Altered Interhemispheric DLPFC Coordination and Cardiac Hyperreactivity During Multisensory Aversive Challenge in Recurrent Pregnancy Loss With Comorbid Anxiety (NEURO-CARD-2)

Altered Interhemispheric DLPFC Coordination and Cardiac Hyperreactivity During Multisensory Aversive Challenge in Recurrent Pregnancy Loss With Comorbid Anxiety: a Multicenter Simultaneous fNIRS-ECG Study (NEURO-CARD-fNIRS)

This exploratory clinical study, NEURO-CARD-2, will use simultaneous functional near-infrared spectroscopy (fNIRS) and electrocardiography (ECG) to investigate interhemispheric dysfunction in the dorsolateral prefrontal cortex (DLPFC) and its association with autonomic sympathetic activation in women with recurrent pregnancy loss (RPL) and comorbid anxiety. Using a standardized multisensory aversive emotional stimulation paradigm, the study will assess cortical and cardiac responses within a Brain-Heart-Emotion interaction framework. The objective will be to identify neurobiological signatures of emotion-autonomic dysregulation in this population and to inform the future development of precision-targeted interventions.

Study Overview

• Status: Completed
• Conditions: Anxiety Disorder; Recurrent Pregnancy Loss Without Current Pregnancy
• Intervention / Treatment: Behavioral: Multisensory aversive emotional challenge task

Detailed Description

Recurrent pregnancy loss (RPL), defined as two or more consecutive pregnancy losses before 24 weeks of gestation, affects an estimated 2% to 5% of reproductive-aged couples worldwide. In addition to its reproductive consequences, RPL is associated with a substantial psychological burden, and approximately 50% of affected women experience chronic anxiety. This emotional burden has been linked to persistent sympathetic activation, including elevated resting heart rate and reduced heart rate variability, which may contribute to cardiovascular and reproductive risk.

Contemporary psycho-cardiology models, including statements from the American Heart Association, emphasize the close relationship between emotional dysregulation and autonomic dysfunction. The neural mechanisms linking altered central emotion regulation to cardiac autonomic outcomes in women with RPL remain insufficiently characterized, particularly in those with comorbid anxiety.

Emerging work in interoceptive neuroscience suggests that higher-order brain regions may provide shared neural substrates for anxiety and sympathetic overactivation. The dorsolateral prefrontal cortex (DLPFC), a key node in the cognitive control network and central autonomic network, is of particular interest. Neuroimaging and neuromodulation studies have shown hemispheric asymmetry within the DLPFC. The right DLPFC has been associated with threat processing, anxiety, and sympathetic arousal, whereas the left DLPFC has been associated with cognitive reappraisal, emotional inhibition, and parasympathetic modulation.

A Brain-Heart-Emotion interaction model underlies this study. Within this framework, effective autonomic adaptation during negative emotional challenge is hypothesized to depend on coordinated bilateral DLPFC recruitment. Functional decoupling, expressed as right-lateralized DLPFC dominance, may weaken emotion regulation capacity and promote sympathetic overactivation. In women with RPL and comorbid anxiety, this pattern is hypothesized to contribute to the convergence of emotional dysregulation and cardiac dysfunction, with potential implications for reproductive and cardiovascular risk.

To evaluate this hypothesis, this prospective exploratory clinical study will use simultaneous fNIRS and ECG during a standardized multisensory emotional provocation paradigm. Patterns of interhemispheric DLPFC activation and their associations with heart rate dynamics will be examined in women with RPL with and without comorbid anxiety within the proposed Brain-Heart-Emotion framework.

If confirmed, the findings may provide mechanistic insight and empirical support for neurobiologically informed precision-targeted interventions. Potential implications may include support for inhibitory neuromodulation targeting the right DLPFC, with possible benefits for emotional regulation, autonomic balance, long-term cardiovascular risk reduction, and reproductive outcomes in this high-risk population.

• Study Type: Observational
• Enrollment (Actual): 70

Contacts and Locations

Study Locations

• China
  • Liaoning
    • Shenyang, Liaoning, China, 110024
      • Central Hospital Affiliated to Shenyang Medical College
    • Shenyang, Liaoning, China, 110001
      • The Second Affiliated Hospital of Shenyang Medical College
    • Shenyang, Liaoning, China, 110045
      • 157 Hospital of Liaoning Health Industry Group
    • Shenyang, Liaoning, China, 110086
      • 242 Hospital Affiliated to Shenyang Medical College

Participation Criteria

Eligibility Criteria

• Ages Eligible for Study: Adult
• Accepts Healthy Volunteers: No

• Sampling Method: Non-Probability Sample

Study Population

Recruitment is expected to occur through obstetrics and gynecology outpatient clinics, as well as community outreach via flyers and media campaigns.

Description

Inclusion Criteria:

• Female, age 18 to 45 years, right-handed;
• Diagnosis of recurrent pregnancy loss, defined as two or more consecutive spontaneous miscarriages before 28 weeks of gestation;
• Not currently pregnant, or diagnosed with missed abortion at the time of assessment;
• Completed a structured psychiatric evaluation conducted by licensed psychiatrists at each center, with diagnostic confirmation according to the Diagnostic and Statistical Manual of Mental Disorders, Fifth Edition (DSM-5);
• For participants assigned to the RPL with anxiety group: meets DSM-5 diagnostic criteria for generalized anxiety disorder, has a Hamilton Anxiety Rating Scale score of at least 14, and has a 17-item Hamilton Depression Rating Scale score of 17 or lower;
• For participants assigned to the RPL without anxiety group: does not meet DSM-5 diagnostic criteria for generalized anxiety disorder, has a Hamilton Anxiety Rating Scale score below 14, and has a 17-item Hamilton Depression Rating Scale score of 17 or lower.

Exclusion Criteria:

• Use of psychotropic medication within the past month, including selective serotonin reuptake inhibitors, serotonin-norepinephrine reuptake inhibitors, tricyclic antidepressants, benzodiazepines, antipsychotics, or mood stabilizers;
• Unstable or uncontrolled blood pressure, defined as systolic blood pressure greater than 180 mmHg or less than 90 mmHg at screening;
• Major comorbid organic conditions that could affect autonomic or neural measurements, including hyperthyroidism, atrial fibrillation, clinically significant valvular heart disease, prior stroke, epilepsy, traumatic brain injury, or chronic pulmonary disease;
• Significant sensory or communication barriers that could impair task performance or stimulus perception, including hearing impairment, language difficulty, or sensory neuropathy;
• High suicide risk or severe psychiatric comorbidity, including psychotic disorders, bipolar disorder, or substance use disorders;
• Marked intolerance to auditory, visual, or cold stimuli based on medical history or pre-test report;
• Any other condition judged by the study physician to interfere with safe participation in the multisensory aversive stimulation protocol.

Study Plan

How is the study designed?

Number of groups / cohorts

2

Cohorts and Interventions

Group / Cohort	Intervention / Treatment
Women with recurrent pregnancy loss and comorbid anxiety; Women with recurrent pregnancy loss who will meet DSM-5 criteria for a comorbid anxiety disorder. Participants in this cohort will undergo the same protocol-assigned multisensory aversive stimulation paradigm during simultaneous fNIRS and ECG recording. This cohort will be compared with women with recurrent pregnancy loss without comorbid anxiety to evaluate DLPFC activation, interhemispheric coherence, heart-rate responses, and brain-heart coupling features.	Behavioral: Multisensory aversive emotional challenge task; Participants will undergo a standardized multisensory aversive stimulation paradigm during simultaneous fNIRS and ECG recording. The protocol will use a block design with 12 stimulation blocks, each comprising a 20 s resting phase followed by a 30 s multisensory stimulation phase. During stimulation, participants will view six negative high-arousal images per block selected from the Geneva Affective Picture Database, while concurrently being exposed to time-locked band-limited white noise calibrated to approximately 90 dB(A) and placing both hands on a 0.5 liter bottle filled with ice water maintained at approximately 0 °C. The auditory stimulus will have spectral energy restricted to 2-6 kHz. This standardized multisensory protocol will be used to elicit negative affect and sympathetic arousal.
Women with recurrent pregnancy loss without comorbid anxiety; Women with recurrent pregnancy loss who will not meet DSM-5 criteria for a comorbid anxiety disorder. Participants in this cohort will undergo the same protocol-assigned multisensory aversive stimulation paradigm during simultaneous fNIRS and ECG recording. This cohort will serve as the comparison group for evaluating DLPFC activation, interhemispheric coherence, heart-rate responses, and brain-heart coupling features.	Behavioral: Multisensory aversive emotional challenge task; Participants will undergo a standardized multisensory aversive stimulation paradigm during simultaneous fNIRS and ECG recording. The protocol will use a block design with 12 stimulation blocks, each comprising a 20 s resting phase followed by a 30 s multisensory stimulation phase. During stimulation, participants will view six negative high-arousal images per block selected from the Geneva Affective Picture Database, while concurrently being exposed to time-locked band-limited white noise calibrated to approximately 90 dB(A) and placing both hands on a 0.5 liter bottle filled with ice water maintained at approximately 0 °C. The auditory stimulus will have spectral energy restricted to 2-6 kHz. This standardized multisensory protocol will be used to elicit negative affect and sympathetic arousal.

What is the study measuring?

Primary Outcome Measures

Outcome Measure	Measure Description	Time Frame
Group-dependent hemispheric asymmetry of DLPFC activation during aversive emotional stimulation (group × hemisphere interaction)	The primary endpoint will be the group-by-hemisphere interaction in task-evoked DLPFC HbO activation during aversive emotional stimulation, comparing women with recurrent pregnancy loss (RPL) with anxiety versus without anxiety. Hemisphere will be defined as right versus left DLPFC, measured within the same participant. The participant-level activation metric will be the DLPFC HbO response estimate (β) derived from the prespecified fNIRS analysis pipeline. The primary hypothesis will be tested using a linear mixed-effects model (LME) fitted by restricted maximum likelihood, with prespecified covariates including age, education, body mass index, miscarriage etiology category, and resting heart rate. The primary confirmatory test will evaluate whether the right-minus-left difference in DLPFC activation differs by group.	Single-session fNIRS-ECG protocol (Day 1)

Secondary Outcome Measures

Outcome Measure	Measure Description	Time Frame
Group difference in interhemispheric synchronization of DLPFC activity	Interhemispheric DLPFC synchronization will be assessed using wavelet transform coherence (WTC) between homologous left and right DLPFC HbO signals. For each participant, coherence values will be averaged across the task-relevant low-frequency band (0.01-0.08 Hz) and across the stimulation period, background-corrected using the 3-minute resting period, and Fisher z-transformed to yield a participant-level synchronization metric. Between-group differences will be summarized descriptively and tested using a two-sample independent t test and a covariate-adjusted linear regression model with prespecified covariates. Prespecified supporting analyses will repeat the same computation and inferential framework across four additional prefrontal subregions to assess spatial specificity.	Single-session fNIRS-ECG protocol (Day 1)
Group difference in mean heart-rate increase during aversive emotional stimulation	Autonomic reactivity will be assessed as the mean heart-rate increase during aversive stimulation relative to the resting baseline, derived from ECG over the prespecified task window using a uniform preprocessing pipeline across participants. Preprocessed R-R intervals will be resampled at 11 Hz, filtered with a fourth-order low-pass Butterworth filter with a 0.1 Hz cutoff, detrended, segmented into 40-second epochs consisting of 10 seconds before stimulus onset and 30 seconds during stimulation, and baseline-corrected using the 10-second prestimulus period. The resulting heart-rate increase will be averaged across the 12 blocks for each participant. Between-group differences will be tested using a two-sample independent t test and a covariate-adjusted linear regression model.	Single-session fNIRS-ECG protocol (Day 1)

Other Outcome Measures

Outcome Measure	Measure Description	Time Frame
Support vector machine discrimination of individual joint WTC-HR response profiles in recurrent pregnancy loss with versus without anxiety	This exploratory outcome will assess whether a support vector machine classifier based on individual joint response profiles defined by interhemispheric WTC in the DLPFC and task-evoked HR change will differentiate RPL participants with comorbid anxiety from those without anxiety during multisensory aversive stimulation. Four prespecified linear support vector machine models will be evaluated: a WTC-only model, an HR-only model, a joint unweighted WTC-HR model, and a joint adaptive-weighted WTC-HR model. Model performance will be derived under leave-one-out cross-validation. The prespecified primary performance metric for model comparison will be balanced accuracy. Agreement between predicted and observed group labels will be assessed using Fisher's exact test, with odds ratios and 95% confidence intervals reported.	Single-session fNIRS-ECG protocol (Day 1)
Comparison of adverse events (AEs) and serious adverse events (SAEs) between groups	AEs will include signs of discomfort, distress, or autonomic instability during multisensory aversive stimulation, including acute anxiety, withdrawal requests, chest tightness, dizziness, nausea, cold-related discomfort, auditory discomfort, headache, or skin irritation at fNIRS or ECG sensor sites. SAEs will include syncope, severe anxiety reactions, or clinically concerning physiological abnormalities requiring clinical intervention. Participants will be monitored continuously during the protocol with real-time ECG and direct observation. Group-level differences in AE and SAE incidence will be compared descriptively and with Fisher's exact test.	Single-session fNIRS-ECG protocol (Day 1)
Interhemispheric DLPFC coordination as a mediator of the association between anxiety status and task-evoked heart-rate response in recurrent pregnancy loss	This exploratory endpoint will assess whether reduced interhemispheric DLPFC coordination mediates the association between anxiety status in recurrent pregnancy loss and the mean task-evoked heart-rate response during multisensory aversive stimulation. The independent variable will be anxiety-group status, the mediator will be interhemispheric DLPFC coordination quantified by WTC, and the dependent variable will be the mean baseline-corrected heart-rate response across epochs. Mediation models will be adjusted for age, educational attainment, body mass index, etiology category, and resting heart rate. The primary mediation outputs will include the indirect effect, direct effect, total effect, and path-specific estimates, with uncertainty assessed using 5,000 bootstrap resamples. The analysis will be prespecified for the DLPFC as the primary mechanistic region of interest, with additional prefrontal subregions evaluated as exploratory regional-specificity analyses.	Single-session fNIRS-ECG protocol (Day 1)

Collaborators and Investigators

• Sponsor: Shenyang Medical College
• Collaborators: Central Hospital Affiliated to Shenyang Medical Collage; The Second Hospital of Shenyang Medical College
• Investigators: Study Chair: Yun-En Liu, MD, Shenyang Medical College; Principal Investigator: Lin Tao, MM, Shenyang Medical College; Study Director: Fei Meng, MD, Central Hospital Affiliated to Shenyang Medical Collage; Study Director: Xiu-Ling Zhang, MD, The Second Hospital of Shenyang Medical College

Study record dates

Study Major Dates

• Study Start (Actual): June 30, 2025
• Primary Completion (Actual): April 25, 2026
• Study Completion (Actual): April 25, 2026

Study Registration Dates

• First Submitted: June 22, 2025
• First Submitted That Met QC Criteria: June 22, 2025
• First Posted (Actual): July 1, 2025

Study Record Updates

• Last Update Posted (Actual): May 28, 2026
• Last Update Submitted That Met QC Criteria: May 27, 2026
• Last Verified: July 1, 2025

More Information

Terms related to this study

• Keywords: anxiety; functional near-infrared spectroscopy; recurrent pregnancy loss; dorsolateral prefrontal cortex; emotion-autonomic dysregulation
• Additional Relevant MeSH Terms: Mental Disorders; Anxiety Disorders
• Other Study ID Numbers: NEURO-CARD-fNIRS

Plan for Individual participant data (IPD)

• Plan to Share Individual Participant Data (IPD)?: YES
• IPD Plan Description: De-dentified individual participant data, together with the full study protocol, MATLAB analysis scripts, and all supporting materials, will be released in full at the time of publication of the final manuscript. All materials will be deposited on one internationally recognized open science repository selected by the research team, such as Open Science Framework (https://osf.io), Zenodo (https://zenodo.org), or GitHub (https://github.com). Access will be granted for non-commercial research purposes. Requests for additional information may be directed to the corresponding author.
• IPD Sharing Time Frame: Available at the time of publication of the final article
• IPD Sharing Access Criteria: Data will be deposited on the Open Science Framework (https://osf.io) and will be accessible for non-commercial research purposes.
• IPD Sharing Supporting Information Type: STUDY_PROTOCOL; ANALYTIC_CODE

Drug and device information, study documents

• Studies a U.S. FDA-regulated drug product: No
• Studies a U.S. FDA-regulated device product: No