, HSP90AA1 and NR3C1. PGR encodes progesterone receptor which mediates the physiological effects of progesterone. Both cervical stromal and epithelial cells express progesterone receptors [54]. Progesterone is known for its role in maintaining pregnancy by preserving uterine quiescence in the latter half of pregnancy, limiting the production of stimulatory prostaglandins, and inhibiting the expression of contraction-associated protein genes within the myometrium. It appears that progesterone also inhibits cervical collagen decomposition [55]. The onset of labour, both at term and preterm, is associated with a functional withdrawal of progesterone activity at the level of the uterus. Progesterone was the first FDA approved supplementation for the prevention of PTB [56] and evidence of the therapeutic utility of progesterone for the prevention of CI and PTB in women at-risk is well documented [57]. In our study, we identified a statistically significant prevalence of rare damaging PGR variants in cases compared to controls. In silico and 3D analysis of the identified variants demonstrated that three variants, one localized within the DNA binding domain and two within the hormone/ligand binding domain, predict variants as pathogenic. In theory, such variants could lead to the reduction of activity (or even loss of function) of the progesterone receptor via an inability/reduced ability to be stimulated by progesterone or an inability/reduced ability to bind the DNA. We speculate that impaired PGR functioning due to pathogenic variants could eventually lead to preterm cervical shortening by two possible pathways: 1) malfunctioning endogenous progesterone effect leading to an abrupt progesterone withdrawal; 2) unsuccessful exogenous progesterone administration. In fact, progesterone therapy is effective only in a subset of patients [58], leaving the rest without a clear understanding of the underlying aetiology. In theory, PGR variants may thus account for a proportion of unsuccessful progesterone administration.

Next, NR3C1 encodes the prednisolone receptor, a steroid hormone receptor whose endogenous agonist is cortisol. This receptor regulates the expression of anti-inflammatory and immunosuppressive effects. Importantly that progesterone, which shows structural similarities to glucocorticoids, can bind the intracellular glucocorticoid receptor, promoting maternal immune tolerance to foetal alloantigens through a wealth of immunomodulatory mechanisms [59]. Literature suggests that variants in NR3C1 are associated with the AD glucocorticoid resistance characterized by impaired cortisol signalling, clinically resulting in hypoglycaemia, hypertension, metabolic alkalosis, chronic fatigue and female infertility [60]. In turn, heat shock protein 90 (HSP90AA1) assists in folding the prednisolone receptor and facilitates its transport into the nucleus [61]. Both genes exhibit significant expression within cervical tissues. Once again, we may cautiously assume that variants in these genes could potentially disrupt the normal prednisolone action/metabolism pathway leading to increased susceptibility to infections or compromising the immunosuppressive effects crucial for maintaining maternal immune system tolerance during pregnancy eventually resulting in preterm cervical shortening and/or PTB.

Notably, rare damaging variants in the steroid hormone receptors pathway reached exome-wide significance in our moderate sample size cohort, pointing to the significance of these variants for the pathophysiology of CI. In a recent study, Wang et al. also discovered a variant burden in genes regulated by the PGR, proposing it as a predictor of responses to progestin treatment for PTB [62]. Hence our findings for the first time corroborate Wang et al.’s suggestion that rare damaging SNVs within the PGR pathway may play a significant role in PTB development.

While our findings suggest intriguing associations between rare damaging variants in PGR, HSP90AA1, NR3C1 and CI, it is crucial to approach these observations with caution. These are early assumptions that require rigorous validation through further investigations. It is noteworthy that standard variant interpretation guidelines do not assess variants in genes lacking interactions with clinical phenotype [63]. Functional studies of the identified variants are necessary to elucidate their specific impact on the progesterone and prednisolone pathways and, by extension, their potential contribution to CI.

Study limitations

While our study represents the largest cohort of comprehensively phenotyped females with CI undergoing WES, it is important to acknowledge the limitations that may impact the generalizability of our findings. The relatively modest sample size, though substantial, is still insufficient to definitively ascertain the spectrum of possible connective tissue disorders resulting from P/LP variants in connective tissue or other genes. Establishing a more comprehensive understanding of the causative role of rare damaging variants, particularly in influencing the complex phenotype of CI and the broader spectrum of connective tissue disorders, necessitates a larger cohort. It is noteworthy that our study relied on control data from our internal WES database, primarily composed of individuals undergoing testing for cardiac and neurological diseases. This targeted selection may introduce a bias, limiting the generalizability of our results to a broader population. Moreover, our study is constrained by the absence of a matched control group that underwent thorough phenotyping in a manner analogous to the study cohort. A meticulously phenotyped control group would have provided a crucial baseline for estimating the prevalence of hypermobility within the CI group.

Concluding remarks and the future perspectives

Number of genes identified as potentially contributing to the CI/PTB are limited [64,65]. The current understanding of the pathophysiology of CI primarily revolves around inflammation and connective tissue dysfunction posing a challenge to unravelling novel pathways and genes contributing to the pathobiology of this condition. Our study provides evidence that unexplored avenues exist. While our primary goal was to elucidate the relationship between CI and compromised connective tissue function, our study has illuminated PGR, NR3C1 and HSP90AA1 –steroid pathway genes not conventionally associated with this condition, but potentially contributing to impaired endogenous progesterone effects leading to CI and/or PTB and/or reduced efficacy of progesterone administration in prolonging pregnancy in clinics (Fig 5). This discovery holds the promise of at least informed clinical decisions and improved management of CI patients; at best, it could open new avenues for therapeutic explorations. We believe that the success of identifying new CI-associated genes is rooted in meticulous patient selection. We strongly advocate for precise phenotyping of PTB patients and subgrouping based on clinical representation as a fundamental key to successful genetic and pathophysiological studies of PTB-related conditions. Collaborative efforts within the scientific community can provide more statistical power and enhance the generalizability of our observations.

10.1371/journal.pone.0310718.g005 Fig 5 Summary of study design and major outcomes.

We were the first to apply the Beighton/Brighton criteria to test the hypothesis linking the connective tissue dysfunction-driven nature of CI to genetics. While our analysis strengthened the association of CI with connective tissue pathways, further research is needed to explore the relationships between subclinical phenotypic expressions of connective tissue disorders and CI. Our next hypothesis is that the use of Beighton/Brighton criteria, along with the connective tissue dysfunction assessment questionnaire developed by our group, can serve as a predictive tool for CI/PTB, at least for a subset of patients. This investigation is currently underway in our group.

Conclusions

CI is not attributed to rare damaging variants in known genes causing connective tissue disorders in a Mendelian fashion, although this finding does not exclude the involvement of the connective tissue dysfunction pathways as a significant mechanism contributing to CI as multifactorial disorder. Rare damaging variants in the progesterone receptor (PGR), glucocorticoid receptor (NR3C1), and heat shock protein HSP 90-alpha (HSP90AA1), may play a role in the pathogenesis of CI and/or PTB by interfering with progesterone’s physiological effects during pregnancy.

Supporting information

S1 Table Pathogenic/Likely pathogenic variants identified in controls.

(XLSX)

S2 Table Genes enriched for rare damaging SNVs.

(XLSX)

S3 Table Connective tissue dysfunction assessment questionnaire.

(PDF)

S4 Table Connective tissue disorder panel genes.

(CSV)

10.1371/journal.pone.0310718.r001
Decision Letter 0
Bayraktar Burak Academic Editor
© 2024 Burak Bayraktar
2024
Burak Bayraktar
https://creativecommons.org/licenses/by/4.0/ This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Submission Version0
3 Apr 2024

PONE-D-24-05772Further Insights into the Genetics of Preterm Cervical Shortening During PregnancyPLOS ONE

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Additional Editor Comments:

Editor:

“We conducted whole exome sequencing (WES) analysis on 114 patients diagnosed with a short cervix, CI or a history of CI in previous pregnancies.” How did you choose the cases? More precisely, was this study a prospective study on short cervix? Or were patients who underwent WES examination for other indications retrospectively investigated for short cervix?

Can you include patient’s demographics? Smoking, uterine anomaly, systemic diseases, etc. was the excluded?

I guess it means you found no significant genome changes?

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Reviewer #1: No

**********

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Reviewer #1: No

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Reviewer #1: No

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Reviewer #1: Study of important and relevant themes for the magazine. The main objective of the study was to deepen discussions about the genetic factors that contribute to IC.

The study brings relevant results, but its biggest problem is that it does not present a methods session. There is a very brief paragraph in the discussion commenting on what was carried out in the study. I strongly suggest that the authors insert a methods session into the study involving study design, setting (where the study was carried out), population studied, inclusion and exclusion criteria, how these participants were recruited, procedures carried out for data collection, analysis of data, ethics committee, etc. Without this session, it is not possible to evaluate the study and say whether it was carried out correctly or whether there were biases.

Another point that I think is important to mention is that the title is not eye-catching and does not arouse the reader's curiosity.

I leave as a suggestion: Unraveling the Genetic Landscape of Cervical Insufficiency: Insights into Connective Tissue Dysfunction and Hormonal Pathways

In the summary, I suggest clearly and objectively stating the objective of the study and also structuring a methods session.

Keywords: lots of keywords. Choose a maximum of five.

In the introduction, it is important to provide a little more detail about the reasons why the PTB is a public health concern. What are its implications?

Furthermore, what were the motivations for conducting the study? What are the hypotheses? What new does the study bring? What knowledge gaps do you aim to fill? The justification for the study needs to be clearer and better defined in the introduction

Regarding the results and discussion, I have no comments at first, I would need to know more details about how the study was conducted (methods) to be able to assess whether the results were reported and discussed appropriately.

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10.1371/journal.pone.0310718.r002
Author response to Decision Letter 0
Submission Version1
17 May 2024

Dear Burak Bayraktar,

Dear Reviewer,

Thank you very much for processing and reviewing our manuscript. We have addressed all the comments and provided point-by-point answers below, incorporating the suggested changes into the manuscript.

The provided line numbers are from the unmarked version of revised paper without tracked changes.

Editor:

Question: “We conducted whole exome sequencing (WES) analysis on 114 patients diagnosed with a short cervix, CI or a history of CI in previous pregnancies.” How did you choose the cases? More precisely, was this study a prospective study on short cervix? Or were patients who underwent WES examination for other indications retrospectively investigated for short cervix?

Answer: The process of enrolling patients in the study is described in the Methods section (lines 483-502). Briefly, patients undergoing routine antenatal care were examined for a short cervix, and those requiring treatment for cervical shortening were recruited. This study was conducted as a prospective longitudinal cohort study, as detailed in the Methods section (lines 484).

Question: Can you include patient’s demographics? Smoking, uterine anomaly, systemic diseases, etc. was the excluded?

Answer: Some patient demographics, including ethnicity, age, and parity, are described in the Results section (lines 128-132). Additional phenotype data was collected using the connective tissue dysfunction assessment questionnaire, as detailed in the Methods section (lines 497-499). We have now supplemented the Methods section with the exclusion criteria, which also includes uterine anomalies. Notably, systemic diseases (unless part of a genetic syndrome) and smoking were not exclusion criteria.

Question: I guess it means you found no significant genome changes?

Answer: None of the patients in our study were found to have monogenic connective tissue disorder. Instead, our main finding was the identification of rare damaging variant enrichment in steroid hormone receptor pathway genes, with variants in PGR, HSP90AA1, and NR3C1 reaching genome-wide statistical significance even after correction for multiple testing (as indicated in lines 262-263 of the Results section).

Reviewer #1:

Question: Study of important and relevant themes for the magazine. The main objective of the study was to deepen discussions about the genetic factors that contribute to IC.

The study brings relevant results, but its biggest problem is that it does not present a methods session. There is a very brief paragraph in the discussion commenting on what was carried out in the study. I strongly suggest that the authors insert a methods session into the study involving study design, setting (where the study was carried out), population studied, inclusion and exclusion criteria, how these participants were recruited, procedures carried out for data collection, analysis of data, ethics committee, etc. Without this session, it is not possible to evaluate the study and say whether it was carried out correctly or whether there were biases.

Answer: Thank you sincerely for dedicating your time and effort to reviewing our manuscript. We apologize for any confusion regarding the Methods section's visibility. You can find the Methods section starting with line 474. The Methods placement after the Discussion is in line with the formatting guidelines stipulated by PLOS One.

Question: Another point that I think is important to mention is that the title is not eye-catching and does not arouse the reader's curiosity. I leave as a suggestion: Unraveling the Genetic Landscape of Cervical Insufficiency: Insights into Connective Tissue Dysfunction and Hormonal Pathways

Answer: Thank you very much for this nice suggestion, with pleasure we are accepting this new title.

Question: In the summary, I suggest clearly and objectively stating the objective of the study and also structuring a methods session.

Answer: The primary aim of our study is stated in lines 115-116, and we have revised it for clarity.

Question: Keywords: lots of keywords. Choose a maximum of five.

Answer: We understand the concern regarding the abundance of keywords. However, PLOS One encourages the inclusion of multiple keywords to enhance the discoverability of published articles. Therefore, we prefer to retain the current keywords.

Question: In the introduction, it is important to provide a little more detail about the reasons why the PTB is a public health concern. What are its implications?

Answer: We have briefly addressed the paragraph on PTB (please see lines 46-50). However, we chose not to significantly expand this paragraph as considerable literature already exists discussing the social and economic concerns of PTB, as referenced in this paragraph. Given that the primary focus of our article is the genetics of cervical insufficiency, we opted not to shift the primary focus of the study.

Question: Furthermore, what were the motivations for conducting the study?

Answer: Our primary motivation for studying the genetics of CI stems from clinical needs, as pregnancy outcomes in this patient group are particularly concerning (see lines 97-99 and 276-278 in the Discussion section). The genetics of PTB and particularly CI remain poorly understood, with existing literature often lacking clinical subgrouping. We aimed to address this gap by focusing specifically on the CI patient group, anticipating insights into specific genes and molecular pathways, particularly related to the connective tissue hypothesis (112-114).

Question: What are the hypotheses?

Answer: As you may understand, the hypothesis for this study stems from our previous pilot study where we identified variants in extracellular matrix-related genes in patients with cervical insufficiency. This led us to hypothesize that CI might represent a subtle form of connective tissue disorder (please refer to lines 108-114).

Question: What new does the study bring?

Answer: While we understand that some studies choose to highlight findings in the Introduction, we have opted to reserve the anticipation of study results for the Results and Discussion sections.

Question: What knowledge gaps do you aim to fill?

Our study aims to address significant knowledge gaps in the field of cervical insufficiency. Currently, there are no aetiological treatments available for this condition, largely due to limited understanding of its molecular mechanisms and the neglect of CI genetics in the scientific community. Our goal is to decipher the molecular pathways underlying CI to contribute to a better understanding of its pathogenesis.

Question: The justification for the study needs to be clearer and better defined in the introduction.

Answer: Thank you very much for the detailed analysis of our Introduction section and providing valuable insights. We have made substantial changes in the Introduction to better clarify the justification for conducting our study.

As outlined in the Introduction, there is limited understanding of the genetics of cervical insufficiency, and our pilot study was among the first to apply next-generation sequencing to this patient group, for the current study we collected the largest cohort of patients for whole exome sequencing. We aimed to validate the long-standing link between connective tissue dysfunction and CI through genetic studies and developed the connective tissue dysfunction assessment as a comprehensive clinical tool for this purpose.

Unexpectedly, our study also revealed significant findings related to the progesterone pathway, despite the modest sample size. We hope you will find this aspect of the study particularly intriguing.

Question: Regarding the results and discussion, I have no comments at first, I would need to know more details about how the study was conducted (methods) to be able to assess whether the results were reported and discussed appropriately.

Answer: Thank you for your initial assessment. We understand that a thorough evaluation of the Results and Discussion sections depends on a comprehensive understanding of the study methods. We hope that our Methods section will provide the necessary details, and we look forward to your feedback on the manuscript once it is complete.

Once again, we hope our clarifications have addressed all current concerns, and we remain open to any further suggestions or feedback you may have.

Thank you once again for your valuable input.

Kind regards,

Authors

Attachment Submitted filename: Response to Reviewers.docx

10.1371/journal.pone.0310718.r003
Decision Letter 1
Bayraktar Burak Academic Editor
© 2024 Burak Bayraktar
2024
Burak Bayraktar
https://creativecommons.org/licenses/by/4.0/ This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Submission Version1
15 Jul 2024

PONE-D-24-05772R1Unravelling the Genetic Landscape of Cervical Insufficiency: Insights into Connective Tissue Dysfunction and Hormonal PathwaysPLOS ONE

Dear Dr. Volozonoka,

Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process.

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We look forward to receiving your revised manuscript.

Kind regards,

Burak Bayraktar

Academic Editor

PLOS ONE

[Note: HTML markup is below. Please do not edit.]

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation.

Reviewer #2: All comments have been addressed

Reviewer #3: All comments have been addressed

**********

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Reviewer #2: Partly

Reviewer #3: Yes

**********

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Reviewer #2: No

Reviewer #3: Yes

**********

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Reviewer #2: Yes

Reviewer #3: Yes

**********

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Reviewer #2: Yes

Reviewer #3: Yes

**********

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Reviewer #2: Dear Authors,

this is an interesting prospective longitudinal cohort study, trying to identify the molecular causes of cervical insufficiency. This one represents one of the origins of the main cause itself of fetal morbidity and mortality in Obstetrics: Preterm Birth. I read with great interest the Manuscript, which falls within the aim of this Journal. It is the revision number two for this journal. Several of the corrections required have been applied. In my honest opinion, some major improvements and corrections are still needed.

Title- I see the corrections applied

Abstract:

The abstract should better be structured for sake of clarity. It should contain background, Objective, methods, Results and Conclusion. More insights regarding the methods are already needed

Introduction:

Improvements have been made according to the previous reviewer advices, but a descriptive paragraph of what preterm birth is, should be written. I have no objections for the rest of the introduction

Results: This paragraph appears too repetitive and it should not just be the list of the collected data. Numbers should be presented in a systematic and goal-oriented manner.

Discussion:

The discussion should elucidate and find possible explanations for the research. It should be revised in the light of the methods.

Conclusions:

Rename this section.

It should be more consistent and summarize your main findings, clinical implications and suggest areas for further research.

Avoid overgeneralization.

Materials and Methods:

There are some improvement compared to the first version, but is still not enough to make the manuscript scientifically sound. There is no chance to promote this kind of study lacking an appropriate Materials and Methods section.

Reviewer #3: Dear authors,

Thank you for revising the manuscript considering the previously suggestions from the reviewers in the first review round.

All the comments of the previously reviewers have been addressed by the authors and the manuscript was improved.

I strongly suggest to the authors to move the Methods, according to the Plos One submission guidelines called "Materials and Methods", after the Introduction section.

The "Materials and Methods" in the Plos One is located in the Middle section, after the introduction.

Please verify the manuscript organization located in this page: https://journals.plos.org/plosone/s/submission-guidelines

I do not have additional comments on the revision in this manuscript version.

**********

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Reviewer #2: No

Reviewer #3: No

**********

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10.1371/journal.pone.0310718.r004
Author response to Decision Letter 1
Submission Version2
21 Aug 2024

Dear Burak Bayraktar,

Dear Reviewers,

Thank you very much for processing and reviewing our manuscript. We have addressed all the comments and provided point-by-point answers below, incorporating the suggested changes into the manuscript.

The provided line numbers are from the unmarked version of revised paper without tracked changes.

Reviewer #2:

Dear Authors,

this is an interesting prospective longitudinal cohort study, trying to identify the molecular causes of cervical insufficiency. This one represents one of the origins of the main cause itself of fetal morbidity and mortality in Obstetrics: Preterm Birth. I read with great interest the Manuscript, which falls within the aim of this Journal. It is the revision number two for this journal. Several of the corrections required have been applied. In my honest opinion, some major improvements and corrections are still needed.

Title- I see the corrections applied

Abstract:

The abstract should better be structured for sake of clarity. It should contain background, Objective, methods, Results and Conclusion. More insights regarding the methods are already needed

Answer: Dear Reviewer, thank you very much for your time and valuable feedback on improving our manuscript. We have restructured the Abstract to include Background, Methods, Results and Conclusion. We have enhanced the text to provide more details and clarity.

Introduction:

Improvements have been made according to the previous reviewer advices, but a descriptive paragraph of what preterm birth is, should be written. I have no objections for the rest of the introduction

Answer: Please refer to lines 48-79 of the Introduction, which includes a sub-section titled ‘Preterm birth: one outcome – multiple aetiologies and distinct molecular pathways’ (line 53) providing prevalence, etiological pathways, contributing factors, and current treatment options of PTB.

Results: This paragraph appears too repetitive and it should not just be the list of the collected data. Numbers should be presented in a systematic and goal-oriented manner.

Answer: We have revised the Results section to minimize repetition and present data systematically. The structure focuses on:

1. Variant analysis in genes associated with connective tissue disorders, based on ACGS guidelines, to explore the hypothesis that CI might reflect a subtle connective tissue disorder. We also correlated genetic findings with connective tissue dysfunction assessment results (lines 317-360).

2. Genome-wide (a hypothesis-free) rare variant burden test that identified PGR, HSP90AA1, and NR3C1 genes as potentially significant in CI development (lines 361-417).

3. Pathway enrichment analyses performed both in a targeted manner and genome-wide (lines 418-433).

We believe our revisions address the concern about repetitiveness and present the data in a clear manner.

Discussion:

The discussion should elucidate and find possible explanations for the research. It should be revised in the light of the methods.

Answer: The Discussion section provides detailed explanations and contextualize our findings based on the methodologies used. The discussion is organized into sub-sections corresponding to the different perspectives of our analysis (i.e., connective tissue perspective, hormonal pathway perspective). Each sub-section addresses our findings in the context of existing literature and aims to elucidate possible explanations.

If Reviewer have specific concerns or inquiries regarding any part of the discussion, we would be happy to address them.

Conclusions:

Rename this section.

It should be more consistent and summarize your main findings, clinical implications and suggest areas for further research.

Avoid overgeneralization.

Answer: We have renamed and revised the Conclusions section to ensure it provides a clear and concise summary of our main findings, avoiding overgeneralization.

Materials and Methods:

There are some improvement compared to the first version, but is still not enough to make the manuscript scientifically sound. There is no chance to promote this kind of study lacking an appropriate Materials and Methods section.

Answer: The Methods section has now been moved after the Introduction and provides a detailed account of all steps performed during the study. If the Reviewer has specific concerns or requires further clarification on any aspect of this section, we would be happy to address them. We have aimed to ensure that all procedures involving patient and genomic data are thoroughly described.

Reviewer #3: Dear authors,

Thank you for revising the manuscript considering the previously suggestions from the reviewers in the first review round.

All the comments of the previously reviewers have been addressed by the authors and the manuscript was improved.

I strongly suggest to the authors to move the Methods, according to the Plos One submission guidelines called "Materials and Methods", after the Introduction section.

The "Materials and Methods" in the Plos One is located in the Middle section, after the introduction.

Please verify the manuscript organization located in this page: https://journals.plos.org/plosone/s/submission-guidelines

I do not have additional comments on the revision in this manuscript version.

Answer: Thank you very much for your time and efforts in reviewing our manuscript. We have moved the Methods section immediately after the Introduction.

Once again, we hope our clarifications have addressed all current concerns, and we remain open to any further suggestions or feedback you may have.

Thank you once again for your valuable input.

Kind regards,

Authors

Attachment Submitted filename: Response to Reviewers_21_08_2024.docx

10.1371/journal.pone.0310718.r005
Decision Letter 2
Bayraktar Burak Academic Editor
© 2024 Burak Bayraktar
2024
Burak Bayraktar
https://creativecommons.org/licenses/by/4.0/ This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Submission Version2
6 Sep 2024

Unravelling the Genetic Landscape of Cervical Insufficiency: Insights into Connective Tissue Dysfunction and Hormonal Pathways

PONE-D-24-05772R2

Dear Dr. Volozonoka,

We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements.

Within one week, you’ll receive an e-mail detailing the required amendments. When these have been addressed, you’ll receive a formal acceptance letter and your manuscript will be scheduled for publication.

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Kind regards,

Burak Bayraktar

Academic Editor

PLOS ONE

10.1371/journal.pone.0310718.r006
Acceptance letter
Bayraktar Burak Academic Editor
© 2024 Burak Bayraktar
2024
Burak Bayraktar
https://creativecommons.org/licenses/by/4.0/ This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
10 Sep 2024

PONE-D-24-05772R2

PLOS ONE

Dear Dr. Volozonoka,

I'm pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now being handed over to our production team.

At this stage, our production department will prepare your paper for publication. This includes ensuring the following:

* All references, tables, and figures are properly cited

* All relevant supporting information is included in the manuscript submission,

* There are no issues that prevent the paper from being properly typeset

If revisions are needed, the production department will contact you directly to resolve them. If no revisions are needed, you will receive an email when the publication date has been set. At this time, we do not offer pre-publication proofs to authors during production of the accepted work. Please keep in mind that we are working through a large volume of accepted articles, so please give us a few weeks to review your paper and let you know the next and final steps.

Lastly, if your institution or institutions have a press office, please let them know about your upcoming paper now to help maximize its impact. If they'll be preparing press materials, please inform our press team within the next 48 hours. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information, please contact onepress@plos.org.

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Thank you for submitting your work to PLOS ONE and supporting open access.

Kind regards,

PLOS ONE Editorial Office Staff

on behalf of

Dr. Burak Bayraktar

Academic Editor

PLOS ONE
==== Refs
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