A Randomised Trial to Compare Antral Follicle Count and Serum Anti-Mullerian Hormone Level for Determination of Gonadotrophin Dosing in In-vitro Fertilisation
Phase: NA
Conditions: Infertility
Sponsor: The University of Hong Kong

 
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RESEARCH PROPOSAL 
 
1. 
General Information 
 
Title: 
A randomised trial to compare antral follicle count and serum anti-Mullerian hormone level 
for determination of gonadotrophin dosing in in-vitro fertilisation 
 
Protocol number: AMH-IVF-2012 (Version 1) 
Protocol date: 29 February 2016 
 
Principle Investigator: 
Dr. LI Hang Wun Raymond 
Associate Professor, Department of Obstetrics and Gynaecology, The University of Hong 
Kong, Queen Mary Hospital 
 
 
Co-Investigators:  
Dr. LEE Chi Yan Vivian 
Associate Consultant, Department of Obstetrics and Gynaecology, The University of Hong 
Kong, Queen Mary Hospital 
 
 
Dr. LAU Yee Lan Estella 
Scientific Officer, Department of Obstetrics and Gynaecology, The University of Hong 
Kong, Queen Mary Hospital 
 
Professor YEUNG Shu Biu William 
Professor, Department of Obstetrics and Gynaecology, The University of Hong Kong, Queen 
Mary Hospital 
 
Professor HO Pak Chung 
Chair Professor, Department of Obstetrics and Gynaecology, The University of Hong Kong, 
Queen Mary Hospital 
 
Professor NG Hung Yu Ernest 
Clinical Professor, Department of Obstetrics and Gynaecology, The University of Hong 
Kong, Queen Mary Hospital 
 
 
 
Correspondence to: 
Dr. LI Hang Wun Raymond 
Department of Obstetrics and Gynaecology, Queen Mary Hospital, 102 Pokfulam Road, 
Hong Kong. 
Tel:  
(852) 22553111 
Fax: 
(852) 25173278 
Email: raymondli@hku.hk 
 
 

 
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2. 
Background 
 
A number of parameters known as ovarian reserve markers, such as serum anti-Mullerian 
hormone (AMH) concentration and antral follicle count (AFC) have been shown to predict 
ovarian responses to gonadotrophin during in-vitro fertilization (IVF) treatment, although it 
was generally revealed that these markers were not good predictors of pregnancy outcome 
(Broekmans et al, 2006).   
 
AMH, also known as Mullerian-inhibiting substance, is a dimeric glycoprotein that belongs 
to the transforming growth factor-beta family.  It acts on tissue growth and differentiation, 
and is involved in the regression of the Mullerian ducts during male fetal development (Lee 
et al, 1996).  In the female, AMH is exclusively produced by the granulosa cells of preantral 
and small antral follicles, and regulates ovarian activity and follicular steriodogenesis.  There 
have been increasing reports on the use of AMH as an ovarian function marker (Ledger, 
2010, Anderson et al, 2012; Nelson et al, 2012).   
 
In IVF cycles, basal serum AMH level was significantly correlated with AFC, the number of 
follicles obtained after stimulation and the number of retrieved oocytes, and has been found 
useful in prediction of suboptimal and excessive ovarian responses upon ovarian stimulation 
(Ficicioglu et al, 2006; Lekamge et al, 2007; Kwee et al, 2008; Broer et al, 2009; Ledger, 
2010; Broer et al, 2011).  It has been suggested that measurement of serum AMH can be used 
for patient-tailored individualization of the dosage of gonadotrophin used in ovarian 
stimulation during IVF treatment (Olivennes et al, 2009; Nelson et al, 2009; Yates et al, 
2011).  Compared to AFC, AMH has the advantage of having less intra- and inter-cycle 
variation (van Disseldorp et al, 2010) and is not operator-dependent, despite its additional 
cost.  However, there is yet no prospective randomized trial to compare the performance of 
AFC and AMH in this regard. 
 
Therefore, we propose this randomized trial to evaluate the role of baseline serum AMH and 
AFC for determination of gonadotrophin dosing in in-vitro fertilization treatment. 
 
 
 
3. 
Objectives: 
 
This is a prospective randomized trial to compare the use of AFC and serum AMH as the 
basis for gonadotrophin dosing in in-vitro fertilization treatment. 
 
The hypothesis is that the use of serum AMH as the criterion for determination of 
gonadotrophin dosing in IVF treatment results in more optimal ovarian response than AFC. 
 
4. 
Study design: 
 
Patients undergoing the first-time IVF treatment cycle using either conventional insemination 
technique or intracytoplasmic sperm injection at Queen Mary Hospital will be invited to 
participate in this study.  Participating subjects will be randomized into either (i) AFC or (ii) 
AMH group, where the gonadotrophin dosing will be determined based on the baseline AFC 
and serum AMH respectively as assessed one month before the IVF treatment.  

 
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Randomisation will be performed according to a computer-generated list which will be read 
by an independent research nurse.  That research nurse will assign the initial gonadotrophin 
dose according to the study protocol.  The clinician and patient will both be blinded to the 
randomization throughout the course of treatment.  The proportion of subjects having 
appropriate ovarian response, defined as the number of oocytes retrieved being 6 to 14 
inclusive (Broer et al, 2010), will be compared between the two arms. 
 
 
5. 
Subject selection and exclusion 
 
5.1 
Inclusion criteria: 
 Subjects undergoing the first IVF cycle during the study period. 
 
5.2 
Exclusion criteria: 
 Body mass index >=30 kg/m2 
 Subjects in repeated IVF cycles 
 Subjects undergoing IVF treatment using donor oocytes 
 Subjects undergoing pre-implantation genetic diagnosis 
 
 
6. 
Treatment of subjects 
Subjects will attend the Assisted Reproduction Clinic on day 2-5 of the menstrual 
cycle preceding their scheduled IVF treatment.  They will undergo transvaginal 
ultrasound examination to determine the AFC and assess for other pelvic 
abnormalities as what would be routinely done before IVF according to our usual 
protocol.  Blood (10 c.c.) will also be taken for measurement of reproductive 
hormones including FSH, LH, E2 and AMH. The subjects will be asked for separate 
consent to archive the residual serum samples for future studies on reproductive 
endocrinology as appropriate (such future studies will be subject to approval by the 
HKU/HA HKWC IRB).  
 
Subjects will be interviewed by a dedicated research nurse to explain on this study.  
Those who give written informed consent will be randomized into either the AMH or 
AFC groups according to a computer-generated randomization list. 
 
(All participants will be blinded to AFC during ultrasound examination. A research 
nurse will keep the ultrasound sheets of all patients separately, which will be filed 
back to the patients’ medical record upon completion of the ovarian stimulation.) 
 
In their IVF treatment cycle, the ovarian stimulation regimen will be determined 
according to the randomization result as per the following table:  
 
Gonadotrophin 
(FSH 
or 
HMG) regimen 
AMH Group 
AFC Group 
300 IU daily 
AMH <= 1.0 ng/ml 
AFC <=5 
225 IU daily 
AMH >1.0 and <=3.3 ng/ml 
AFC >5 and <=15 
150 IU daily 
AMH >3.3 ng/ml 
AFC >15 
 
The AMH cut-off values are based on review of 214 patients previously treated in our 
centre who had AMH and AFC determined before ovarian stimulation.  By regression 

 
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analysis, an AMH value of 1.0 ng/ml and 3.3 ng/ml corresponded to AFC of 5 and 15 
respectively. Both linear and quadratic regression models gave roughly the same 
results. 
 
The dose will be charted by the research nurse, and both the clinician and the patient 
will be blinded to the assigned group. 
 
All subjects will be assessed by transvaginal ultrasound scan on the 7th day of ovarian 
stimulation, with the dose of gonadotrophin adjusted according to the ovarian 
response as detailed in the following table: 
 
Response category 
Criteria based on the number 
of follicles >10 mm (F10) 
Action on gonadotrophin 
dose 
Under-response 
F10 <=5  
Step-up the dose: 
150  225 IU daily, or 
225  300 IU daily 
Normal response 
F10 >5 and <=15 
Keep at same dose 
Excessive response 
F10 >15 
Step-down the dose: 
225  150 IU daily 
150  112.5 IU daily 
 
The dose adjustment will be made for once only throughout the treatment cycle.  
 
All subjects will be treated on a GnRH antagonist protocol for pituitary down-
regulation.  They will receive cetrorelix or ganirelix 250 microgram subcutaneous 
injection starting from the 6th day of ovarian stimulation until the day of hCG trigger, 
according to our current clinic protocol. 
 
Human chorionic gonadotrophin (hCG) will be administered for ovulatory trigger 
when the leading follicle reaches 18 mm, with preferably two or more other follicles 
reaching 16 mm in mean diameter.  Oocyte retrieval will be carried out 34-36 hours 
post-hCG, where all follicles >10 mm will be aspirated. 
 
All subjects will proceed to hCG trigger and oocyte retrieval except for those with 
zero follicle developing to beyond 10 mm after 12 days of stimulation, or when these 
are contraindicated for other medical reasons.  Any events of ovarian 
hyperstimulation syndrome (OHSS) or anticipated risk of OHSS will be managed 
according to current practice. 
 
 
 
7. 
Study outcomes 
 
Primary outcome:  
 
Percentage of subjects having appropriate ovarian response (defined as number 
of oocytes retrieved being between 6 and 14). 
 
Secondary outcome: 
 Percentage of subjects requiring step-up or step-down of gonadotrophin dose upon 
first ultrasound tracking. 
 

 
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Hormone assay: 
AMH concentration will be determined with ELISA using a commercial automated 
method (Access AMH Assay, Beckman-Coulter, Texas, USA), which has a limit of 
detection of 0.02 ng/ml. 
 
 
8. 
Statistics 
 
8.1 
Statistical tests: 
 
The percentage of subjects having appropriate ovarian response, defined as number of 
oocytes retrieved between 6 and 14 (Broer et al, 2010), as well as the percentage 
requiring step-up and step-down of gonadotrophin dose, will be compared between 
the AMH and AFC groups using χ2 test or Fisher’s Exact test as appropriate. 
Statistical analyses will be performed using the SPSS software. 
 
8.2 
Sample size estimation: 
 
According to review of our Centre’s data (unpublished), 50% of our patients have 
appropriate ovarian response when using AFC to determine gonadotrophin dosing as 
in current practice.  Assuming that an increase to 70% of appropriate response in the 
AMH group will be clinically significant, we require a minimum of 93 subjects to 
demonstrate a significant difference between the AMH and AFC groups with 80% 
power and type I error of 0.05.  To allow for drop-outs, a total of 200 subjects (i.e. 
100 per group) will be recruited.  
 
9. 
Assessment of safety 
 
There is no safety concern as the study does not involve any extra medical 
intervention to the patients concerned.  It only involves the comparison of two 
assessment tools for determination of gonadotrophin dosing.  The use of AFC for this 
purpose is the current practice in our Centre, whereas the use of AMH for this 
purpose is well supported in recent literature. 
 
 
10. 
Direct access to source data / documents: 
 
Trial-related monitoring, audits and regulatory inspections are allowed.  
 
 
11. 
Quality control and quality assurance 
 
This shall follow the general quality control policy established for our clinic as well as 
for our hormone assay laboratory. 
 
12. 
Ethics: 
 
Ethics approval will be applied for before commencement of study. 
 
 

 
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13. Data Handling and record keeping 
 
All data will be stored and analysed using SPSS. All the investigators will be 
responsible for data management including data coding, monitoring and verification. 
 
14. Financing and insurance 
 
 
The study will be funded by the Department of Obstetrics and Gynaecology, 
University of Hong Kong. 
 
15. Publication policy 
 
The findings of this study will be submitted for consideration for publication in peer-
reviewed scientific journal. 
 
16. Supplements 
 
Nil 
 
References: 
 
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the assessment of ovarian reserve: when and for whom is it indicated?  Maturitas 
2012; 71(1): 28-33. 
2. Broekmans FJ, Kwee J, Hendriks DJ, Mol BW, Lambalk CB.  A systematic review of 
tests predicting ovarian reserve and IVF outcome.  Hum Reprod Update 2006; 12: 
685-718. 
3. Broer SL, Mol B, Dolleman M, Fauser BC, Broekmans FJM.  The role of anti-
Mullerian hormone assessment in assisted reproductive technology outcome.  Curr 
Opin Obstet Gynecol 2010; 22: 193-201. 
4. Broer SL, Mol BWJ, Hendriks D, Broekmans FJM.  The role of antimullerian 
hormone in prediction of outcome after IVF: comparison with the antral follicle 
count.  Fertil Steril 2009; 91(3):705-714. 
5. Broer SL, Dólleman M, Opmeer BC, Fauser BC, Mol BW, Broekmans FJ.  AMH and 
AFC as predictors of excessive response in controlled ovarian hyperstimulation: a 
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6. Ficicioglu C, Kutlu T, Baglam E, Bakacak Z.  Early follicular antimullerian hormone 
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8. Ledger WL.  Clinical utility of measurement of anti-Mullierian hormone in 
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9. Lee MM, Donahoe PK, Hasegawa T, Silverman B, Crist GB, Best S et al.  Mullerian 
inhibiting substance in humans: normal levels from infancy to adulthood.  J Clin 
Endocrinol Metab 1996: 81: 571-576. 
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Anti-Mullerian hormone: clairvoyance or crystal clear?  Hum Reprod 2012; 27(3): 
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