Comparison of hhCG With hCG+ß in the Early Prediction of Ongoing Pregnancy After In-vitro Fertilization and Embryo Transfer.
Phase: NA
Conditions: Pregnancy Test
Sponsor: Instituto Valenciano de Infertilidad, IVI VALENCIA

IVI Valencia, Dr. Ernesto Bosch, 1701-VLC-011-EB 
 
 
Study Protocol 
 
Comparison of hhCG with hCG+ß in the early 
prediction of ongoing pregnancy after in-vitro 
fertilization and embryo transfer. 
(hyperPOC) 
 
A monocentric, prospective, open, biomedical study 
Code: 1701-VLC-011-EB (CIM RD002786) 
 
May 09, 2017 
 
 
 
Responsibilities and Signature:  
Dr. Ernesto Bosch, Principal Investigator of the biomedical study “Comparison of hhCG with 
hCG+ß in the early prediction of ongoing pregnancy after in-vitro fertilization and embryo 
transfer (hyperPOC)”,  
- confirms that this study respects the ethical and legal norms applicable to this type of study 
and will fulfill the norms of Good Clinical practices throughout the study.  
- has the necessary material and human resources to carry out the study, and this will not 
interfere with other studies or other practices which take place habitually.  
- is committed to assure that all the subjects in the study are treated according to that which 
is established in the study protocol which has approval from the corresponding Ethics 
Committee.  
- confirms that all the collaborators partaking in the undertaking of this study are qualified 
and trained to do so.  
Principal Investigator:  
 
 
 
 
 
 
 
 
 
 
 
 
Dr. Ernesto Bosch 
Medical Director IVI Valencia  
 
 
Date 
 
11701-VLC-011-EB (CIM RD002786), Protocol Version No. 2.0  
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IVI VALENCIA, Dr. Ernesto Bosch, 1701-VLC-011-EB 
 
Name/Function 
Date 
Signature 
Authors 
Dr. Ernesto Bosch, MD, PhD, 
Gynecologist 
Medical Director of  
IVI Clinic, Valencia 
Dr. Martin Hund, Global Clinical 
Leader, Roche Centralised and Point 
of Care Solutions 
Dr. Reinhard van der Does,  
Medical Director,  
IST GmbH 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
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IVI VALENCIA, Dr. Ernesto Bosch, 1701-VLC-011-EB 
REVISION HISTORY  
 
Version 
Changes 
Date 
Responsible 
1 
New document 
Jan 31, 
2017 
Ernesto Bosch, 
MD, PhD 
Martin Hund, 
PhD 
Reinhard van 
der Does, MD 
1.1 
Shift of documentation of number of embryos 
transferred to Screening assessment 
Definition of clinical pregnancy on day 35 (+3) as 
detection of embryo sac and subsequent adaptations 
including the definitions of pregnancy categories 
March 
10, 2017 
Ernesto Bosch, 
MD, PhD 
Martin Hund, 
PhD 
Reinhard van 
der Does, MD 
2.0 
Changed recruitment time to: 
June 1, 2017 to February 28, 2018 
Visit dates: now counted from date of embryo 
transfer instead of date of oocyte pick-up, leading to a 
reduction of figures by 5 for all visit dates, as day-5 
blastocysts are being transferred. 
New visit (Visit 4) with US inserted at day 21 (+4/-3), 
range of visit at day 28 (Visit 5) changed to (+4/-3), at 
Visit 6 documentation of pregnancy category. 
Inclusion criteria: Also frozen day-5 blastocysts and 
egg donation permitted, maximum age increased to 
45 years. 
Subsequently for exclusion criteria: egg donation 
deleted; term 'gestational surrogacy' specified.  
May 09, 
2017 
Ernesto Bosch, 
MD, PhD 
Martin Hund, 
PhD 
Reinhard van 
der Does, MD 
1701-VLC-011-EB (CIM RD002786), Protocol Version No. 2.0  
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IVI VALENCIA, Dr. Ernesto Bosch, 1701-VLC-011-EB 
GLOSSARY OF ABBREVIATIONS 
 
AE 
Adverse Event 
AUC 
Area Under the Curve 
eCRF 
Electronic Case Report Form 
ET 
Embryo Transfer 
EU 
European 
hhCG 
Hyperglycosylated human chorionic gonadotropin 
IVF 
In Vitro Fertilization 
ROC 
Receiver Operator Curve 
US  
Ultrasound 
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IVI VALENCIA, Dr. Ernesto Bosch, 1701-VLC-011-EB 
SYNOPSIS OF PROTOCOL 
 
ACRONYM 
HyperPOC (hyperglycosylated hCG Proof Of Concept study) 
TITLE 
Comparison of hhCG with hCG+ß in the early prediction of 
ongoing pregnancy after in-vitro fertilization and embryo 
transfer 
STUDY NUMBER 
1701-VLC-011-EB (CIM RD002786) 
TRIAL CLASSIFICATION 
Biomedical study  
SPONSOR 
IVI Valencia 
STUDY AUTHORS 
Dr. Ernesto Bosch 
Director del Área de Medicina Reproductiva 
Unidad de Medicina Reproductiva, IVI Valencia 
Plaza de la Policía Local, 46015 Valencia, Spain 
E-mail:  Ernesto.bosch@ivi.es 
Business Phone: +34 96 305 09 00 (ext 11594) 
Dr. Martin Hund 
Gobal Clinical Leader, Roche Diagnostics Centralised and 
Point of Care Solutions 
Forrenstrasse 2, CH-6343 Rotkreuz, Switzerland 
Email: martin.hund@roche.com 
Business Phone: +41 41 798 57 20 
Dr. Reinhard van der Does, Medical Director, IST GmbH 
Soldnerstrasse 1, D-68219 Mannheim, Germany 
Email: reinhard.vanderdoes@istgmbh.com 
Business Phone: +49 621 87986 10 
RESPONSIBLE CONTACT 
PERSONS 
Principal Investigator: 
Dr. Ernesto Bosch 
Dr. Martin Hund, Roche Diagnostics Centralised and Point of 
Care Solutions 
Dr. Reinhard van der Does, Medical Director, IST GmbH 
SUPPORT FUNCTIONS 
Biostatistics: Silke Ahlers, Prometris GmbH, Mannheim, 
Germany 
PRODUCTS 
hhCG (Quest Diagnostics), Elecsys® hCG+ß  
INDICATION 
Prediction of ongoing pregnancy in women undergoing IVF 
STUDY DESIGN 
Monocentric, prospective, open, biomedical study 
STUDY PURPOSE 
SUMMARY 
Proof of concept for the prediction of ongoing pregnancy in 
women undergoing IVF using hhCG 
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IVI VALENCIA, Dr. Ernesto Bosch, 1701-VLC-011-EB 
PRIMARY PURPOSE 
Utility of the hhCG assay (Quest Diagnostics), early after In-
Vitro Fertilization and fresh or frozen Embryo Transfer (IVF-
ET), as aid in the prediction of ongoing pregnancy  
STUDY OBJECTIVES 
Primary objective:  
To determine, based on the AUCs of the ROC curves, whether 
serum hyperglycosylated human chorionic gonadotropin 
(hhCG) measured on day 4 (+1) after ET* is superior to 
hCG+ß measured on day 4 (+1) in predicting clinical 
pregnancy on day 21 (+4/-3) (=6 weeks after last menstruation, 
with detection of embryo sac by US). 
*ET= Embryo Transfer; all days mentioned in the following are counted from ET 
(=Day 0).  
 
Secondary objectives*: 
1. To determine, based on the AUC of the ROC curves, 
whether hhCG measured on day 7 (+1) and day 11 (+2) is 
superior to hCG+ß measured on the same days in 
predicting clinical pregnancy on day 21 (+4/-3)  
2. To determine, based on the AUC of the ROC curve, 
whether hhCG measured on day 4 (+1), day 7 (+1) and day 
11 (+2) is superior to hCG+ß measured on the same days 
in predicting ongoing pregnancy on day 65 (+7) . 
3. To determine, based on the AUC of the ROC curve, 
whether hhCG measured on day 4 (+1) and day 7 (+1) is 
superior to hCG+ß measured on the same days in 
predicting pregnancy on day 11 (+2) defined by serum 
hCG+ß. 
4. To compare the sensitivity and specificity and the positive 
and negative predictive value of hhCG with hCG+ß 
measured on days 4 (+1), 7 (+1) and 11 (+2) for clinical 
pregnancy on day 21 (+4/-3) and ongoing pregnancy on 
day 65 (+7) and to identify cut-off values. 
5. To compare the sensitivity and specificity and the positive 
and negative predictive value of hhCG with hCG+ß 
measured on days 4 (+1) and 7 (+1) for pregnancy on day 
11 (+2) defined by serum hCG+ß and to identify cut-off 
values. 
6. To determine, based on the AUCs of the ROC curves, 
whether changes in hhCG on day 7 (+1) vs. day 4 (+1) and 
day 11 (+2) vs. day 7 (+1) are superior to corresponding 
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IVI VALENCIA, Dr. Ernesto Bosch, 1701-VLC-011-EB 
changes in hCG+ß in predicting clinical pregnancy on day 
21 (+4/-3) and ongoing pregnancy on day 65 (+7). 
7. To determine, based on the AUCs of the ROC curves, 
whether changes in hhCG on day 7 (+1) vs. day 4 (+1) are 
superior to corresponding changes in hCG+ß in predicting 
pregnancy on day 11 (+2) defined by serum hCG+ß. 
8. To compare the sensitivity and specificity and the positive 
and negative predictive value of changes in hhCG with 
those of hCG+ß on day 7 (+1) vs. day 4 (+1) and day 11 
(+2) vs. day 7 (+1) for clinical pregnancy on day 21 (+4/-3) 
and ongoing pregnancy on day 65 (+7). 
9. To compare the sensitivity and specificity and the positive 
and negative predictive value of changes in hhCG with 
those of hCG+ß on day 7 (+1) vs. day 4 (+1) for pregnancy 
on day 11 (+2) defined by serum hCG+ß.  
10. To determine the course of hhCG and hCG+ß from day 
4(+1) through day 11(+2)  
11. To determine the usefulness of absolute values and changes 
in hhCG over time in distinguishing between the 5 disjunct 
pregnancy categories of 1) negative pregnancy, 2) ectopic 
pregnancy, 3) 1st trimester miscarriage 4) ongoing 
pregnancy on day 65 (+7) and 5) biochemical pregnancy. 
12. To investigate into differences in the course of hhCG 
values between single and multiple pregnancies (if 
warranted by the number of multiple pregnancies) 
13. To determine incidents and indirect harms. 
 
*All days mentioned above are counted from ET (=Day 0). 
TARGET STUDY 
POPULATION 
Inclusion criteria:  
• Written informed consent 
• Patients undergoing their first or second IVF-ET cycle  
• Age 18-45 years  
• Use of Ovitrelle for final follicular maturation and 
luteinization in the case of fresh ET  
• Fresh or frozen day-5 blastocysts transfer (autologous or 
egg donation) 
 
 
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IVI VALENCIA, Dr. Ernesto Bosch, 1701-VLC-011-EB 
 
Exclusion criteria:  
• Gestational surrogacy (patient's eggs used for pregnancy in 
a surrogate mother) 
• Canceled IVF cycles 
• GnRH agonist triggering cycles  in the case of fresh ET 
STUDY DURATION 
PLANNED  
Clinical part of the study: from June 2017 to April 2018 
Patient recruitment time: from June 1, 2017 to Dec 31, 2017 
STUDY POPULATION & 
TOTAL NUMBER OF 
PATIENTS 
A sample size of n= 136 patients eligible for the per-protocol 
analysis has been calculated of whom n=61 are expected to 
present with clinical pregnancy on day 21* (+4/-3). Based on an 
assumed rate of 10% patients enrolled but ineligible for the per-
protocol analysis, n=152 patients are planned to be included in 
the study.  
 
*Counted from ET (=Day 0). 
NO. OF SITES 
Monocentric study 
SCHEDULE OF 
ASSESSMENTS 
See Schedule of Assessments below 
SAFETY 
As the only impact on patient safety by the study is from taking 
two additional blood samples, a general AE collection and 
assessment will not be carried out. Any AEs occurring in 
temporal relationship to drawing the two blood samples in 
addition to the study site’s clinical routine will be documented 
observing the rights of patients' compensation for damage 
through participation in the study and the requirements of the 
insurance policy taken out. Incidents in connection with the 
diagnostic test will be documented following EU and local 
regulations and guidelines. Indirect harms to the patients are 
not possible to occur as the test result will only be known after 
individual study end. Hence, any incorrect results of the test 
can have no consequence for the further course of the patient 
during the study.   
COMPLIANCE STATEMENT 
The study will be conducted in compliance with the Guidelines 
for Good Pharmacoepidemiology Practices (GPP) and 
applicable regulatory requirements. 
 
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IVI VALENCIA, Dr. Ernesto Bosch, 1701-VLC-011-EB 
Schedule of Assessments 
 
1 Embryo Transfer = day 0 
2 Leaving the patient ample time for consideration after patient information has taken place  
3 Including previous pregnancies and complications/outcomes 
4 Determination of hCG+ß by study site and asservation of aliquots (0.5 mL of serum for determination of hhCG by Quest 
Diagnostics, 0.5 mL for potential future biomarker analysis by IVI and 2.0 mL for Roche Diagnostics) (aliquots to be kept 
frozen at -800C and shipped in batches of patients having all of their samples completed). hhCG to be analyzed by Quest 
Diagnostics in batches per patient, i.e. same lot of test used for all samples of a patient.  
5 Detection of embryo sac, heart beat or ectopic pregnancy. 
6 Final documentation may be earlier in case of premature termination. Centrally measured hhCG values will be entered as 
soon as available. 
 
 
Screening 
Visit 1 
Visit 2 
Visit 3 
Visit 4 
Visit 5 
Visit 6  
Days from ET1 
 
Day 4 (+1) 
Day 7 (+1) 
Day 11 (+2) 
Day 21 (+4/-3) 
Day 28 (+4/-3) 
Day 65 (+7) 
Patient 
information 
 
 
 
 
 
 
 
Informed 
consent 2 
 
 
 
 
 
 
 
Demogr. data, 
Med. hist.3 
 
 
 
 
 
 
 
Body weight 
and height 
 
 
 
 
 
 
 
Concomitant 
diseases 
 
 
 
 
 
 
 
Concomitant 
medication 
 
 
 
 
 
 
 
Incl. / excl. 
criteria 
 
 
 
 
 
 
 
Number of 
embryos 
transferred 
 
 
 
 
 
 
 
Serum 
estradiol, 
progesterone 
 
 
 
 
 
 
 
(Serum) hhCG, 
hCG+ß 4 
 
 
 
 
 
 
 
Ultrasound5 
 
 
 
 
 
 
 
Pregnancy 
category 
 
 
 
 
 
 
 
Incidents and 
indirect harms 
 
 
 
 
 
 
 
Final 
documentation6 
 
 
 
 
 
 
 
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IVI VALENCIA, Dr. Ernesto Bosch, 1701-VLC-011-EB 
TABLE OF CONTENTS 
 
1 
INTRODUCTION 
11 
1.1 hhCG Method Description 
11 
1.2 Background and Study Rationale 
11 
1.2.1 
Background 
11 
1.2.2 
Study Rationale 
12 
1.3 Study Objectives 
12 
2 
STUDY DESIGN AND ASSESSMENTS 
12 
2.1 Overview of Study Design 
12 
2.1.1 
Blinding of Study 
12 
2.1.2 
Scope of Study 
12 
2.2 In-/Exclusion Criteria 
12 
2.3 Patient Recruitment 
13 
2.4 Data Collection 
13 
2.5 Endpoints of the Study 
15 
2.5.1 
Primary Endpoint 
15 
2.5.2 
Secondary Endpoints 
15 
2.5.3 
Safety Variables 
16 
2.6 Known and Potential Risks and Benefits 
16 
3 
MATERIALS 
17 
4 
EVALUATION REQUIREMENTS 
17 
4.1 Data Management Plan 
17 
4.2 Safety of Specimen Handling 
17 
4.3 Safety Assessment 
17 
4.3.1 
Adverse Events 
17 
4.3.2 
Incidents and Indirect Harms 
17 
4.4 Documentation of Medication 
18 
5 
DIAGNOSTIC CRITERIA AND DEFINITION OF ANALYSIS GROUPS 
18 
5.1 Pregnancy Endpoints 
18 
5.2 Patient Group for the Primary Analysis 
19 
6 
PATIENT INSURANCE 
19 
7 
SAMPLE SIZE AND STATISTICAL ANALYSIS 
20 
7.1 Sample Size Calculation 
20 
7.2 Targeted Accrual 
20 
7.3 Groups for Biostatistical Analysis 
20 
7.4 General Biostatistical Approaches 
21 
7.5 Descriptive Statistics 
22 
7.6 Analysis by Study Objectives 
22 
7.6.1 
Primary study Objective 
22 
7.6.2 
Secondary Study Objectives 
22 
8 
SAMPLE COLLECTION AND PROCESSING 
23 
9 
SECRECY AGREEMENT 
24 
10 
PROTOCOL REFERENCES 
25 
 
 
 
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IVI VALENCIA, Dr. Ernesto Bosch, 1701-VLC-011-EB 
 
1 INTRODUCTION  
1.1 hhCG Method Description 
Serum samples will be collected and shipped on dry ice to Quest Diagnostics (Quest 
Diagnostics, Nichols Institute, San Juan Capistrano, CA) for measurements of hhCG. The 
ultrasensitive hhCG assay will be performed using a procedure described by Pandian et al. [1]. 
hhCG will be measured using an electrochemiluminescence (ECL) technique on 96-well plates 
from Meso Scale Discovery. This immunometric assay uses the hhCG speciﬁc antibody B-152 
as the coating antibody and the hCG+ß beta speciﬁc antibody B-207 as the labeled antibody 
with Sulfo-Tag (Meso Scale Discovery). The light signal (relative light unit, RLU) generated by 
the antibody-antigen and antibody reaction will be measured by a luminometer (Meso Scale 
Discovery). The RLU is directly proportional to the concentration of hhCG. 
1.2 Background and Study Rationale 
1.2.1 Background 
Hyperglycosylated human chorionic gonadotropin (hhCG) as it was later called was first 
isolated from the urine of women with invasive trophoblastic disease [2]. It is a 
hyperglycosylated isoform of human chorionic gonadotropin (hCG) with more extensive and 
more complex carbohydrate moieties than hCG during the initial 3 weeks of pregnancy hhCG 
is the primary hCG isoform [3–9]. It is synthesized primarily by cytotrophoblasts, whereas 
hCG is produced by syncytiotrophoblasts [12, 13]. hhCG is supposed to be of relevance 
during implantation into the uterine wall [13–15]. The monoclonal antibody against hhCG, 
B152, has a specificity for hhCG of >99 % relative to hCG [4, 8, 10], whereas many other 
antibodies against hCG show significant cross reactivity with hhCG [11]. 
Urinary hhCG levels during pregnancy were shown to rise 3 days sooner on average than 
those of hCG. The ratio of hhCG to hCG may allow to differentiate between clinical and 
biochemical pregnancy [5]. 
In a retrospective monocenter evaluation in 112 women with in vitro fertilization (IVF), fresh 
embryo transfer (ET) and day-9 and day-16 after egg retrieval (equals days 4 and 11 after ET) 
serum samples carried out by Chuan et al. it was found that a day-9 hhCG level of >110 
pg/mL was 96% specific for ongoing pregnancy, yielding a positive predictive value of 94%. 
Compared with the day-9 hCG level, hhCG was more sensitive and had a larger area under 
the curve (0.87 vs. 0.67, respectively). In contrast, the diagnostic test characteristics were 
similar between the day-16 hhCG and hCG levels [16]. In a prospective blinded clinical trial 
by Strom et al. enrolling 58 IVF-ET patients a single serum or urine hhCG measurement 
identified pregnancies (both biochemical and clinical) on day 6 post ET with 100% sensitivity 
and specificity, whereby it was possible to identify biochemical pregnancies by their lower 
hhCG values [17]. 
 
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IVI VALENCIA, Dr. Ernesto Bosch, 1701-VLC-011-EB 
1.2.2 Study Rationale 
Proceeding on the results of previous clinical studies and particularly of the two studies by 
Chuan and Strom mentioned above [16, 17], it is a logical further step to try and validate the 
hypothesis of a superior predictive value of hhCG vs. hCG early after IVF-ET for clinical and 
ongoing pregnancy in a prospective study sufficiently powered based on existing data for the 
two diagnostic tests. The nature of the study is therefore that of a proof of concept trial. 
1.3 Study Objectives 
See Study Synopsis. 
2 STUDY DESIGN AND ASSESSMENTS 
2.1 Overview of Study Design 
The study has been planned considering the principles of evidence based medicine using the 
PICO criteria [18]. It is a monocenter, open, biomedical study in patients undergoing in-vitro 
fertilization. The purpose of proof of concept requires keeping variability within very narrow 
limits. Accordingly, there is a strict definition of the selection criteria for patients to be 
enrolled, and the primary analysis will be carried out in the per protocol population. 
It is planned to include a total of 152 patients. The study protocol is based on routine 
procedures at the site, with the only additional measures stipulated being the taking of 6-mL 
venous blood samples on visits 1 and 2 and the asservation of another 6-mL of venous blood 
on visit 3 when blood is taken anyhow within the scope of clinical routine. The three 6-mL 
blood samples will be processed for central assessment of hhCG. Neither the patient nor the 
investigator will know the result of the centralized analysis prior to individual study 
completion. Apart from the two blood samples mentioned there are only two further 
procedures involving the patients directly. This refers to the ultrasound assessments at visit 4 
and the final visit for the assessment of clinical and ongoing pregnancy (both part of the study 
site’s routine measures). 
2.1.1 Blinding of Study 
The study will be conducted in an open fashion. However, results of the hhCG assessments 
will only become available after study end.  
2.1.2 Scope of Study 
The study data will serve for a proof-of-concept evaluation. 
2.2 In-/Exclusion Criteria  
See Study Synopsis. 
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2.3 Patient Recruitment 
Patients will be screened in a consecutive, unselected manner, and, after written informed 
consent has been obtained, will be eligible to be enrolled in the study if all inclusion and none 
of the exclusion criteria are met.  
2.4 Data Collection 
For an overview see Schedule of Assessments in the Protocol Synopsis. 
 
Patients will only be included into the study after they have given their informed consent in 
writing. The study will begin with a Screening phase starting with Patient information and 
Patient's Informed Consent (leaving the patient ample time for consideration after Patient 
Information) and followed by the documentation of general patient data. On the basis of 
available data the decision as to the patient's eligibility to participate in the study will then be 
made considering the inclusion/exclusion criteria. In the case of eligible patients a total of five 
visits (planned in accordance with clinical routine) will follow during a period of a maximum 
of 68 days starting with day 4(+1) after ET (= Day 0).  
 
As compared with clinical routine an additional 6 mL of blood is planned to be taken on each 
visit 1-3 (on Visit 3 a blood sample is drawn anyhow following clinical routine). Hence, the 
total amount of blood taken for the purpose of the study will be 18 mL (not considering the 
small amounts of blood needed for measuring serum estradiol and progesterone by IVI for 
visits 1 and 2). During visits 1 - 3 an aliquot of 0.5 mL of serum will be stored for the central 
assessment of hhCG by Quest Diagnostics, 0.5 mL for potential future biomarker analyses by 
IVI and another 2.0 mL for shipment to Roche potentially to be used for repeat measurements 
or measurements within the scope of developing a new hhCG immunoassay. Apart from this, 
there will be no further study-related measures with a potential impact on the patient's health 
and well-being. The US assessments at Visit 4 and 5 are part of clinical routine assessments.  
Therefore, a general AE collection and assessment will not be carried out. Any AEs occurring 
in temporal relationship to drawing the two blood samples in addition to the study site’s 
clinical routine will be documented on paper forms observing the rights of patients’ 
compensation for damage through participation in the study and the requirements of the 
insurance policy taken out. Concomitant diseases and medication will be documented at each 
visit. Any changes from previous visits found, however, will not be followed up as a possible 
indication of an adverse event. Incidents in connection with the diagnostic test will be 
documented following EU and local regulations and guidelines. This will be done on a paper 
form and the information will not be entered into the eCRF. Indirect harms to the patients are 
not possible to occur as results of the central hhCG analyses will only be known to the study 
site after study end. Hence, any incorrect results of the hhCG test can have no consequences 
for the further course of the patient during the study. 
 
 
 
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IVI VALENCIA, Dr. Ernesto Bosch, 1701-VLC-011-EB 
Screening Phase 
• Patient information 
• Patient's informed consent 
• Demographic data (race, ethnicity, age) 
• Medical history including history of previous pregnancies and their outcomes and 
complications 
• Body weight and height 
• Concomitant diseases and medication 
• Check of inclusion and exclusion criteria 
• Number of embryos transferred and dates of oocyte pick-up, fertilization and ET 
Visit 1 
At Visit 1 the following will be done and documented in the eCRF:  
• Measurement and documentation of serum estradiol, progesterone and hCG+ß values  
by the study site 
• Taking of 6 mL of venous blood for asservation of 0.5 mL of serum (to be kept in the 
freezer at -800C with temperature log and shipped in batches for the central analysis of 
hhCG), of a further 0.5 mL for IVI kept in the freezer at -80°C and another 2.0 mL 
serum kept in the freezer at -800C to be shipped to Roche at the end of the study.  
• Concomitant diseases and medication. 
Visit 2 
At Visit 2 the following will be done and documented in the eCRF: 
• Measurement and documentation of serum estradiol, progesterone and hCG+ß values 
by the study site 
• Taking of a further 6 mL of venous blood for asservation of 0.5 mL of serum (to be 
kept in the freezer at -800C with temperature log and shipped in batches for the central 
analysis of hhCG), of a further 0.5 mL for IVI kept in the freezer at -800C and of 
another of 2.0 mL kept in the freezer at -800C  for to be shipped to Roche at the end of 
the study.  
• Concomitant diseases and medication. 
Visit 3 
At Visit 3 the following will be done and documented in the eCRF:  
• Measurement and documentation of serum estradiol, progesterone and hCG+ß by the 
study site 
• Separation of 6 mL of blood taken in addition to routine blood sampling for 
asservation of 0.5 mL of serum (to be kept in the freezer at -800C with temperature log 
and shipped in batches for the central analysis of hhCG), of a further 0.5 mL for IVI 
kept in the freezer at -800C and of another 2.0 mL kept in the freezer at -800C for to be 
shipped to Roche at the end of the study. 
• Concomitant diseases and medication. 
 
 
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IVI VALENCIA, Dr. Ernesto Bosch, 1701-VLC-011-EB 
Visit 4 
At Visit 4 the following will be done and documented in the eCRF:  
• Concomitant diseases and medication 
• Ultrasound for detection of embryo sac, (heart beat) or ectopic pregnancy. 
Visit 5 
At Visit 4 the following will be done and documented in the eCRF: 
• Concomitant diseases and medication 
• Ultrasound for detection of embryo sac, heart beat or ectopic pregnancy. 
Visit 6  
At Visit 5 the following will be done and documented in the eCRF:  
• Concomitant diseases and medication 
• Documentation of pregnancy category: negative pregnancy, ectopic pregnancy, 1st 
trimester miscarriage, ongoing pregnancy (single/multiple) on day 65 (+7), 
biochemical pregnancy (see section 5.1) 
• Incidents and indirect harms 
• Final documentation. 
2.5 Endpoints of the Study 
2.5.1 Primary Endpoint 
AUC of the ROC curve for hhCG and hCG+ß on day 4 (+1) after ET for the prediction of 
clinical pregnancy on day 21 (+4/-3) after ET (=6 weeks after last menstruation, detection of 
embryo sac by US). 
2.5.2 Secondary Endpoints 
See also Section 5 for definitions. Secondary endpoints of the study are*:  
1. AUC of the ROC curve for hhCG and hCG+ß on days 7 (+1) and 11 (+2) for the 
prediction of clinical pregnancy on day 21 (+4/-3) 
2. AUC of the ROC curve for hhCG and hCG+ß on days 4 (+1), 7 (+1) and 11 (+2) for the 
prediction of ongoing pregnancy on day 65 (+7) 
3. AUC of the ROC curve for hhCG and hCG+ß on days 4 (+1) and 7 (+1) for the 
prediction of pregnancy on day 11 (+2) defined by serum hCG+ß. 
4. Sensitivity and specificity and positive and negative predictive value of hhCG and 
hCG+ß on days 4 (+1), 7 (+1) and 11 (+2) for clinical pregnancy on day 21 (+4/-3) 
5. Sensitivity and specificity and positive and negative predictive value of hhCG and 
hCG+ß on days 4 (+1), 7 (+1) and 11 (+2) for ongoing pregnancy on day 65 (+7)  
6. Sensitivity and specificity and positive and negative predictive value of hhCG and 
hCG+ß on days 4 (+1) and 7 (+1) for pregnancy on day 11 (+2) defined by serum 
hCG+ß  
7. AUC of the ROC curve for changes in hhCG and hCG+ß on day 7 (+1) vs. day 4 (+1) 
and on day 11 (+2) vs. day 7 (+1) for the prediction of clinical pregnancy on day 21 
(+4/-3)  
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8. AUC of the ROC curve for changes in hhCG and hCG+ß on day 7 (+1) vs. day 4 (+1) 
and on day 11 (+2) vs. day 7 (+1) for the prediction of ongoing pregnancy on day 65 
(+7) 
9. AUC of the ROC curve for changes in hhCG and hCG+ß on day 7 (+1) vs. day 4 (+1) 
for the prediction of pregnancy on day 11 (+2) defined by serum hCG+ß 
10. Sensitivity and specificity and positive and negative predictive value of changes in hhCG 
and hCG+ß on day 7 (+1) vs. day 4 (+1) and on day 11 (+2) vs. day 7 (+1) for the 
prediction of clinical pregnancy on day 21 (+4/-3) 
11. Sensitivity and specificity and positive and negative predictive value of changes in hhCG 
and hCG+ß on day 7 (+1) vs. day 4 (+1) and on day 11 (+2) vs. day 7 (+1) for the 
prediction of ongoing pregnancy on day 65 (+7) 
12. Sensitivity and specificity and positive and negative predictive value of changes in hhCG 
and hCG+ß on day 11 (+2) vs. day 7 (+1) for the prediction of pregnancy on day 11 (+2) 
defined by serum hCG+ß 
13. Absolute values and changes vs. previous visits of hhCG and hCG+ß for course of the 
variables during the study 
14. Absolute values and changes vs. previous visits of hhCG and hCG+ß for the distinction 
of outcomes 1) negative pregnancy, 2) ectopic pregnancy, 3) 1st trimester miscarriage 4) 
ongoing pregnancy on day 65 (+7) and 5) biochemical pregnancy. 
15. Absolute values and changes vs. previous visits of hhCG and hCG+ß for the distinction 
between single and multiple pregnancies 
16. Course of serum estradiol and progesterone during the study 
17. Any AEs with a temporal relationship with drawing the two blood samples in addition 
to the site's clinical routine 
18. Incidents involved with hhCG testing. 
*All days mentioned in this section are counted from ET (=Day 0). 
2.5.3 Safety Variables 
AEs are only recorded if they occur in a temporal relationship with drawing of the two blood 
samples on visits 1 and 2 as these are the only measures carried out in addition to the routine 
procedures at the site and, hence, for the sole purpose of the study (interventions). A causal 
relationship of any adverse events with hhCG testing is impossible as a patient's test results 
will not be made known during the course of the study. Hence, the patients' course during and 
after the study cannot be influenced by any inaccurate test values or the interpretation of the 
hhCG values (accurate or inaccurate). 
 
Incidents will be documented following the guidance provided in MEDDEV 2.12.-1 rev. 6 and 
local regulations and guidelines. Indirect harms are not possible to occur as discussed in 
section 4.3.2. 
2.6 Known and Potential Risks and Benefits 
Apart from two additional blood drawings on visits 1 and 2 there will be no further measures 
(interventions) during the study in addition to such procedures as are part of the clinical 
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routine at the site. Except for the known and generally accepted risks involved with drawing 
blood samples, no further risks nor any benefits for the patients are involved. 
3 MATERIALS 
The project is supported by Roche Diagnostics Centralised and Point of Care Solutions within 
the scope of a research agreement between IVI Valencia and Roche Diagnostics Centralised 
and Point of Care Solutions.  
4 EVALUATION REQUIREMENTS 
4.1 Data Management Plan 
Clinical, laboratory and test result data will be entered into an eCRF. A limited range of 
validation checks will be detailed in a manual and implemented into the eCRF as appropriate. 
For data analysis SAS software will be used. The study database shall be made available to 
Roche Diagnostics and the study site in an agreed upon format and data transfer mode. 
4.2 Safety of Specimen Handling  
For specimen handling the local safety regulation will apply to rule out any possible biological 
hazard. 
4.3 Safety Assessment 
4.3.1 Adverse Events 
AEs are only recorded if they occur in a temporal relationship with the blood drawings on 
visits 1 and 2 as these are the only measures carried out in addition to the routine procedures 
at the site and, hence, for the sole purpose of the study (interventions). A causal relationship 
of any adverse events with hhCG testing is impossible as a patient's test results will not be 
made known during the course of the study. Hence, the patients' course during and after the 
study cannot be influenced by any inaccurate test values or the interpretation of the hhCG 
values (accurate or inaccurate). 
4.3.2 Incidents and Indirect Harms 
The documentation and reporting of incidents will follow the guidance provided in MEDDEV 
2.12.-1 rev. 6 and local regulations and guidelines.  
 
An incident is any malfunction or deterioration in the characteristics and/or performance of a 
device, as well as any inadequacy in the labelling or the instructions for use which, directly or 
indirectly, might lead to or might have led to the death of a subject, or user or of other persons 
or to a serious deterioration in their state of health. 
 
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Indirect harms to the patients are not possible to occur as the test result will only be known 
after individual study end. Hence, any incorrect results of the test can have no consequence 
for the further course of the patient during the study. 
 
All incidents are to be recorded on a separate paper form. A description of the event, 
including the start date, resolution date, action taken, and the outcome should be provided. As 
a general principle, there should be a pre-disposition to report rather than not to report in 
case of doubt on whether or not to report an incident. Any report should not be unduly 
delayed because of incomplete information.  
4.4 Documentation of Medication 
Concomitant medication will be documented at each visit. Any changes from previous visits 
found, however, will not be followed up as a possible indication of an adverse event. 
5 Diagnostic Criteria and Definition of Analysis Groups 
5.1 Pregnancy Endpoints 
The study will have to use various pregnancy endpoints for various analyses. For the analysis 
of the primary objective and some of the secondary objectives the endpoint will be clinical 
pregnancy on day 21* (+4/-3) defined as detection of embryo sac by US, regardless of whether 
or not there will be a miscarriage during the later course of the 1st trimester. For some other 
secondary objectives the endpoint will be ongoing pregnancy on day 65 (+7) defined as 
detection of fetal heart beat by US at this time point or later). Detection of embryo sac/fetal 
heart beat on day 65 (+7) replaces missing detection of embryo sac on day 21 (+4/-3) for the 
definition of clinical pregnancy.  
 
For pregnancy on day 11 (+2) as an individual endpoint and for the allocation of patients to 
categories as described below (negative and biochemical pregnancy in particular) a cut-off 
value for hCG+ß on day 11 (+2) of >10 mIU/mL will be used as per practice at the study site. 
 
For the purposes of the analysis with respect to secondary objective no. 11 there will be 5 
disjunct pregnancy categories: 
1) Negative pregnancy 
hCG+ß on day 11 (+2) ≤10 mIU/mL, unless clinical pregnancy, ectopic pregnancy, ongoing 
pregnancy or 1st trimester miscarriage are detected at a later stage 
2) Ectopic pregnancy 
Rules out the remaining categories 3), 4) and 5) 
3) 1st trimester miscarriage  
Rules out (supersedes) categories 4) and 5) 
4) Ongoing pregnancy on day 65 (+7) 
5) Biochemical pregnancy 
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hCG+ß >10 mIU/mL on day 11 (+2) and no clinical/ongoing pregnancy at any stage and no 
ectopic pregnancy and no signs or symptoms of a 1st trimester miscarriage. 
 
The following patients are ineligible for the analysis with respect to secondary objective no. 11: 
hCG+ß on day 11 (+2) >10 mIU/mL, missing data on ectopic pregnancy/ presence of embryo 
sac, fetal heart beat and no 1st trimester miscarriage.  
1st trimester miscarriage will always be assumed in cases with detection of embryo sac on day 
21 (+4/-3) and no ongoing pregnancy on day 65 (+7). It should be noted that per-protocol 
patients with this constellation will be included in the group of subjects with clinical 
pregnancy on day 21 (+4/-3)  for the primary analysis of the study.  
*All days mentioned in this section are counted from ET (=Day 0). 
5.2 Patient Group for the Primary Analysis 
The primary analysis of the study will be carried out in the per-protocol group. This will 
include patients who have met all inclusion criteria, violated no exclusion criterion and for 
whom the data for the evaluation of the primary objective have been determined in 
compliance with the stipulations of this protocol (i.e. valid results for hhCG and hCG+ß on 
day 4 (+1)* and for US on day 21* (+4/-3).Ongoing pregnancy on day 65 (+7) replaces 
missing US data for day 21 (+4/-3) as to the question of ongoing pregnancy on this day. 
*Days mentioned in this section are counted from ET (=Day 0). 
6 Patient Insurance 
IVI Valencia undertakes to insure liability for potential damage caused to a subject in 
connection with the trial and provides insurance policy for all study subjects. 
 
 
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7 SAMPLE SIZE AND STATISTICAL ANALYSIS 
7.1 Sample Size Calculation 
The two tests hhCG and hCG+β will be compared using receiver operating characteristics 
(ROC) curves by analyzing the area under the curve (AUC). Based on the data by Chuan S et 
al. [16], an AUC of 0.67 is assumed for the hCG+β test and an AUC of 0.79 for the hhCG test. 
Under the assumption that 45% of all patients with IVF will present with clinical pregnancy 
on day 21* (+4/-3), 136 patients are necessary to achieve 80% power for a one-sided z-test 
with significance level alpha = 2.5%.  
Power 
N+ * 
N- ** 
AUC1 
AUC2 
Correlation 
0.8022 
61 
75 
0.6700 
0.7900 
0.6 
* Pregnancies 
** No Pregnancies 
The results are for tests with continuous values and are based on the methodology of Hanley 
and McNeil (1983). The calculations are further based on the binormal model and the 
assumption of a correlation of 0.6 between the two tests for both the positive and negative 
groups (N+ and N-). Allowing for 10% of patients being non-evaluable, 152 patients are 
planned to be enrolled. 
*Counted from ET (=Day 0). 
 
7.2 Targeted Accrual 
The 152 patients are planned to be enrolled over a period of 9 months, from June 1, 2017 at 
the earliest to February 28, 2018. 
7.3 Groups for Biostatistical Analysis 
The population of patients enrolled will include all patients who gave informed consent. 
 
The patients treated population will consist of all patients for whom the IVF-ET was 
performed. 
 
The per-protocol population will consist of all eligible patients treated, i.e. will be restricted to 
those patients of the patients treated population who did not violate any eligibility criteria and 
who have available measurements for both hhCG and hCG+β on day 4* (+1) and an 
assessment of clinical pregnancy on day 21* (+4/-3). A missing assessment of clinical 
pregnancy on day 21 (+4/-3) can be replaced by an assessment of ongoing pregnancy on day 
28 (+4/-3) and/or 65 (+7). 
In case of sufficiently high numbers of multiple pregnancies, subgroup analyses may be 
performed for patients with multiple and single pregnancies. Further subgroups of interest, if 
any, will be defined in the Statistical Analysis Plan. 
*All days mentioned in this section are counted from ET (=Day 0). 
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7.4 General Biostatistical Approaches 
For the pregnancy tests based on hhCG and hCG+β levels, ROC analyses will be performed. 
The predicted results of the test (positive or negative, i.e. pregnant or not pregnant) will be 
compared to the actual presence/absence of pregnancy as observed (e.g. clinical pregnancy on 
day 21* (+4/-3) or ongoing pregnancy on day 65 (+7)). There are the following possibilities 
for the test results and observed results: 
 
Observed 
Positive 
Negative 
Test 
Positive 
True positive (TP) 
False positive (FP) 
Negative 
False negative (FN) 
True negative (TN) 
 
For the test concerned, the rate of true positives and true negatives can be regarded in two 
different ways: in relation to the observed positives and negatives and in relation to the 
predicted positives and negatives. 
Based on the observed positives and negatives, the rate of true positives is called the 
sensitivity and the rate of true negatives is called specificity, i.e.: 
Sensitivity = TP / (TP + FN) 
Specificity = TN / (TN + FP) 
 
Based on the predicted positives and negatives, the rate of true positives is called the positive 
predictive value (PPV), and the rate of true negatives is called the negative predictive value 
(NPV), i.e.: 
PPV = TP / (TP + FP) 
NPV = TN / (FN + TN) 
 
Sensitivity, specificity, positive and negative predictive values are all dependent on the 
definition of the test concerned. Receiver operating characteristics (ROC) curves are used to 
visualize sensitivity and specificity for various thresholds for the pertinent test: Sensitivity is 
plotted against 1-specificity and thus displays the test’s characteristics for various cut-offs. 
Tests can be compared by looking at summary measures for the ROC curves, e.g. the area 
under the curve (AUC). Since high sensitivity and specificity are wanted in a test, a bigger area 
under the curve is desirable. 
To define a test for practical use, a threshold has to be chosen, i.e. a cut-off that defines that 
values below are considered as negative test results and values above as positive test results. A 
threshold should be chosen as a point that optimizes the combination of sensitivity and 
specificity which can be done via the ROC curve by choosing the point with the least distance 
to the upper left corner which stands for specificity = 100% and sensitivity = 100%. 
 
All other data will be summarized by descriptive statistics in case of continuous variables or by 
absolute and relative frequencies. Laboratory data will be displayed over time for each visit as 
will ultrasound results. Concomitant diseases and medications will be listed for the complete 
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duration of the study. Incidents and indirect harms will be summarized by absolute and 
relative frequencies. 
*Counted from ET (=Day 0). 
 
7.5 Descriptive Statistics 
Continuous variables will be summarized by descriptive statistics (mean, standard deviation, 
median, Q1, Q3, minimum, maximum, number of available data); categorical variables will be 
summarized by absolute and relative frequencies and number of available data. The relative 
frequencies will be based on the number of available data if not mentioned otherwise. 
7.6 Analysis by Study Objectives 
7.6.1 Primary study Objective 
The primary study objective is to determine, based on the AUCs of the ROC curves, whether 
serum hyperglycosylated human chorionic gonadotropin (hhCG) measured on day 4 (+1) 
after embryo transfer (ET) is superior to hCG+ß measured on day 4* (+1) in predicting 
clinical pregnancy on day 21* (+4/-3) (=6 weeks after last menstruation, detection of embryo 
sac by US).  
The empirical AUCs, pertinent standard errors and 95% confidence intervals will be estimated 
for both the hhCG test and the hCG+β test. Furthermore, the empirical AUCs of the two tests 
will be compared using a logistic regression model and the difference in AUCs will be 
estimated including 95% confidence intervals and chi-square test for the contrast. hhCG 
measured on day 4 (+1) after ET is considered superior to hCG+ß if the null hypothesis  
 
 
H0:     AUC (hhCG) ≤ AUC (hCG+β )  
can be rejected at an one-sided significance level of 0.025. 
 
The empirical ROC curves will be plotted to provide a visualization that supports the 
comparisons. 
*Counted from ET (=Day 0). 
7.6.2 Secondary Study Objectives 
In addition to the comparison of the AUCs under the ROC curves for day 4*, the same 
analyses will be performed for the hhCG and hCG + β tests at days 7 and 11 with respect to 
predicting clinical pregnancy on day 21*; at days 4, 7 and 11 with respect to predicting 
pregnancy on day 65; at days 4 and 7 with respect to predicting pregnancy on day 11. The 
same methods as for the analysis of the primary objective will be applied here. 
 
For the comparisons of sensitivity and specificity and the positive and negative predictive 
values at various time points with respect to pregnancy at various later time points, the 
following approach will be used: 
Optimal thresholds for the two tests and the various time points will be identified from the 
pertinent ROC curves for the time point (day 4, 7 or 11) and endpoint (pregnancy at day 65, 
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21 or 11) considered. The optimal threshold for the time point and test at hand will be found 
using Youden’s index (sensitivity + specificity -1), choosing the point for which the index is 
maximal. 
The thresholds and the corresponding sensitivities and specificities will be summarized for 
each time point, pregnancy endpoint and test. Pertinent positive and negative predictive 
values will be shown for the same thresholds for the various time points.  
 
The course of hhCG and hCG + β over time will be summarized by descriptive summary 
statistics (mean, standard error of the mean, median, Q1, Q3, minimum and maximum) for 
the disjoint pregnancy outcome categories as defined in 5.1. If there are sufficiently many 
multiple pregnancies, the same analysis will be performed for the categories of multiple and 
single pregnancies (irrespective of pregnancy outcome). 
 
Incidents and indirect harms will be summarized by absolute and relative frequencies. 
*All days mentioned in this section are counted from ET (=Day 0). 
8 SAMPLE COLLECTION AND PROCESSING 
Sample collection and processing for estradiol, progesterone and hCG+ß will be done 
according to local study site procedures and regulations. Serum obtained for the assessment of 
hhCG (0.5 mL at each occasion) will be stored in a freezer at -800C till shipped kept in dry ice 
for analysis to Quest Diagnostics (Quest Diagnostics, Nichols Institute, San Juan Capistrano, 
CA) in batches of complete samples of patients with the provision that in any particular 
patient the same batch of the diagnostic will be used. Another 0.5 mL of serum will be kept 
frozen for future potential biomarker analyses by IVI and 2.0 mL of serum from each occasion 
will be stored in a freezer at -800C and shipped kept on dry ice to Roche. 
 
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9 SECRECY AGREEMENT 
Secrecy agreement: 
The Principal Investigator agrees to handle all information and documentation he may have 
received from Roche Diagnostics confidentially and ensures that all other persons involved in 
this project will keep secrecy, too. 
 
Principal Investigator of the study: 
 
 
Location: 
IVI Clinic, Valencia.  
Plaza de la Policía Local, 3 
46015 Valencia 
Name:  
Ernesto Bosch, MD, PhD, Gynecologist 
Medical Director of IVI Clinic Valencia 
Date / Signature: 
 
 
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10 PROTOCOL REFERENCES 
1. 
Pandian R, Lu J, Ossolinska-Plewnia J. Fully automated chemiluminometric assay for 
hyperglycosylated human chorionic gonadotropin (invasive trophoblast antigen). Clin 
Chem 2003;49:808–10. 
2. 
Elliott MM, Kardana A, Lustbader JW, Cole LA. Carbohydrate and Peptide structure of 
the α- and β-subunits of human chorionic gonadotropin from normal and aberrant 
pregnancy and choriocarcinoma. Endocrine. 1997;7:15–32.  
3. 
Valmu L, Alfthan H, Hotakainen K, Birken S, Stenman UH. Sitespecific glycan analysis of 
human chorionic gonadotropin {beta}- subunit from malignancies and pregnancy by 
liquid chromatography - electrospray mass spectrometry. Glycobiology. 2006;16:1207–18.  
4. 
Cole LA. Hyperglycosylated hCG. Placenta. 2007;28:977–86. 
5. 
O’Connor JF, Ellish N, Kakuma T, Schlatterer J, Kovalevskaya G. Differential urinary 
gonadotrophin profiles in early pregnancy and early pregnancy loss. Prenat Diagn. 
1998;18:1232–40.  
6. 
Davies S, Byrn F, Cole LA. Testing for early pregnancy viability and complications. 
Clinics Lab Med. 2003;23:257–64.  
7. 
Cole LA, Khanlian SA, Sutton JM, Davies S, Stephens N. Hyperglycosylated hCG 
(Invasive Trophoblast Antigen, ITA) a key antigen for early pregnancy detection. Clin 
Biochem. 2003;36:647– 55.  
8. 
Sutton-Riley JM, Khanlian SA, Byrn FW, Cole LA. A single serum test for measuring 
early pregnancy outcome with high predictive value. Clin Biochem. 2006;39:682–7.  
9. 
Kovalevskaya G, Birken S, Kakuma T, Ozaki N, Sauer M, Lindheim S, CohenM, Kelly A, 
Schlatterer J, O’Connor JF. Differential expression of human chorionic gonadotropin 
(hCG) glycosylation isoforms in failing and continuing pregnancies: preliminary 
characterization of the hyperglycosylated hCG epitope. J Endocrinol. 2002;172:497–506.  
10. Cole LA, Dai D, Butler SA, Leslie KK, Kohorn EI. Gestational trophoblastic diseases: 1. 
Pathophysiology of hyperglycosylated hCG-regulated neoplasia. Gynecol Oncol. 
2006;102:144–9.  
11. Cole LA, Sutton JM, Higgins TN, Cembrowski GS. Between method variation in human 
chorionic gonadotropin test results. Clin Chem. 2004;50:874–82.  
12. Kovalevskaya G, Kakuma T, Schlatterer J, O’Connor JF. Hyperglycosylate HCG 
expression in pregnancy: cellular origin and clinical applications. Mol Cell Endo. 
2007;260–262:237–43.  
13. Guibourdenche J, Handschuh K, Tsatsaris V, Gerbaud MC, Legul F, Muller D, Evain-
Brion D, Fournier T. Hyperglycosylated hCG is a marker of early human trophoblast 
invasion. J Clin Endocrinol Metab. 2010;95:E240–4.  
14. Cole LA, Khanlian SA, Riley JM, Butler SA. Hyperglycosylated hCG (hCG-H) in 
gestational implantation, and in choriocarcinoma and testicular germ cell malignancy 
tumorigenesis. J Reprod Med. 2006;51:919–29. 
15. Handschuh K, Guibourdenche J, Tsatsari V, Guesnon M, Laurendeau I, Evain-Brion D, 
Fournier T. Human chorionic gonadotropin produced by the invasive trophoblast but not 
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the villous trophoblast promotes cell invasion and is downregulated by peroxisome 
proliferator-activated receptor-gamma. Endocrinology. 2007;148:5011–9.  
16. Chuan S, Homer M, Pandian R, Conway D, Garzo G, Yeo L,Su HI. Hyperglycosylated 
human chorionic gonadotropin as an early predictor of pregnancy outcomes after in vitro 
fertilization. Fertil Steril. 2014;101:392-8. 
17.  Strom CM, Bonilla-Guererro R, Zhang K, Doody KJ, Tourgeman D, Alvero R, Cedars MI, 
Crossley B, Pandian R, Sharma R, Neidich J, Salazar D. The sensitivity and specificity of 
hyperglycosylated hCG (hhCG) levels to reliably diagnose clinical IVF pregnancies at 6 
days following embryo transfer. J Assist Reprod Genet. 2012; 29:609–614. 
18. Sackett DL, Straus SE, Richardson WS, Rosenberg W and Haynes RB: How to Practice 
and Teach EBM. 2000. 2nd edition. New York: Churchill Livingstone. 
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