Efficacy of dehydroepiandrosterone toovercome the effect of ovarian ageing(DITTO): a proof of principlerandomised controlled trial protocol

Kannamannadiar Jayaprakasan,1,2,3 Amarin Narkwichean,1 Walid E Maalouf,1

Bruce K Campbell1

To cite: Jayaprakasan K,Narkwichean A, Maalouf WE,et al. Efficacy ofdehydroepiandrosterone toovercome the effect ofovarian ageing (DITTO): aproof of principle randomisedcontrolled trial protocol. BMJOpen 2014;4:e005767.doi:10.1136/bmjopen-2014-005767

▸ Prepublication history forthis paper is available online.To view these files pleasevisit the journal online(http://dx.doi.org/10.1136/bmjopen-2014-005767).

Received 23 May 2014Revised 4 September 2014Accepted 5 September 2014

1Division of Child Health,Obstetrics and Gynaecology,School of Medicine, Facultyof Medicine and HealthSciences, University ofNottingham, Nottingham, UK2Nottingham UniversityResearch and Treatment Unitin Reproduction (NURTURE),Queen’s Medical Centre,University of Nottingham,Nottingham, UK3Derby Fertility Unit, RoyalDerby Hospital, Derby, UK

Correspondence toDr KannamannadiarJayaprakasan;k.jayaprakasan@nottingham.ac.uk

ABSTRACTIntroduction: Dehydroepiandrosterone (DHEA) hasbeen proposed to improve pregnancy rates in womenwith diminished ovarian reserve undergoing in vitrofertilisation (IVF) treatment. However, evidenceregarding its efficacy is supported by a limited numberof randomised controlled trials (RCTs). This double-blinded RCT aims to measure the effect of DHEAsupplementation prior to and during controlled ovarianhyperstimulation on ovarian response prior to IVFtreatment in women predicted to have poor ovarianreserve.Methods and analysis: Sixty women with ovarianantral follicle count ≤10 and serum anti-Mullerianhormone ≤5 pmol/L undergoing IVF/intracytoplasmicsperm injection (ICSI) treatment at the Nurture fertilityclinic, Nottingham will be recruited. They will berandomised to either receive DHEA capsule 75 mg/dayor placebo for at least 12 weeks before egg collection.All participants will undergo standard long downregulation protocol using human menopausalgonadotropin 300 IU/day. Serum samples and follicularfluids at the time of egg collection will be collected forhormonal immunoassays. For ICSI participants,cumulus cells stripped from oocyte will be collected forcumulus gene expression analyses regarding oocytecompetence. Microdrops of oocyte culture mediabefore the time of ICSI will be assessed for glucose,pyruvate and lactate utilisation. Embryo transfer will beperformed on day 2, 3 or 5 based on the number andquality of the embryos available. Pregnancy will bedefined as urine pregnancy test positive (biochemicalpregnancy) and 6–8 weeks ultrasound scan with fetalheart beat (clinical pregnancy) and live birth. It isplanned to perform the molecular and nutritionalfingerprint analyses in batches after finishing theclinical phase of the study.Ethics and dissemination: The approval of the studywas granted by the NHS Research Ethics Committee(Ref number NRES 12/EM/0002), the Medicines andHealthcare products Regulatory Agency (MHRA), andthe Nottingham University Hospitals Trust Research andDevelopment department. All participants shall providewritten informed consent before being randomised intoallocated treatment groups.

Trial registration number: Protocol V.2.0; EudraCTnumber: 2011-002425-21; http://www.clinicaltrials.gov;NCT01572025; CTA reference: 03057/0053/001-0002

INTRODUCTIONOne of the major changes in the societiesnoted worldwide over the past few decades ispostponement of childbearing due to womenpursuing higher education and a successfulcareer. Currently, large numbers of womendefer attempting to conceive until theirmid-30s or 40s.1 For instance, in the UK, therehas been a rise in childlessness at the age of35 from 12% for those born in 1941 to 25%for those born in 1971.2 An important issuethat has arisen consequently from this trend isthe marked increase in the incidence ofwomen with infertility who seek medical inter-ventions to overcome the involuntary childless-ness incurred as a result of ovarian ageing.Ovarian ageing, dictated by a decline in

the quantity and quality of oocytes within theovaries,3 4 is responsible for the well-established observation of age-related decline

Strengths and limitations of this study

▪ The study is a double-blinded placebo-controlledrandomised controlled trial. Randomisationnumbers will be generated by the computersystem, and therefore the patient and the health-care team will not know which treatment (dehy-droepiandrosterone (DHEA) or placebo) thepatient receives.

▪ The authors also plan to use molecular studiesof the cumulus gene expression and media nutri-tional fingerprint to elucidate the mechanism ofDHEA on the oocyte development.

▪ As this is a pilot study, the intended sample sizeis small and plans to recruit only 60 participants.

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in fertility5–8 and of age-related increase in adversereproductive events such as miscarriages9 10 and aneu-ploid pregnancies.11–13 While the age-related decline infertility cannot be overcome with conventional in vitrofertilisation (IVF) treatment, reduced ovarian reservesecondary to ovarian ageing is one of the major factorsdetermining the success of IVF outcome. Further, loss ofovarian function can have additional health conse-quences through effects on metabolism, cardiovascularfunction, cognition, response to stress, bone strength14–

16 and risk of malignancies.17

While chronological age is an important determinantof ovarian ageing, there is a considerable interindividualvariation in the rate of the ageing process, possibly influ-enced by genetic and environmental factors. The paceat which ovarian ageing occurs is determined by the rateof primordial follicle initiation, the rate of follicle turn-over or loss and the rate of follicle and oocyte matur-ation.18 19 These processes are regulated byintrafollicular and interfollicular interactions mediatedby pituitary derived gonadotropins and oocytes andsomatic cell-derived local growth regulators, which maybe liable to therapeutic manipulation with drugs such asdehydroepiandrosterone (DHEA).DHEA is a weak androgenic steroid secreted mainly

from the adrenal glands but also from ovaries and byperipheral conversion. The serum levels of DHEAdecline markedly with advancing age. It has been pro-posed that the oral administration of DHEA in womenmay have antiageing effects and improve well-being andsexual function. A few observational studies reportedthat DHEA improves ovarian response and pregnancyrates in women with reduced ovarian reserve followingassisted reproduction treatment (ART).20–26 In one ofthe only three randomised controlled trials (RCTs) pub-lished so far, a group of 33 women with reduced ovarianreserve were randomised to receive either 75 mg DHEAper day orally for at least 6 weeks before starting the firstcycle of ovulation induction or no pretreatment.Participants who did not conceive and continued to thesecond cycle took DHEA for at least 16 weeks, while theaverage duration of DHEA intake by the study group was13.5 weeks. While there was no significant difference interms of mean number of retrieved oocytes and fertilisa-tion rates, the cumulative live birth rates over two IVFcycles (23.1% vs 4%; p=0.05) were significantly higher inthe DHEA group compared with the control group.26 Itwas suggested that the benefit of DHEA treatmentwould be most effective if it was supplemented for atleast 3 months, which is equal to the time needed toaffect the gonadotropin-responsive follicle pool.Therefore, they believed that DHEA acts in ovarianrecruitment and early folliculogenesis.27 However, therewere few methodological issues with that trial. It was notblinded. It was registered at the ClinicalTrials.gov as con-trolled clinical trial (CCT) on 3 May 2010 after the trialwas completed in July 2009, in fact after the trial reporthad been submitted for publication in February 2012.

The authors freely admitted that they had no prespeci-fied sample size or primary end point, and were inspect-ing the results as data accumulated and stopped whenthey achieved a nominally significant difference in preg-nancy rate.While subsequent RCTs failed to demonstrate signifi-

cant improvement after DHEA supplementation,28 29

the other controlled studies reported in the literature sofar suggest that DHEA may improve quantitative andqualitative ovarian response. It is important to note theweak methods as they were not blinded and employedonly small sample sizes.30 Further investigation into therole of DHEA on ovarian ageing is required and womenundergoing ART provide an ideal model with whom wecan evaluate the developmental competence of oocytesboth in vivo and in vitro. Hence, this warrants a well-designed and large RCT to examine the effect of DHEAin women with diminished ovarian reserve undergoingART treatments. While there are many ovarian reservetests with varying predictive abilities reported, antral fol-licle count (AFC) and anti-Mullerian hormone (AMH)have consistently been found to have the best diagnosticaccuracy to predict poor ovarian reserve.31

We are proposing a pilot randomised, double-blinded,placebo controlled trial to examine the feasibility of con-ducting a large trial, in addition to exploring the effectof DHEA on oocyte quantity and quality. The principalobjective of this study is to evaluate the effect of DHEAon the ovarian response to gonadotropins (oocyte quan-tity) during controlled ovarian stimulation and thedevelopmental competence of oocytes (oocyte quality)by using molecular and clinical markers in women withaged ovaries, identified by using reported AFC andAMH thresholds.32–34

MATERIALS AND METHODSDesign, setting and participantsThis study is planned as a double-blind, placebo-controlled, randomised pilot trial, being conducted at auniversity-based tertiary fertility centre (NottinghamUniversity Research and Treatment Unit inReproduction (NURTURE), University of Nottingham,UK). An expected number of 60 patients undergoingeither IVF or intracytoplasmic sperm injection (ICSI)and predicted to have poor ovarian response will berecruited to this RCT. The study participants will receiveoral capsules of either 75 mg of DHEA or placebo for atleast 12 weeks before starting ovarian stimulation withgonadotropin during IVF/ICSI treatment. Participantswill be provided with a maximum 20-week supply ofmedication. A DHEA capsule is made from active 75 mgDHEA powder encapsulated in the white gelatinecapsule by the licensed pharmaceutical companyauthorised by the Medicines and Healthcare productsRegulatory Agency (MHRA). Placebo is also producedfrom the capsule with similar size, colour and appear-ance but without the DHEA powder.

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Inclusion criteria1. Women aged 23–43 years with diminished ovarian

reserve (predicted to be poor responders), definedas AFC scan ≤10 and/or serum AMH ≤5 pmol/Lundergoing IVF or ICSI treatment.

2. Women must have a regular spontaneous menstrualcycle of 21–35 days.

3. Women must be willing and comply with scheduledvisits, the treatment plan and laboratory tests andgive written informed consent.

Exclusion criteria1. Women with body mass index >35 kg/m2;2. Women with a single ovary;3. Women with untreated hydrosalpinx/submucous

fibroid/endometrial polyp at the start of treatment;4. Women with any history of seizure disorders;5. Women with previous participation in this trial in an

earlier treatment cycle;6. Women with any known endocrine disorders such as

congenital adrenal hyperplasia, thyroid diseases,hyperprolactinaemia;

7. Known allergy to DHEA;8. Diabetic women on insulin, as insulin lessens the

DHEA levels and might reduce the effectiveness ofDHEA supplements.

Recruitment and randomisationAt the initial visit, women seeking IVF/ICSI treatmentwill undergo an ultrasound scan for AFC as well as anAMH blood test. If a woman fulfils the eligibility criteria,she will be invited to participate in this study. After

written signed consent is obtained, participants will berandomised to receive oral capsules of either 75 mgDHEA or placebo (once daily) for at least 12 weeks (12–20 weeks) before starting ovarian stimulation and contin-ued through stimulation until the day before egg collec-tion (figure 1). The randomisation will be based on acomputer-generated pseudo-random code using randompermuted blocks of randomly varying size, created by theUniversity of Nottingham Clinical Trials Unit (CTU). Therandomisation will be stratified by age. At randomisation,the computer will then issue a trial number, which will bethe unique identifier for the trial participant. The corre-sponding treatment allocation will be transmitted to theunblinded pharmacy staff, who will dispense the allocatedtreatment (trial medication or placebo) to theparticipant.The randomisation code will be maintained by the

CTU. The pharmacy will have the list of participantswith their allocated treatment. The code will be brokenonly if any participants develop a serious adverse event,in which case the chief investigator will be notified.

Primary objectivesThis study aims to evaluate the hypothesis that DHEAsupplementation for at least 12 weeks prior to andduring controlled ovarian hyperstimulation in patientspredicted to be poor responders increases the oocytequantity (number of oocytes retrieved). The feasibility ofconducting a large trial is also to be assessed by evaluat-ing the recruitment rates and compliance of therecruited participants with DHEA/placebo intake andfollow-up rates.

Figure 1 Participant flow diagram (IVF, in vitro fertilization; ICSI, intracytoplasmic sperm injection; DHEA,

dehydroepiandrosterone).

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Secondary objectivesOocyte quality will be determined by expressions of estab-lished molecular markers. The expression levels ofvarious cumulus markers including gonadotropin recep-tors, pentraxin 3, cyclooxygenase 2, hyaluronan synthase2, bone morphogenetic protein (BMP) antagonist—gremlin and epidermal growth factor-like signalling mole-cules will be measured to define oocyte developmentalcompetence using real-time PCR. Nutritional fingerprint-ing by measuring glucose, pyruvate and lactate utilisationas the energy consumption from the oocyte culturemedia will be evaluated to assess the developmental com-petence of the fertilised oocytes. Finally, we will also beassessing aneuploidy rates in immature oocytes to see anybeneficial effect of DHEA on oocyte quality. Biochemical(positive urine human chorionic gonadotropin (hCG) at2 weeks following embryo transfer) and clinical (viablepregnancy on ultrasound at 5 weeks following embryotransfer) pregnancy rates will also be reported as second-ary outcomes. However, the present study is underpow-ered to measure plausible effect sizes.

ProcedureAll participants will undergo a standard long downregu-lation protocol (figure 2) starting with the GnRHagonist, either nafarelin (Synarel; Pharmacia, UK) orbuserelin (Aventis Pharma, Kent, UK), 7 days earlierthan the expected first date of the next menstrual cycle.Pituitary downregulation will be confirmed by both atransvaginal ultrasound scan (quiescent ovaries with fol-licles <10 mm diameter, endometrium ≤5 mm in thick-ness) and serum oestradiol (<200 pmol/L) on day 2after the first day of the menstruation period. A sampleof serum from the downregulation blood sample will bestored for serum DHEA and AMH estimation. On con-firmation of downregulation, ovarian stimulation will bestarted with Human Menopausal Gonadotropin (HMG,Menopur, Ferring, UK) 300 IU daily by subcutaneous

injection. From day 8 of stimulation, participants will bemonitored for their ovarian response using transvaginalultrasound and serum oestradiol levels. Once they meetthe criteria for oocyte retrieval (≥3 leading follicles are≥17 mm), 10 000 IU of hCG, Pregnyl (OrganonLaboratories Ltd, Cambs, UK) will be administered sub-cutaneously to trigger oocyte maturation. The trial inter-vention, gonadotropin injections and GnRH agonist willbe continued until hCG administration. Thirty-six hoursfollowing HCG administration, oocyte retrieval fromovarian follicles will be performed (figure 2).Extra blood samples of serum DHEA and AMH con-

centrations will be collected on the day of egg collec-tion. Conventional insemination (IVF) or ICSI treatmentwill be appropriately selected for individual patients byembryologists depending on the partner’s semen ana-lysis and previous treatment history. Prior to ICSI, theoocytes will be denuded prior to sperm injection accord-ing to standard clinical practices. Expression levels of apanel of nine cumulus markers of oocyte competence asaforementioned will be quantified by real-time PCR incumulus cells removed from one cumulus oocytecomplex (COC)/patient. The metaphase II oocytesfrom these selected COCs will be inseminated accordingto standard clinical practices. We estimate that the ICSItreatment rate will be approximately 50% of totalpatients, and thus will be able to have cumulus markersdata from about 30 patients. Microdrop culture will beused to track individual zygotes throughout embryodevelopment in vitro, until transfer on day 2, 3 to 5 orcryopreservation. Oocyte developmental competencewill then be assessed indirectly by measuring glucose(G), pyruvate (P) and lactate (L) utilisation from theculture media as the energy consumption by the oocytefrom the medium (‘nutritional finger printing’) will varydepending on its ability to develop to the blastocyststage. The fluid within the follicle from which the aboveoocytes are collected will be stored and analysed for

Figure 2 IVF/ICSI treatment protocol used in the study (IVF, in vitro fertilization; ICSI, intracytoplasmic sperm injection; DHEA,

dehydroepiandrosterone; hCG, human chorionic gonadotropin).

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DHEA levels. Chromosomal assessment of the immatureoocytes recovered at denudation while doing ICSI willbe performed by using comparative genomic hybridisa-tion. Participants will be reassured that the oocytes thatare utilised for the research would have no further rolein their treatment; therefore, taking part in the studywould not affect their treatment outcome.Embryo transfer is performed 2, 3 or 5 days following

the day of egg collection, based on the number andquality of embryos available. One or two embryos will betransferred depending on the embryo quality andcouple preference. The maximum number of embryostransferred will be two as per the Human Fertilisationand Embryology Authority (HFEA) regulations. Thecouples will be advised by the embryologists regardingblastocyst culture and transfer if there are ≥4 fertilisedoocytes at fertilisation check. Couples will be advised forsingle embryo transfer if top quality blastocysts are avail-able for transfer. All women undergoing IVF treatmentwill be advised to self-administer progesterone pessaries(Cyclogest; Alpharma, UK) 400 mg twice daily startingon the second day after egg collection until the day ofthe pregnancy test, which will then be continued until8 weeks of pregnancy if the treatment is successful. Aurine pregnancy test will be performed approximately14 days from the day of embryo transfer. All womenhaving IVF/ICSI treatment will have an early pregnancyscan at around 7 weeks of gestation (clinical pregnancy;figure 2). All the stimulation and outcome data will becollected and recorded on to the database. Side effectsreported by women will be recorded.The project will be performed over a period of 2 years

and the last follow-up visit of the participants will be theday of the first early pregnancy scans. While we are plan-ning to do the molecular analysis and chromosomal ana-lysis during the last phase of the trial, we willprospectively collect the baseline and clinical outcomedata as participants go through the trial and undergoIVF treatment.

Statistical analysisThe trial is designed to test the ‘proof of principle’ thatDHEA priming improves the ovarian response to supero-vulation. The primary outcome is the total number ofoocytes retrieved. Only the first cycle of treatment fol-lowing randomisation will be analysed. If treatmentcycles are cancelled during the stimulation phase due topoor ovarian response or if no eggs are retrieved at eggcollection, the number of eggs collected will berecorded as zero. All participants will be advised to use abarrier contraception while taking trial medications.However, if participants spontaneously conceive duringDHEA/placebo treatment, for the primary analysis wewill assign the number of the patient’s oocyte count withthe highest observed number in the rest of the studywhile this patient will be included into the positive preg-nancy outcome group for secondary (pregnancy)outcome analysis. The secondary outcome will include

(1) biochemical/clinical pregnancy rates, (2) live birthrates, (3) relative quotients of nine cumulus geneexpressions using real-time PCR, (4) rates of glucose,pyruvate and lactate consumption, (5) rates of aneu-ploidy in immature oocytes, (6) serum oestradiol onhCG day and (7) serum AMH on the sample collectedat egg collection (after treatment).As this is a pilot study, we estimated the sample size

based on the number of participants that we couldrecruit over a period of 18 months. We anticipateapproximately 600 women undergoing IVF/ICSI treat-ment at NURTURE over an 18-month period.Considering 20% as our predicted poor respondersbased on the inclusion criteria and 50% recruitmentrate, we will be able to recruit 60 participant during thestudy period. We anticipate that the size of the effect onthe primary outcome based on the data generated fromthis trial will enable us to estimate the sample size for alarge multicentre trial.Statistical analysis will be performed by using the

Statistical Package for the Social Sciences (SPSS,Chicago, Illinois, USA). Assuming that the oocytenumbers in a study population are usually not normallydistributed, the difference in the primary outcomebetween groups will be tested using the Mann-WhitneyU test. A two-tailed p value of <0.05 will be taken to indi-cate statistical significance. Analyses will be performedbetween the study and control participants who havecomplied with the trial as per protocol and not just onthe intention to treat basis.

Ethics and disseminationThe side effects associated with the proposed dose ofDHEA are rare. The published cohort studies and theRCT have not reported any significant adverse or andro-genic side effects.20–23 26 However, the potential sideeffects are related to androgenic effects, and theseinclude acne, facial hair growth and rarely a deepeningof the voice.35 If women report any of these side effects,they will be advised to discontinue the trial medication.The notes of all pregnant participants will be retrospect-ively reviewed to ensure the normality of the childrenborn.This study has received appropriate approval from the

NHS Research Ethics Committee (East Midlands-Derby1ethics committee; Ref number NRES 12/EM/0002), theMedicines and Healthcare products Regulatory Agency(MHRA) for use of investigational medicinal products,and the Nottingham University Hospitals Trust Researchand Development department. It is being conducted inaccordance with the good clinical practice (GCP) princi-ples, the ethical principles that are in agreement withthe Declaration of Helsinki 1996, the Medicines forHuman Use Regulations, Statutory Instrument 2004, andall involved UK laws and regulations. All participantsshall provide written informed consent before being ran-domised into allocated treatment groups. The

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participants will be informed which treatment theyreceived after the trial is completed.We are planning to disseminate the study through

peer-reviewed journal publications and scientific confer-ences. The data that will be produced from this researchwill have the potential to influence clinical practice infertility clinics worldwide. If the planned intervention isfound to be effective, it provides a low cost, safe andeasily administrable therapy for ovarian ageing.Furthermore, the overall results will give us more appre-ciation in understanding the underlying mechanisms ofhow DHEA combats reproductive ageing. This providesa mechanistic framework for translational research onmechanisms of ovarian ageing and drug interventions toslow down the ovarian ageing process and subsequentadverse consequences.

Acknowledgements The authors would like to thank Professor Jim Thorntonfor his help with the critical review and editing of the manuscript.

Collaborators Nicholas Raine-Fenning.

Contributors KJ, AN, WEM and BKC were responsible for the conception,design and analytical plan. Critical evaluation was performed by all authors.KJ, AN and BKC were responsible for writing the first draft and the finalmanuscript was read and approved by all authors.

Funding This work has been supported by the University of Nottinghamthrough the Early Career Research and Knowledge Transfer scheme andNottingham University Hospital (NUH) Charity; Project code: A2RHD6.

Competing interests None.

Patient consent Obtained.

Ethics approval NHS Research Ethics Committee (Ref number NRES:12/EM/0002).

Provenance and peer review Not commissioned; externally peer reviewed.

Open Access This is an Open Access article distributed in accordance withthe Creative Commons Attribution Non Commercial (CC BY-NC 4.0) license,which permits others to distribute, remix, adapt, build upon this work non-commercially, and license their derivative works on different terms, providedthe original work is properly cited and the use is non-commercial. See: http://creativecommons.org/licenses/by-nc/4.0/

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