STRENGTHS AND LIMITATIONS OF THIS STUDY
-
This is a large-scale randomised controlled trial (sample size: 400) investigating acupuncture therapy in women with diminished ovarian reserve (DOR), aiming to provide a high level of clinical evidence.
-
Sham acupuncture will be used as the control, and participants, outcome assessors and statisticians will be blinded to group allocations.
-
Women with DOR, who desire for natural conception or in vitro fertilisation embryo transfer, will be included, which may increase the applicability of the results to clinical practice.
-
Multiple outcome data will be collected at multiple time points, which can provide diverse clinical evidence.
-
One limitation of this study will be inclusion of patients from multiple centres, which leads to higher requirements for quality control.
Introduction
As the global population ages at an accelerated pace, women’s reproductive health has emerged as a prominent issue in the field of public health. Diminished ovarian reserve (DOR) is both a cause and a process of infertility in women. Although there is no unified definition for DOR, researchers and clinicians have identified specific characteristics, including a reduction in the number and quality of the remaining oocytes in the ovary, as well as changes in ovarian reserve related indicators such as anti-Mullerian hormone (AMH), follicle-stimulating hormone (FSH), luteinising hormone (LH) and antral follicle count (AFC).1 A retrospective cohort study based on the Society for Assisted Reproductive Technology Clinic Outcomes Reporting System found that DOR prevalence increased from 19% to 26% in the assisted reproductive technology population in USA from 2004 to 2011.2 Additionally, results of a meta-analysis including 3082 women at risk of recurrent pregnancy loss (RPL) showed that DOR was significantly associated with RPL, and low levels of AMH and AFC could predict higher odds of pregnancy loss.3
Currently, the treatment for DOR includes a range of approaches such as promoting general health, providing psychological counselling, offering contraception and sexual health guidance, implementing ovulation promotion and in vitro fertilisation embryo transfer (IVF-ET) treatment, using traditional Chinese medicine (TCM), as well as other therapies such as in vitro activation, bone marrow-derived stem cells, gene therapy, and platelet-rich plasma.4 Among these treatment options, acupuncture, as a form of TCM therapy, has the advantages of minimal side effects and high feasibility.
In previous basic research studies, acupuncture has improved ovarian reserve by regulating several signalling pathways (eg, PI3K/Akt, Nrf2/ARE, Bcl-2/Caspase-3).5 6 Lu et al conducted a microRNA (miRNA) profiling study7 of acupuncture on rats with DOR. Results suggested that the expression of rno-miR-92b-3p, mdo-miR-26b-5p_R+1_1ss10TC and bta-miR-7857–3 p_R-1 in ovarian tissues could provide relevant cues to alleviate DOR-mediated oxidative stress. Fang et al reported increased serum estradiol 2 (E2), superoxide dismutase contents, expressions of Bax and Bcl-2 protein, and messenger RNA (mRNA), as well as decreased serum FSH, LH and malondialdehyde levels in female SD rats receiving acupuncture compared with the blank controls.6
Moreover, a few clinical trials have investigated the effectiveness and safety of acupuncture in improving ovarian function. In a randomised controlled trial (RCT) applying acupuncture to 60 patients with DOR infertility, Wen et al demonstrated a reduction in FSH levels, an increase in AFC levels, AMH levels and the number of available embryos, with no adverse events (AEs) reported.8 These results were consistent with an earlier RCT of acupuncture on 20 patients with DOR, which showed reduced FSH levels and increased AFC.9
However, despite these findings, there is insufficient clinical evidence and theoretical basis to support the validity of applying acupuncture to patients with DOR. Therefore, in this study, a multicentre RCT of acupuncture for DOR will be conducted at seven hospitals, with an expected enrolment of 400 patients, to verify the efficacy and safety of acupuncture for DOR. As per our current knowledge, this study will serve as the inaugural multicentre RCT with a substantial sample size, comparing acupuncture to sham acupuncture. This research expects to add valuable evidence to evidence-based medicine and provide insights into the use of acupuncture in DOR.
Methods and analysis
Study design
A multicentre RCT will be conducted on the efficacy and safety of acupuncture for DOR. 400 women with DOR will be randomised in a 1:1 ratio to the acupuncture group or the sham acupuncture group. This study protocol has received approval from the relevant ethics committees and will adhere rigorously to the principles outlined in the Declaration of Helsinki. A flow diagram of the trial is shown in figure 1. The schedule of enrolment, interventions and assessments of the trial is provided in figure 2.
Flow diagram of the trial. AFC, antral follicle count; AMH, anti-Mullerian hormone; CPR, clinical pregnancy rate; DOR, diminished ovarian reserve; FSH, follicle-stimulating hormone; IVF-ET, in vitro fertilisation-embryo transfer; SAS, Self-Rating Anxiety Scale.
Schedule of enrolment, interventions and assessments of the trial. AEs, adverse events; AFC, antral follicle count; AMH, anti-Mullerian hormone; CPR, clinical pregnancy rate; FSH, follicle-stimulating hormone; IVF-ET, in vitro fertilisation-embryo transfer; SAS, Self-Rating Anxiety Scale. *Patients adopting an IVF-ET plan should take acupuncture/sham acupuncture preintervention for at least 8 weeks.
Recruitment
Women with DOR will be recruited at seven hospitals in China: Acupuncture and Moxibustion Hospital of China Academy of Chinese Medical Sciences (CACMS); Chengdu Xinan Gynaecology Hospital; The Affiliated Hospital of Shandong University of TCM; Jiangsu Province Hospital of Chinese Medicine; Guang’anmen Hospital of CACMS; Wang Jing Hospital of CACMS; Nanjing Jinling Hospital.
Participants
The inclusion criteria are: (1) Female individuals aged between 20 years and 40 years; (2) AMH<1.2 ng/mL; (3) AFC <5 in two separate tests, conducted at least 4 weeks apart (tested on days 2–4 of the menstrual cycle);10 (4) Desire for natural conception or have an IVF-ET plan with an antagonist protocol for ovulation as evaluated by reproductive physicians; (5) Voluntary participation in the trial and provision of signed informed consent.
The exclusion criteria are: (1) Presence of other conditions that may impede conception, such as congenital developmental abnormalities of the reproductive organs, untreated endometrial polyps, untreated uterine infection, etc; (2) Diagnosis of polycystic ovarian syndrome (PCOS); (3) Coexistence of uncorrected hyperprolactinaemia, hyperandrogenaemia, abnormal thyroid function, chronic adrenal hypofunction and other metabolic endocrine diseases; (4) Comorbidity with severe cardiovascular, cerebrovascular, hepatic, renal, malignant tumours, haematopoietic system or mental health conditions; (5) Receipt of acupuncture treatment aimed at enhancing fertility within the previous 12 weeks; (6) Anticipation of an IVF-ET plan within 8 weeks of enrolment.
Sample size
The sample size calculation was performed using Power Analysis & Sample Size (PASS) V.15 software and was based on the primary outcome.11 According to our pilot study,9 we hypothesised that AFC will be increased by 2.5 and 0.4 with a SD of 2.2 in the acupuncture group and sham acupuncture group, respectively. For sample size calculation, the procedure “Superiority by a Margin Tests for the Difference Between Two Means” was run with a superiority margin (SM) of 1.5, a one-sided 5% level of significance and a power of 80%. According to the calculation results, a sample size of 167 for each group will be sufficient. Considering a 20% withdrawal rate, a total of 400 participants (200 in each group) are expected to be enrolled in this study.
Randomisation and allocation concealment
Eligible participants will be randomly allocated into the acupuncture group or the sham acupuncture group in a 1:1 ratio via the web response system of the Central Randomisation System for Clinical Research. Acupuncturists will be responsible for randomisation at each centre. Participants, outcome assessors and statisticians will be blinded to group allocations. Unblinding should not be required during the study as it will not have clinical relevance to treatment decisions.
Interventions
Acupuncture or sham acupuncture will be administered by certified acupuncturists who have at least 5 years of clinical experience and have received prior standardised training in operation. Different specifications (Φ=0.25*25 mm, Φ=0.25*40 mm, Φ=0.30*75 mm) of disposable sterile acupuncture needles (Huatuo brand made in Suzhou Medical Appliance Factory, Jiangsu, China) will be used. Participants will be informed of the details of the study, including but not limited to the study objectives, interventions, potential benefits and risks, and so on. If participants agree to take part in this trial, they will be required to submit a signed informed consent form (online supplemental material). Regular reminders via WeChat or telephone will be used to improve participant adherence. If any of the following events occur in the enrolled patients, their intervention will be terminated forcibly: (1) Serious complications or other serious illnesses requiring emergency measures; (2) Pregnancy; (3) Withdrawal of informed consent for any reason. There is no post-trial intervention or care for patients in this trial.
Supplemental material
Acupuncture group
In TCM acupuncture, two sets of acupoints (group A and group B) comprising 14 selected points will be manually applied to patients, alternately, starting with group A (number of needle insertions per patients per session: 6 or 20). Acupoints of group A include Baihui (GV20), Shenting (GV24), bilateral Benshen (GB13), Zhongwan (CV12), bilateral Tianshu (ST25), Guanyuan (CV4), bilateral Zigong (EX-CA1), bilateral Dahe (KI12), bilateral Zusanli (ST36), bilateral Sanyinjiao (SP6) and bilateral Taichong (LR3). Group B consists of three acupoints: bilateral Shenshu (BL23), bilateral Zhongliao (BL33) and bilateral Taixi (KI3). The locations of each of the acupoints (according to the ‘WHO Standard Acupuncture Point Locations in the Western Pacific Region’12 are listed in table 1.
Locations of acupoints
Acupuncture needles will be inserted perpendicularly into a depth of 10–20 mm for GV20, GV24 and GB13, obliquely towards Yongquan (KI1) into a depth of 10–20 mm for LR3, deeply into a depth of 60–70 mm into the third posterior sacral foramina for BL33, and perpendicularly into a depth of 30–40 mm for the remaining acupoints. Needle manipulation techniques such as lifting, thrusting or twirling will be applied to promote ‘deqi’ (a sensation experienced by patients at the acupoint location, characterised by soreness, numbness, distention, etc). Needle retention time will last for 20 min per session, once every other day, 3 sessions a week for 12 weeks, totalling 36 treatment sessions.
Sham acupuncture group
In this trial, as a kind of commonly used sham acupuncture method in clinical trials, sham acupuncture with superficial needling at a non-acupoint (NA)13 14 will be manually used as the control. Two sets of NAs (group C and group D) will be alternately applied to patients, starting with group C (number of needle insertions per patients per session: 6 or 14). Group C consists of 9 NAs (No. 1–9), group D includes 3 NAs (No. 10–12). The locations of each NA are listed in table 2. Each NA should be shallowly pierced approximately 2–3 mm into the skin without eliciting ‘deqi’. The needle retention time, frequency and duration of treatment sessions will be the same as the acupuncture group.
Locations of non-acupoints (NAs)
Outcomes
The primary outcome will be the change in AFC at week 12 from baseline, measured by transvaginal ultrasonography on days 2–5 of the menstrual cycle.
The secondary outcomes are AFC at week 24, the serum levels of basal FSH, AMH, Scores of the Self-Rating Anxiety Scale (SAS) at weeks 12 and 24, clinical pregnancy rate (CPR), and IVF-ET related indicators (number of follicles over 14 mm on human chorionic gonadotropin (HCG) day, E2 level on HCG day, endometrial thickness on HCG day, number of mature metaphase II-arrested (MII) eggs, the number and rate of two-pronuclei zygotes, the availability and quality of embryo, embryo implantation rate, cycle cancellation rate, biochemical pregnancy rate, early pregnancy loss rate, sustained pregnancy rate and live birth rate). Blood samples of participants in this trial will be sent to the laboratory for testing and destroyed after use.
Safety evaluation
AEs will be monitored throughout the trial. All AEs will be systematically collected via semistructured and open-ended questions by acupuncturists. AEs will be recorded in detail in the electronic case report forms (eCRFs).
Statistical analysis
Statistical analysis will be conducted using SAS V.9.3 software. All data of randomised subjects will be statistically analysed according to the principle of intention-to-treat analysis. The missing data will be imputed using multiple imputation methods, followed by sensitivity analysis of the data sets before and after imputation. All statistical tests will be conducted as two-sided, and a value of p<0.05 will be considered statistically significant. Continuous data will be described with mean and SD, median, maximum and minimum value, or IQR, while categorical data will be presented as count and percentage. For comparison with baselines, a paired t-test or non-parametrical test will be used, respectively, in continuous data of normal or non-normal distributions. For comparisons between groups, the independent t test or non-parametrical test will be used for continuous data, χ2 test or Fisher’s exact test for categorical data, and non-parametrical test for ranked data. No interim analysis is planned. No additional studies using the data generated in this trial are planned.
Data collection and management
Data collection will be conducted through eCRFs in a pre-established network data management system of this trial. With the support of ‘alert’ function of eCRFs, dynamic management will be implemented to ensure that data are collected in a complete, timely and accurate manner. For participants who discontinue or deviate from intervention protocols, researchers responsible for data collection will record relevant outcome data as much as possible via returning visit. Data will be locked at the end of the trial cycle. At that time, researchers will not be able to modify the data. All documents of participants in this trial will be confidential and will be kept and managed by the research centres to ensure the security of the data. Self-inspection will be conducted once a month by each centre. In addition, an independent data and safety monitoring board (DSMB) will be established to annually audit and evaluate cumulative study data, including participant safety, study conduct and progress of each centre, and make recommendations regarding continuation, modification or termination of the trial. There is no conflict of interest between the DSMB and the funding sponsors.
Trial status
This study is currently in progress. The study was launched on 31 March 2022, and participant recruitment began on 15 October 2022. As of 4 July 2024, 200 participants have been recruited, and the anticipated end date is 31 October 2024.
Patient and public involvement
None.
Ethics and dissemination
The study protocol has been approved by the Institute of Acupuncture and Moxibustion, CACMS (Ethics approval number: 2022-03-31-1); Chengdu Xinan Gynaecology Hospital (Ethics approval number: 2021–039); The Affiliated Hospital of Shandong University of Traditional Chinese Medicine (Ethics approval number: 2022–097-KY); Jiangsu Province Hospital of Chinese Medicine (Ethics approval number: 2022 NL-187-02); Guang’anmen Hospital of CACMS (Ethics approval number: 2023–011-KY-01); Wang Jing Hospital of CACMS (Ethics approval number: WJEC-KT-2022–045 P001); and Nanjing Jinling Hospital (Ethics approval number: 2022DZKY-066–01) . Written informed consent will be obtained prior to participant enrolment. The results of this study will be published in peer-reviewed publications.
Discussion
As women age, they experience a decline in fertility, which is a normal physiological phenomenon of DOR associated with advanced age, particularly in those over 35 years old. In addition to age-related DOR, some women may develop pathological DOR due to genetic factors, medical factors (including history of reproductive surgery and radiotherapy), autoimmune factors, infections, and environmental and psychosocial factors. Both types (age-related or pathological) of DOR could lead to reduced fertility, menstrual disorders, and several symptoms related to sex hormones deficiencies or fluctuations, significantly impacting the quality of life. Among these patients with fertility needs, some may be unable to conceive naturally and might have to resort to IVF-ET, which can impose a considerable financial burden and stress on them. In this trial, we will focus on woman seeking fertility through either spontaneous conception or IVF-ET.
Multiple outcomes will be evaluated in this trial. AFC refers to the number of follicles between 2 mm and 10 mm identified via ultrasonography during the early follicular phase of the ovaries. Due to its stability across menstrual cycles and ability to accurately predict IVF stimulation response, AFC will be measured to assess the overall status of ovarian reserve in women15 as the primary outcome in this trial. Serum biochemical assessments of FSH and AMH will also be conducted in accordance with the recommendations of the Practice Committee of the American Society for Reproductive Medicine published in 2020.16 FSH is a glycoprotein hormone secreted by basophilic cells of the anterior pituitary gland, primarily functioning to promote follicle development and maturation in the ovary. In committee opinions, FSH is considered the most commonly used screening test for DOR. The serum level of basal FSH is tested on days 2–5 of the menstrual cycle and is manifested as increased with advancing reproductive age. However, FSH testing has significant variability that limits its reliability, potentially yielding more false-positive results in women at low risk for DOR. In contrast, AMH is produced by granulosa cells from small growing follicles in the ovary, and exhibits less variability as a marker for ovarian reserve.17 Consequently, combined observation of AFC, FSH and AMH could comprehensively evaluate ovarian reserve function. CPR and IVF-ET related indicators will also be recorded in this trial, so as to directly reflect the pregnancy-related status. In addition, most patients with DOR manifest varying degrees of anxiety. Therefore, the SAS as a subjective rating scale consisting of 20 items will be employed in this study to assess nervousness, headaches, stomach-aches and other related symptoms.18
TCM posits that the onset of ‘deqi’ after the needles are inserted into the acupoint at a specific depth is essential for the effectiveness of acupuncture. Sham acupuncture of NA shallow insertion was a commonly used comparison to acupuncture in previous RCTs.14 19 Consequently, this study will select 12 NAs with shallow insertion applied on the regions of head, abdomen, lumbar, sacrum, legs, feet, and ankles in the controls.
In clinical trials, acupuncture has increasingly been employed in treating ovarian conditions, such as PCOS, primary ovarian insufficiency and premature ovarian failure (POF), and has shown a positive effect on promoting ovarian function. For instance, Xu et al discovered that in 30 patients who underwent acupuncture on GV24, CV4, SP6, BL23 and other acupoints, their FSH levels and SAS Scores were lower than those receiving Western medicine.20 Chen et al treated POF with acupuncture at GV20, GV24, GB13, CV4 and other acupoints, and found out that acupuncture reduced serums FSH, LH levels and SAS Scores while increasing E2 levels.21 However, there is limited research on acupuncture for improving DOR, and existing studies suffer from small sample sizes. Nowadays, acupuncture has also been involved in the field of assisted reproduction, mainly as a preintervention approach.22 In the USA, 111 of 456 (24.3%) assisted reproductive clinics recommend and have used acupuncture and moxibustion therapy.23 A scoping review found that 34 studies and 10 studies, respectively, reported positive effects of acupuncture on CPR and live birth rate in infertile women undergoing IVF.24 But no studies have reported the impact of acupuncture for IVF-ET outcomes in women with DOR.
Therefore, this study will explore the efficacy and safety of acupuncture for DOR by conducting a large-scale RCT (400 participants) across seven research centres, using sham acupuncture as a control. As the participants in this study will be from multiple centres, the ‘alert’ function of eCRFs, dynamic management and establishment of DSMB will be implemented, so as to ensure the standardisation of data collection and management and the quality control of the entire trial progress. The results of this study will be expected to provide high-quality evidence.25 Given acupuncture’s affordability compared with other common treatments, if positive, it has the potential to reduce the economic burden on patients and their families.
Ethics statements
Patient consent for publication
This post was originally published on https://bmjopen.bmj.com