Infertility is a disorder that impacts an individual’s psychological and social aspects (1). The treatment of infertility is mainly focused on its cause. With the advancement in medical sciences, assisted reproductive technique (ART) can now overcome major issues causing infertility (2). But the successful outcome of all these treatment modalities is completely based on one’s ovarian reserve. Ovarian reserve depicts the quality and quantity of oocytes remaining in the ovaries (3). It thus gives an idea about ovarian function or reproductive age at a given time. Because of aging, ovarian reserve diminish physiologically. But many women face a non-physiologic reduction of ovarian reserve, irrespective of age. The non-physiological reduction may result from multiple factors like autoimmune diseases, environmental pollution, psychological factors, genetic factors, endometriosis, pelvic inflammatory disease and chemotherapy (3). It has been reported that DOR is one of the leading reasons for seeking ART, with approximately 26% of patients undergoing ART due to this condition (4). DOR may be associated with irregular menstruation, hypomenorrhea, amenorrhea, short follicular phase, and poor ovarian response (5). Clinical assessments of DOR are done through ultrasonography by counting the number of ovarian antral follicles (AFC), and serum anti-mullerian hormone (AMH) test (6). DOR patients are being managed through conventional medicine by different stimulation protocols, Hormone replacement therapy (HRT), and Dehydroepiandrosterone (DHEA) as pre-treatment to IVF. It was observed that even after ovarian hyperstimulation with high-dose gonadotrophin, DOR patients have a smaller number of oocytes, a low fertilization rate, and a higher cycle cancellation rate (7). Most patients are ultimately left with the only option of IVF with a donor egg (8). But proper evidence is lacking to support their role in DOR (9). Also, the mode of action of these medicines is still unknown (10). Hence, identifying effective treatment modalities for DOR becomes an area of great clinical relevance.

Although there is no direct explanation of DOR in Ayurvedic texts, the clinical manifestations of certain gynaecological conditions show remarkable similarity. One such disorder is ārtavakṣaya, a menstrual abnormality arising from the depletion (kṣaya) of ārtava upadhātu (the supportive component of reproductive system), which closely parallels DOR in both its etiology and presentation. The clinical presentations of ārtavakṣaya is also evident in several endocrine disorders such as polycystic ovarian syndrome (PCOS), hyperprolactinemia, and thyroid dysfunction, as well as in physiological states like the premenopausal period, DOR, and premature ovarian failure (POF). Regardless of the underlying cause, these conditions share a common pathophysiological mechanism which involves dysregulation of the hypothalamo- pituitary -ovarian axis resulting in oligo or anovulation and manifesting clinically as oligomenorrhea or hypomenorrhea.

Ayurveda similarly attributes ārtavakṣaya to either qualitative or quantitative reduction of ārtava which possess agneya guna (fire element) characterized by yadocitakāla adarśana (irregular menstruation/oligomenorrhea), alpata (scanty menstruation/hypomenorrhea), and yonivedana (pain in vagina). The therapeutic approach in Ayurveda primarily aims to restore the function of ārtava. Management is two-fold, śodhana karma (cleansing therapy) and śamana karma (medicines for restoring normalcy or pacifying action). Śodhana may be achieved through vamana karma (therapeutic emesis) or virecana karma (therapeutic purgation), depending on the patient’s condition, followed by śamana therapy using medicines possessing agneya guṇa (to enhance fire element) (11). Ayurveda also emphasizes comprehensive preconception care which also includes basti karma (therapeutic enema) including uttarbasti (administration of therapeutic oil or ghee via vagina) after śodhana karma to optimize the quality of strībījam (ovum) and prepare the woman for healthy conception (12). In our clinical experience, several women diagnosed with DOR who underwent Ayurvedic interventions based on ārtavakṣaya cikitsā and preconception care protocols at our centre demonstrated notable improvement in serum AMH levels, subsequently becoming eligible for ART using their own oocytes (13). A few even achieved spontaneous conception during treatment. The group of formulations selected in this protocol was chosen for their established indications in restoring ārtava and facilitating preconception care. The specific medicines were selected based on each patient’s clinical condition and presentation. These encouraging outcomes underscore the potential role of Ayurveda in the management of DOR and highlight the need for generating the preliminary evidence for a safe and feasible Ayurveda treatment protocol. In the future, it will help to plan further clinical studies which may benefit DOR patients to have ART procedures with their eggs, thus opening a wide scope for research in integrated medicine.

In addition, the possibility of identifying detailed pharmacological mechanisms that might improve the therapeutic outcomes in DOR patients can be explored by incorporating multi-omics analysis. The role of proteomics, metabolomics, and transcriptomics in the analysis of human follicular fluid has been reported previously by several groups (14–16). Reports suggest that the factors influencing oocyte maturation can be determined by analysing the follicular fluid proteome (14, 17). Proteomics analysis of follicular fluid indicated the pharmacological mechanisms of Chinese patented drugs for improving DOR (18). Similarly, various groups have also identified serum metabolite markers for predicting poor ovarian reserve (19, 20) Also, transcriptomics analysis indicated that several genes are modulated in DOR patients (21). All these indicated that the pharmacological mechanisms induced by Ayurvedic treatment could be studied using multi-omics analysis to identify therapeutic targets. All these give an overall idea of how genes and proteins influence the quality of embryo and oocyte maturation, thereby improving therapeutic outcomes. However, the impact of Ayurvedic interventions in improving therapeutic outcomes has yet to be explored in detail. The pharmacological mechanisms induced by Ayurvedic treatment could be studied using multi-omics analysis to identify therapeutic targets, such as genes/proteins involved in oocyte development including steroidogenesis, oxidative stress, cumulus-oocyte signalling. Thus, the aim of this study is to explore the feasibility and within-subject changes in AMH/AFC and generate mechanistic hypotheses. The information gained through the study might be useful in planning future research which may help to incorporate a feasible Ayurvedic pre-conceptional care for DOR patients undergoing ART procedures. Similarly, the possibility of identifying therapeutic targets through multi-omics analysis can be studied.

This is a prospective, exploratory, single-arm, open-label, single-centred study, coordinated by Amrita Fertility Centre, Amrita School of Ayurveda, and Amrita School of Biotechnology, India with two assessment points (baseline and post-intervention). Participants are women diagnosed with DOR undergoing fertility treatments. Women under the age of 40 satisfying all the eligibility criteria and willing to provide informed consent will be recruited from Amrita Fertility Centre, Kochi. Recruited participants will be admitted at Amrita School of Ayurveda, Kollam to undergo a standardized Ayurvedic treatment protocol for a maximum period of 21 days. Following the inpatient treatment, participants will be advised to continue prescribed internal medications for an additional 1 month. After undergoing Ayurveda treatment, participants will be sent back to Amrita Fertility Centre for post-assessment and further fertility treatments, during which follicular fluid will be collected for multi-omics analysis. Figure 1 depicts the trial’s participant flow. The Standard Protocol Items: Recommendations for Intervention Trials (SPIRIT) declaration is followed while reporting the protocol (22). The institutional ethical committee approved the study and is prospectively registered under ctri.nic.in with the number CTRI/2023/11/059872 on November 15, 2023.

Hypothesis: an Ayurvedic treatment protocol might be able to change the ovarian reserve in women diagnosed with DOR, and the pharmacological mechanisms could be investigated through multi-omics approaches.

Exploratory primary objectives:

Exploratory secondary objectives:

Exploratory primary outcome

1 (a). Change in serum AMH.

Time frame:

1 (b). Improvement in ultrasonography count of ovarian antral follicles, AFC.

Time frame:

Exploratory secondary outcomes.

Multi omics analysis of follicular fluid—(Treatment group—10; after 2 months of Ayurveda treatment and Control group—10).

Time frame: day of oocyte retrieval of the IVF cycle.

On recruitment, participant consent will be obtained and documented in an Informed consent form (ICF) prepared in both Malayalam and English language. The basic demographic data, detailed medical history, baseline assessment details and all other relevant data will be collected and documented in an analog case report form (CRF). These will be done by the project research scientist at Amrita School of Ayurveda.

Clinical data collection: The baseline and post intervention clinical data for analysis will be collected through a blood test and ultrasound scan by experts at Amrita Fertility Centre and laboratory associated with the centre. To ensure consistency, reduces inter observer variability and improves reliability the same radiologist will perform the sonography and laboratory samples will be analysed at the same lab. To ensure quality assurance and reproducibility, standard operating procedures (SOPs) will be followed at every level of data collection and handling. Laboratory technician evaluating the biological samples and radiologist, will continue to be blind to the treatment schedules and clinical results to further enhance scientific rigour.

Follicular fluid collection: The follicular fluid will be collected from 10 participants who visit Amrita Fertility Centre after Ayurvedic treatment. Follicular fluid from 10 participants who did not avail of Ayurvedic treatment protocols undergoing IVF treatment will also be taken as control. During oocyte retrieval procedure, the follicular fluid will be aspirated from multiple follicles by the principal investigator, a Professor of Reproductive Medicine at Amrita Fertility Centre. The follicular fluid collected, after removing the cell debris through centrifugation (700 g for 10 min), will be immediately transferred to liquid nitrogen contained in the cryocan and transported to Amrita School of Biotechnology, by the project research scientist who is trained on handling and pre-processing the follicular fluid samples for performing multi-omics analysis. These samples will be transported to Amrita School of Biotechnology using a cryocan (with liquid nitrogen). Further, the follicular fluid samples will be divided into multiple aliquots in 5 mL Eppendorf tubes to avoid repeated freeze–thaw cycles and these aliquots will be stored in −80 °C till further analysis.

To enable simple and accurate data entry and processing, hard copy of the CRFs will feature numbered and coded items. The principal investigators will review versions of the forms before they are finalized. To guarantee thorough data collection, standard operating procedures along with the templates will be created, and the research scientists working as field personnel will receive the necessary training. This will involve assessing their own performance through quality control, which involves looking for any missing information or improbable answers. Additionally, after data collection, principal investigators will do additional quality control checks to ensure that the collected data are complete, consistent and comprehensive. Before the CRFs are used for data documentation necessary corrections/modifications will be made.

All participant data will be internally documented using unique identifier codes to maintain confidentiality. Access to these identifiers will be restricted to the core research team, including the Principal Investigator, Co-Investigators, and Project Scientists directly involved in the study. Other than the hardcopy the data will be encrypted on a hard drive with a password. None of the details that reveal the identity of the participant will be stored in the database. All data will be accessed using the anonymous codes that are assigned to each participants who are involved in the clinical trial. Additionally, the follicular fluid samples collected from these individuals will be assigned with the same coding and these will be recorded in a paper and stored in the server with passwords. The data collected after the follicular fluid data analysis will be given utmost security, that none of these would be made available in public domain. If the data generated is used for publication process, the identity of the participant will be kept anonymous and none of the personnel data will be shared. However, few data including the participant’s previous IVF history and demographic information will be used in the publication on a case basis. Nevertheless, all the sensitive information regarding the participant and the samples collected will be secured.

After completing the study, all the files related to the study will be stored in a server and in an encrypted hard drive. The analog CRFs will be kept locked in cupboards for storing it for long term. Both the electronic and hard copy of all the data will be stored for a minimum of 10 years after the study completion and frequent checks will be done to ensure that the data is accessible and available. The multiomics data of the collected samples will be stored in a encrypted storage device. The raw proteomics mass spectrometry data will be submitted to ProteomeXchange consortium (32) via the PRIDE (33) partner repository. After submitting the raw data, a dataset identifier number will be provided, and it will be available with a accession number in the PRIDE repository.

The AMH and AFC changes within the 30 participants who underwent Ayurveda treatment will be assessed using paired t-tests (if the data is normally distributed) or Wilcoxon signed-rank test (if the data is non-normal), as determined by Shapiro–Wilk test. Statistical significance is considered as p < 0.05 for all two-sided tests. Results will be reported as mean ± SD or median (IQR), with effect sizes (Cohen’s d) for pre-post changes. As a sensitivity analysis, ANCOVA will be utilized for adjusting post-treatment data for baseline AMH/AFC and variables such as age, BMI, and ovarian response.

Follicular fluid from 10 control participants and 10 Ayurvedic-treated participants chosen from the 30-participants treatment arm and matched according to age and baseline AMH/AFC will be obtained for the secondary, exploratory multi-omics analysis. All the laboratory experiments will be performed in triplicates (of each biological samples) and statistical analysis will be done using GraphPad Prism software. The statistical analysis of collected proteome data will be performed using Proteome Scaffold software (version 4). Peptide identifications will be accepted if they can be established at greater than 95.0% probability. The Scaffold Local FDR algorithm and peptide prophet algorithm with scaffold delta-mass correction will assign peptide probabilities. Protein identifications will be accepted if they can be established at greater than 99.0% probability and contain at least two peptides representing the protein family. The protein prophet algorithm will assign protein probabilities (34, 35). For metabolomics analysis, after data generation, the heatmaps, principal component analysis, and multivariate statistical analysis will be performed using R (Version 4.5.1) and MetaboAnalyst 6.0. To evaluate the differences between the treatment and control groups in transcriptomics, proteomics, and metabolomics, t-tests (for normally distributed data) or Mann–Whitney U tests (for non-normal data) will be used for each gene, protein, or metabolite. To account for multiple comparisons, the Benjamini–Hochberg FDR correction will be used; an FDR of less than 0.05 will be considered significant. As described under each section, specific assays will be used to further validate the most important genes and proteins. No formal sample size calculation was performed for the exploratory multi-omics analysis, as it is hypothesis-generating, and the 10 treatment samples were selected based on matching and feasibility.

Diminished ovarian reserve characterized by reduction in oocyte quality and number often results in poor reproductive outcomes, posing a great challenge in successful assisted reproductive techniques. It was observed that DOR is the most common reason for selecting ART in fertility treatments (4). Women with DOR tend to exhibit poor ovarian response to gonadotropins, requiring higher doses of medication and prolonged stimulation protocols, yet still yielding suboptimal results. Even though various stimulation protocols (36) and other options are being tried, patients become poor responders due to low quality or quantity of oocytes. Currently, there is no established standard of care that can effectively restore the ovarian reserve, hence underscoring the need for novel and integrative treatment approaches. In recent years, traditional medicine systems like Ayurveda, Traditional Chinese medicine have attracted scientific interest for their potential in enhancing and improving various aspects thereby alleviating the disease conditions including PCOS and DOR. The importance of investigating the role and impact of traditional medicines has been highlighted through a study that used an Ayurveda treatment protocol in improving AMH values in DOR conditions (13). Another study has highlighted the importance of traditional Chinese medicine that includes herbal treatment along with acupuncture in improving the AFC and FSH levels in women with DOR (37). Similarly, the cell signalling pathways modulated in granulosa cells using Er Zhi Tian Gui Formula suggested its potential (38). The impact of few formulations including Gengnianchun and Bushen Jianpi Tiaoxue Decoction on mTOR and estrogen signaling pathways in reducing apoptosis and oxidative stress in granulosa cells has been demonstrated (39, 40). A recent meta-analysis has also shown the impact of certain East Asian interventions including herbal and acupuncture therapies in improving the live birth and clinical pregnancy rates with minimal adverse events (41). Following these approaches, this study protocol aims to investigate the feasibility and potential of an Ayurvedic treatment in improving the ovarian reserve markers in DOR patients. Through integrative approaches this study might be able to predict the advantages of traditional Ayurvedic treatment approaches in improving the ovarian reserve markers and the overall ovarian microenvironment.