Impact of Day 2 Versus Day 3 Fresh Embryo Transfer on Live Birth Rate in Poor-Prognosis POSEIDON Group 4 Patients: A Single-Center, Parallel-Group, Open-Label Randomized Controlled Trial
Phase: NA
Conditions: POSEIDON Category 4, Low Prognosis in Assisted Reproductive Technology, Female Infertility Due to Diminished Ovarian Reserve
Sponsor: Shanghai Ninth People's Hospital Affiliated to Shanghai Jiao Tong University

Clinical Research Protocol V1.1 
(English Version) 
Shanghai Jiao Tong University School of Medicine Affiliated Ninth People's Hospital 
Clinical Research Protocol 
 
 
 
 
Study title: Effect of Day 2 vs. Day 3 Fresh Embryo 
Transfer on Live Birth Rate in Patients with POSEIDON 
Category 4 Low Prognosis: A Single-Center, Parallel-
Group, Open-Label Randomized Controlled Trial 
 
NCT number: / 
 
Document Date: 2026-03-02 
 
 

I. Protocol Summary 
Project Name 
Effect of Day 2 vs. Day 3 Fresh Embryo Transfer on Live Birth 
Rate in Patients with POSEIDON Category 4 Low Prognosis: A 
Single-Center, Parallel-Group, Open-Label Randomized 
Controlled Trial 
Version No. 
V1.1 
Sponsoring and 
Participating 
Units 
Department of Assisted Reproduction, Shanghai Jiao Tong 
University School of Medicine Affiliated Ninth People's Hospital 
Research 
Nature 
Randomized Controlled Clinical Trial 
Research 
Objectives 
This trial aims to compare the efficacy of Day 2 (D2) and Day 3 
(D3) fresh embryo transfer in patients with POSEIDON Category 
4, and clarify the optimal transfer time for this population. 
Research 
Subjects 
Eligible patients with POSEIDON Category 4 (first or second 
IVF/ICSI cycle) 
Research 
Methods 
Single-center, parallel-group, open-label RCT, with 1:1 stratified 
block randomization allocation 
Inclusion 
Criteria 
1. Meeting the definition of POSEIDON Category 4: age ≥ 35 
years, and antral follicle count (AFC) < 5 or anti-Müllerian 
hormone (AMH) < 1.2 ng/mL [1].2. First or second IVF/ICSI 
cycle.3. Controlled ovarian hyperstimulation (COH) using 
gonadotropin-releasing hormone (GnRH) antagonist protocol, with 
≥ 1 oocyte retrieved.4. Signed written informed consent form. 
Exclusion 
Criteria 
1. Use of donor oocytes/sperm, or planned preimplantation genetic 
testing (PGT).2. Severe immune and chromosomal 
abnormalities.3. Uterine cavity abnormalities (i.e., submucosal 
fibroids, complete uterine septum, severe intrauterine adhesions, 
etc.) or untreated and ultrasound-visible hydrosalpinx.4. 
Complicated with severe underlying diseases (such as 
uncontrolled hypertension/diabetes, active malignant tumors).5. A 
history of repeated implantation failure (≥ 2 cycles) or recurrent 
miscarriage (≥ 2 times).6. Progesterone level > 1.5 ng/mL on the 
day of human chorionic gonadotropin (hCG) trigger. 

Research 
Progress Plan 
Patient enrollment will last for 18 months, with on-site monitoring 
every 6 months, and the entire study is expected to last 36 
months. 
Statistical 
Analysis 
Methods 
Analysis Populations: ITT analysis includes all randomized 
patients (primary analysis), and missing data is handled by 
multiple imputation (MICE); PP analysis includes patients who 
comply with the protocol (sensitivity analysis).Continuous 
Variables: Normally distributed data are expressed as mean ± 
standard deviation, and t-test is used for inter-group comparison; 
non-normally distributed data are expressed as median 
(interquartile range), and Mann-Whitney U test is used for inter-
group comparison.Categorical Variables: Expressed as frequency 
(percentage), and χ² test or Fisher's exact test (when expected 
frequency < 5) is used for inter-group comparison.Subgroup 
Analysis: Preset subgroup analyses by age (< 40 years and ≥ 40 
years), AMH level (< 0.5 ng/mL and 0.5-1.2 ng/mL), and number of 
oocytes retrieved (< 4 and ≥ 4).Statistical Software: SAS 9.4 and 
R 4.3.0 software are used, with P < 0.05 (two-sided) considered 
statistically significant. 
Form of 
Research 
Results 
Publication 
The research results will be published in peer-reviewed journals 
and presented at international conferences. A plain language 
summary of the research results will be provided to participants 
(sent during offline follow-up or via email). 
 
 
 
Principal 
Investigato
r 
Jiang 
Shutia
n 
Profession
al Title 
Attending 
Physician 
Date of 
Birth 
October 1991 
Key 
Members 
of the 
Research 
Team 
Name 
Date of 
Birth 
Professional 
Title 
GCP 
Certifica
te 
Project Division 

 
Jiang 
Shutia
n 
October 
1991 
Attending 
Physician 
Yes 
Principal Investigator 
 
Li 
Wenzh
i 
May 1989 
Chief 
Technician 
Yes 
Clinical Execution 
(Embryologist) 
 
Guo 
Haiyan 
October 
1981 
Associate 
Chief 
Physician 
Yes 
Clinical Execution 
(Clinician) 
 
Lyu 
Qifeng 
September 
1970 
Researcher 
Yes 
Quality Control 
 
Jiang 
Xueyi 
January 
1999 
Physician 
No 
Statistical Analysis 
 
Li 
Danjun 
February 
1995 
Physician 
No 
Data Management 
 
Mi Yan 
August 
1999 
Graduate 
Student 
No 
Data 
Management/Resear
ch Coordinator 
II. Research Background 
In in vitro fertilization-embryo transfer (IVF-ET), patients with low prognosis, 
especially those with POSEIDON Category 4 (age ≥ 35 years combined with 
diminished ovarian reserve), face problems such as a small number of oocytes, high 
embryo aneuploidy rate, and decreased endometrial receptivity, resulting in 
extremely low treatment success rates [1]. A 2021 real-world study showed that the 
cumulative live birth rate after a single oocyte retrieval in patients with POSEIDON 
Category 4 was only 12.5%, and the live birth rate after a single fresh transfer was 
only 16.7%, which was the lowest among all low-prognosis populations (meeting 
POSEIDON criteria) [2]. Other studies have reached similar conclusions [3, 4]. 
Therefore, how to improve the live birth rate of this type of patients has become an 
urgent problem to be solved in the field of assisted reproduction (ART) in recent 
years [5]. 
Embryo transfer time is a key factor affecting IVF-ET outcomes. For patients with 
POSEIDON Category 4, prolonging in vitro culture to Day 3 may increase embryo 
stress and reduce the number of available embryos [6]; while Day 2 transfer can 
reduce the time of embryo exposure in vitro, but limits the screening of embryo 

developmental potential. Both transfer strategies have their advantages and 
disadvantages, and it is worth exploring which time to choose for transfer in low-
prognosis patients. In addition, a recent RCT on low-prognosis patients [7] confirmed 
that fresh embryo transfer is superior to the full embryo cryopreservation strategy 
(but did not explore the difference between Day 2 and Day 3 transfer). Therefore, 
comparing the live birth rates of Day 2 fresh transfer and Day 3 fresh transfer 
strategies in patients with POSEIDON Category 4 is helpful to improve the IVF-ET 
outcomes of this type of patients. 
In 2006, Bahceci et al. [8] conducted a randomized controlled trial in poor responders 
(defined as ≤ 5 follicles ≥ 13 mm) and found that the clinical pregnancy rate (38.9% 
vs 24.1%) and ongoing pregnancy rate (29.0% vs 18.3%) of Day 2 transfer were 
significantly higher than those of Day 3 transfer. However, this study did not adopt the 
latest POSEIDON criteria (released in 2016), had high population heterogeneity, and 
did not report the final outcome of ART - live birth rate. In 2018, Sacha et al. 
retrospectively analyzed the outcomes of Day 2 and Day 3 fresh transfer in patients 
with ≤ 2 normal fertilized eggs and found no statistically significant difference in 
clinical pregnancy rate between the two groups. However, the endpoint indicator of 
this study also did not use live birth rate, and the baseline data of the two groups 
were unbalanced without controlling for confounding variables, resulting in low 
comparability of outcome variables [9]. Since then, a 2024 retrospective study 
observed the outcomes of Day 2 and Day 3 fresh transfer in patients with only one 
normal fertilized egg and found no statistically significant difference in live birth rate 
between the two groups [10]. However, more than 50% of the patients in this study 
were young women under 35 years old (with better prognosis than patients with 
POSEIDON Category 4), and no subgroup analysis for those over 35 years old was 
performed. Considering that both studies are small-sample retrospective analyses, 
their reference value is limited. Therefore, there is currently no RCT study targeting 
this high-risk subgroup, and there is a lack of high-quality evidence-based medical 
evidence, leading to no unified standard for the choice of transfer time in clinical 
practice. 
This study intends to take patients with POSEIDON Category 4 as the research 
objects and carry out a prospective randomized controlled study to compare the live 
birth rates of Day 2 fresh transfer and Day 3 fresh transfer, in order to provide a basis 
for the choice of transfer time for low-prognosis patients, thereby improving the 
success rate of this type of patients. 
III. Trial Objectives 
3.1 Primary Objective 
To compare the live birth rate of Day 2 (D2) vs. Day 3 (D3) fresh embryo transfer in 
patients with POSEIDON Category 4. 
3.2 Secondary Objectives 

Secondary outcomes include comparing the clinical pregnancy rate, ongoing 
pregnancy rate, implantation rate, embryo utilization rate (number of available 
embryos / number of fertilized embryos), rate of no available embryos, miscarriage 
rate, ectopic pregnancy rate, incidence of moderate/severe ovarian hyperstimulation 
syndrome (OHSS), and incidence of adverse neonatal outcomes (preterm birth rate, 
low birth weight rate, congenital malformation rate) between the two groups. 
IV. Trial Methods 
4.1 Overall Trial Design 
This study is a single-center prospective study. The trial follows the 2025 SPIRIT 
Guidelines [11] and the 2025 CONSORT Guidelines [12], and adopts a parallel 
randomized controlled design to compare the live birth rates of Day 2 fresh transfer 
and Day 3 fresh transfer, in order to provide a basis for the choice of transfer time for 
low-prognosis patients, thereby improving the success rate of this type of patients. 
Eligible patients with POSEIDON Category 4 (first or second IVF/ICSI cycle) are 
randomly assigned to the Day 2 or Day 3 fresh embryo transfer group at a ratio of 1:1, 
using stratified block randomization (variable block size of 4 or 6) by age (< 40 years, 
≥ 40 years). Patient enrollment will last for 18 months, with on-site monitoring every 6 
months to verify data integrity. The DMC reviews adverse event (AE) reports every 
quarter, and the entire study is expected to be completed within 36 months. 
4.2 Sample Size 
Based on the following assumptions: 
• 
Live birth rate in the Day 3 group: 10% (prognostic characteristics of patients 
with POSEIDON Category 4), live birth rate in the Day 2 group: 20% (a 10% absolute 
difference is set as clinically significant). 
• 
Power: 90%, α = 0.05 (two-sided test), loss to follow-up rate: 10%. 
• 
Calculated by PASS 16.0 software, 470 patients need to be included (235 cases 
in each group). 
4.3 Randomization 
1. 
Sequence Generation: SAS 9.4 software is used to generate the random 
allocation sequence, stratified by age (< 40 years, ≥ 40 years), and variable block 
sizes (4 or 6) are used to avoid allocation prediction. 
2. 
Implementation Process: The coordinator informs the patient, clinician and 
embryologist of the grouping result, and all three are informed of the grouping 
information on the day of oocyte retrieval (to facilitate the arrangement of embryo 
culture and transfer time). 

4.4 Control and Blinding 
4.4.1 Control Selection 
Allocation concealment is achieved through a third-party hosted central 
randomization system. On the day of oocyte retrieval, the research center 
coordinator logs into the system and enters the patient's information (age) to obtain 
the grouping result (Day 2/Day 3 transfer). 
4.4.2 Blinding Implementation 
An open-label design is adopted. The patient, clinician and embryologist are all 
aware of the grouping information to manage the embryo culture time and arrange 
the transfer surgery (unblinded). The outcome assessor (independent obstetrician 
and gynecologist) is unaware of the grouping throughout the process to ensure the 
objectivity of outcome judgment. 
4.5 Follow-up Time Points 
• 
Research Design Phase: Collect opinions through offline seminars (6 patients 
with POSEIDON Category 4, 2 public representatives) to optimize the follow-up 
process (add online follow-up options) and the expression of the informed consent 
form (simplify professional terms). 
• 
Research Implementation Phase: Feedback the research progress (such as the 
number of recruited patients, occurrence of serious adverse events, etc.) to patient 
representatives every 6 months and answer patients' questions. 
• 
After the Research: Present the research results to participants and the public in 
the form of a plain language graphic report, explaining the clinical significance. 
Patient enrollment will last for 18 months, and it is expected to be completed from 
March 2026 to September 2027. Follow-up is carried out in stages according to 
"follow-up on the day of transfer - follow-up after transfer - follow-up during 
pregnancy - follow-up after delivery". 
Follow-up Phase 
Follow-up Time Point 
Follow-up Examinations 
Follow-up on the Day of 
Transfer 
Day of transfer 
Number of embryos, 
embryo grade, number of 
transferred embryos 
Post-transfer Follow-up 1 
12-15 days after embryo 
transfer 
Blood β-hCG test to 
determine biochemical 
pregnancy 
Post-transfer Follow-up 2 
28 days after embryo 
Transvaginal ultrasound 

transfer 
to determine pregnancy 
type (clinical pregnancy 
(intrauterine gestational 
sac)/ectopic pregnancy, 
number of gestational 
sacs) 
Pregnancy Follow-up 1 
12 weeks ± 7 days of 
gestation 
Ultrasound to confirm 
intrauterine live fetus and 
number (ongoing 
pregnancy/early 
miscarriage) 
Pregnancy Follow-up 2 
28 weeks ± 7 days of 
gestation 
Ultrasound to confirm 
intrauterine live fetus and 
number (ongoing 
pregnancy/mid-trimester 
miscarriage) 
Post-delivery Follow-up 
1 month ± 7 days after 
delivery 
Delivery mode, 
gestational age, neonatal 
vital signs and health 
status, birth weight, 
obstetric complications, 
pregnancy complications 
(live birth outcome, 
neonatal outcome) 
V. Selection, Withdrawal and Management of 
Subjects 
5.1 Diagnostic Criteria 
Eligible patients with POSEIDON Category 4 (first or second IVF/ICSI cycle) who 
plan to use Day 2 or Day 3 fresh embryo transfer. 
5.2 Inclusion Criteria 
1. 
Meeting the definition of POSEIDON Category 4: age ≥ 35 years, and antral 
follicle count (AFC) < 5 or anti-Müllerian hormone (AMH) < 1.2 ng/mL [1]. 
2. 
First or second IVF/ICSI cycle. 
3. 
Controlled ovarian hyperstimulation (COH) using gonadotropin-releasing 
hormone (GnRH) antagonist protocol, with ≥ 1 oocyte retrieved. 

4. 
Signed written informed consent form. 
5.3 Exclusion Criteria 
1. 
Use of donor oocytes/sperm, or planned preimplantation genetic testing (PGT). 
2. 
Severe immune and chromosomal abnormalities. 
3. 
Uterine cavity abnormalities (i.e., submucosal fibroids, complete uterine septum, 
severe intrauterine adhesions, etc.) or untreated and ultrasound-visible hydrosalpinx. 
4. 
Complicated with severe underlying diseases (such as uncontrolled 
hypertension/diabetes, active malignant tumors). 
5. 
A history of repeated implantation failure (≥ 2 cycles) or recurrent miscarriage (≥ 
2 times). 
6. 
Progesterone level > 1.5 ng/mL on the day of human chorionic gonadotropin 
(hCG) trigger. 
5.4 Exclusion and Withdrawal Criteria 
5.4.1 Exclusion Criteria 
1. 
Violation of important inclusion criteria; 
2. 
Subjects did not receive trial treatment; 
3. 
No observation data after randomization. 
5.4.2 Withdrawal Criteria 
1. 
During the trial, the subject developed certain comorbidities, complications or 
special physiological changes that made it inappropriate to continue receiving the 
trial; 
2. 
Voluntary withdrawal by the subject. 
5.4.3 Data Handling of Withdrawn Subjects 
Regardless of the reason, the complete clinical data of subjects who withdraw from 
the trial shall be retained. For all withdrawn subjects, the trial conclusion form and the 
reason for withdrawal shall be filled in the case report form. Generally, there are 6 
reasons, namely occurrence of adverse events (including adverse drug reactions and 
allergic reactions), lack of efficacy (disease progression or occurrence of 
complications), violation of the trial protocol (including poor compliance), loss to 
follow-up (including voluntary withdrawal of the patient from the trial), termination by 
the sponsor, or others. 
5.5 Conditions for Terminating the Trial 

1. 
The clinical trial is completely stopped midway before the protocol is completed. 
2. 
If serious safety issues occur during the trial, it shall be terminated in a timely 
manner; 
3. 
If major mistakes in the trial protocol formulation or major deviations in 
implementation are found during the trial, making it difficult to evaluate the drug effect; 
4. 
Other situations that require termination. 
5.6 Subject Management 
5.6.1 Subject Recruitment Methods 
The identification of research candidates will be carried out in collaboration with 
laboratory and clinical staff. All couples of POSEIDON Category 4 patients planning 
to undergo fresh embryo transfer will be considered for inclusion. Women who agree 
to participate will be required to sign a written informed consent form, and they will 
receive a copy signed by the researcher. Any waste or exclusion due to failure to 
meet the inclusion criteria will be recorded. On the day of treatment, laboratory staff 
will assess the presence of a signed consent form, and if present, randomization will 
be performed. 
5.6.2 Informed Consent Process 
Adequate information shall be provided, and a written informed consent form shall be 
signed. 
VI. Trial Process 
6.1 Introduction of Trial Drugs/Devices 
1. 
In vitro fertilization culture medium (G-IVF™ PLUS, Vitrolife Group); 
2. 
Intracytoplasmic sperm injection (ICSI) operating fluid (PVP™, Vitrolife Group); 
3. 
Embryo culture oil (Ovoil™, Vitrolife Group); 
4. 
Hyaluronidase solution (Hyase™, Vitrolife Group); 
5. 
Embryo transfer catheter (Cook Medical, USA); 
6. 
Hormonal drugs: recombinant human follicle-stimulating hormone (rFSH), 
gonadotropin-releasing hormone agonist/antagonist, human chorionic gonadotropin 
(hCG). 
6.2 Drug/Device Administration Method 
 (1) Ovarian Stimulation, Oocyte Collection and Fertilization 

A personalized ovarian stimulation plan will be formulated according to the patient's 
baseline conditions, combined with ultrasound monitoring of follicular development: 
GnRH antagonist protocol: Gonadotropins (urinary gonadotropin) will be used 
starting from Day 2-4 of the menstrual cycle; when the follicle diameter is ≥ 12mm or 
on Day 6 of stimulation, GnRH antagonist (Ganirelix) will be injected subcutaneously 
until the trigger day. 
When at least 1 dominant follicle reaches a diameter of 18mm, triggering will be 
performed (dual trigger with 0.1mg Triptorelin + 5000 IU HCG in the antagonist 
protocol); oocyte retrieval will be performed under transvaginal ultrasound guidance 
36-38 hours after triggering. 
The collected oocytes will be treated with hyaluronidase solution to remove 
surrounding granulosa cells, and mature oocytes (MII stage) will be selected. The 
fertilization method will be selected according to semen parameters: conventional in 
vitro fertilization will be used for patients with normal semen quality, and oocytes will 
be co-cultured with processed sperm in G-IVF™ PLUS culture medium; ICSI will be 
used for patients with severe oligoasthenoteratozoospermia, and a single sperm will 
be injected into the oocyte cytoplasm under a micromanipulator. Fertilization will be 
observed 16-18 hours after fertilization, and normal fertilization is defined as the 
appearance of two pronuclei (2PN). 
(2) Embryo Culture and Transfer 
Day 2 (D2) Transfer Group: Embryos will be cultured for 44-48 hours after oocyte 
retrieval (D2), and embryos with ≥ 2 cells, grade II or above, and no multinucleation 
will be selected for transfer, with 1-2 embryos transferred; Day 3 (D3) Transfer Group: 
Embryos will be cultured for 68-72 hours after oocyte retrieval (D3), and embryos 
with ≥ 6 cells, grade II or above, and no multinucleation will be selected for transfer, 
with 1-2 embryos transferred. Before transfer, the selected high-quality embryos will 
be transferred to pre-warmed culture medium for equilibration for 10-20 minutes. 
Before transfer, the patient will have a moderately full bladder, and 1-2 embryos (in 
line with Chinese legal regulations) will be slowly transferred to the middle of the 
uterine cavity using a Cook embryo transfer catheter under transvaginal ultrasound 
guidance. During the transfer process, avoid the catheter touching the uterine wall to 
ensure the embryos are placed stably. After transfer, the patient will rest in bed for 
20-30 minutes, then receive routine luteal support treatment (such as oral or vaginal 
progesterone administration), and be informed of post-operative precautions and 
follow-up time. 
6.3 Treatment Course and Follow-up Points 
• 
Research Design Phase: Collect opinions through offline seminars (6 
POSEIDON Category 4 patients, 2 public representatives) to optimize the follow-up 
process (add online follow-up options) and the expression of the informed consent 
form (simplify professional terms). 
• 
Research Implementation Phase: Feedback the research progress (such as the 

number of recruited patients, occurrence of serious adverse events, etc.) to patient 
representatives every 6 months and answer patients' questions. 
• 
After the Research: After the 1-month follow-up of the last patient's delivery, de-
identified individual participant data (including data dictionary), statistical codes and 
original embryo evaluation records (anonymized) will be stored in MedPro. 
Researchers can submit an application to the Data Access Committee, stating the 
research purpose and data use scope, and obtain the data after approval. The 
access authority will be open for 1 year after the research results are published, and 
the data will be retained for 10 years. 
Patient enrollment will last for 18 months, and it is expected to be completed from 
March 2026 to September 2027. Follow-up will be carried out in stages according to 
"follow-up on the day of transfer - follow-up after transfer - follow-up during 
pregnancy - follow-up after delivery". On-site monitoring will be conducted every 6 
months to verify data integrity. The entire study is expected to be completed within 36 
months. 
VII. Evaluation Indicators 
7.1 Baseline Indicators 
1. 
Demographic data: Subject's unique identification code, gender, age, BMI, 
duration of infertility, cause of infertility, ovarian reserve status, etc.; 
2. 
General clinical data: Endometrial preparation plan, endometrial thickness, 
hormone level on the day of transfer, comorbid diseases and concurrent medications, 
etc.; 
3. 
Embryological data: Embryo stage, embryo grade, number of embryos, etc. 
7.2 Efficacy Evaluation 
7.2.1 Primary Evaluation Indicator 
The primary outcome of this study will be the live birth rate, defined as the delivery of 
at least one neonate with heartbeat and breathing at ≥ 28 weeks of gestation, 
recorded at the time of delivery. 
7.2.2 Secondary Evaluation Indicators 
Secondary outcomes include comparing the clinical pregnancy rate, ongoing 
pregnancy rate, implantation rate, embryo utilization rate (number of available 
embryos / number of fertilized embryos), miscarriage rate, ectopic pregnancy rate, 
incidence of moderate/severe ovarian hyperstimulation syndrome (OHSS), and 
incidence of adverse neonatal outcomes (preterm birth rate, low birth weight rate, 
congenital malformation rate) between the two groups. 

Outcome 
Indicator 
Definition 
Measurement 
Time 
Live Birth Rate 
Number of live births (delivery of at least 
one neonate with heartbeat and 
breathing at ≥ 28 weeks of gestation) / 
Number of subjects enrolled in the study 
× 100% 
At delivery 
Biochemical 
Pregnancy Rate 
Number of biochemical pregnancies 
(blood HCG ≥ 25 IU/L measured 14/15 
days after transfer) / Number of subjects 
enrolled in the study × 100% 
14 days (D3) or 15 
days (D2) after 
transfer 
Clinical 
Pregnancy Rate 
Number of clinical pregnancies 
(intrauterine gestational sac confirmed 
by ultrasound 28 days after transfer) / 
Number of subjects enrolled in the study 
× 100% 
28 days after 
transfer 
Ongoing 
Pregnancy Rate 
Ongoing pregnancy (intrauterine live 
fetus confirmed by ultrasound at 12 
weeks of gestation) / Number of subjects 
enrolled in the study × 100% 
12 weeks of 
gestation 
Implantation 
Rate 
Number of intrauterine gestational sacs / 
Number of transferred embryos × 100% 
28 days after 
transfer 
Embryo 
Utilization Rate 
Number of embryos available for transfer 
/ Number of fertilized embryos (number 
of 2PN fertilized eggs) × 100% 
Day of transfer 
Rate of No 
Available 
Embryos 
Number of subjects with no available 
embryos / Number of subjects enrolled in 
the study × 100% 
Day of 
transfer/day of 
embryo 
observation 
Miscarriage Rate 
Number of spontaneous pregnancy 
losses before 28 weeks of gestation after 
clinical pregnancy / Number of clinical 
pregnancies × 100% 
≤ 28 weeks of 
gestation 
Incidence of 
Moderate/Severe 
Number of OHSS cases (meeting the 
Royal College of Obstetricians and 
From oocyte 
retrieval to 12 

OHSS 
Gynaecologists (RCOG) criteria: 
abdominal distension, ovarian 
enlargement, electrolyte disturbance, 
etc.) / Total number of oocyte retrieval 
cycles × 100% 
weeks of gestation 
Preterm Birth 
Rate 
Number of deliveries at 28-36⁺⁶ weeks of 
gestation / Total number of deliveries × 
100% 
At delivery 
Low Birth Weight 
Rate 
Number of neonates with birth weight < 
2500g / Total number of neonates × 
100% 
At delivery 
Congenital 
Malformation 
Rate 
Number of neonates with congenital 
malformations (structural or functional 
abnormalities diagnosed according to the 
International Classification of Diseases 
(ICD-10)) / Total number of neonates × 
100% 
1 month after 
delivery 
7.3 Safety Evaluation 
7.3.1 Vital Signs 
Changes in blood pressure, pulse, body temperature, respiration, and heart rate 
before and after treatment. 
7.3.2 Laboratory Examinations 
Laboratory examination results, changes in normal/abnormal status before and after 
the trial, and the relationship between abnormal changes and trial drugs when they 
occur. For those with abnormal examination results after medication, close follow-up 
and observation shall be conducted until they return to normal, stable level or pre-
medication level. 
7.3.3 Adverse Events 
Serious adverse events (such as ectopic pregnancy) shall be reported to the DMC, 
Ethics Committee and sponsor within 24 hours; all adverse events shall be recorded 
in the EDC system. 
VIII. Data Quality Assurance 

8.1 Quality Control and Assurance 
Dual data entry will be adopted, and original data will be verified through medical 
record review. 
8.2 Data Management 
1. 
Data Collection: Electronic Data Capture (EDC) system (such as MedPro) will be 
used to collect baseline data (age, AFC, AMH, etc.), COH data (gonadotropin dose, 
follicle size, etc.), embryo data (fertilization rate, embryo grading, etc.) and outcome 
data (live birth rate, clinical pregnancy rate, etc.). Original data will be verified through 
medical record review. 
2. 
Data Management: Dual data entry, range check and query resolution 
mechanism will be adopted. Data will be stored on an encrypted server, and only 
authorized personnel can access it. 
3. 
Trial Monitoring: On-site monitoring will be conducted every 6 months to verify 
data integrity; the DMC will review Adverse Event (AE) reports every quarter. 
4. 
The handling of personal data will comply with relevant national regulations. 
IX. Statistical Analysis 
9.1 Analysis Datasets 
ITT analysis includes all randomized patients (primary analysis), and missing data is 
handled by multiple imputation (MICE); PP analysis includes patients who comply 
with the protocol (sensitivity analysis). 
9.2 Statistical Methods 
1. 
Continuous Variables: Normally distributed data are expressed as mean ± 
standard deviation, and t-test is used for inter-group comparison; non-normally 
distributed data are expressed as median (interquartile range), and Mann-Whitney U 
test is used for inter-group comparison. 
2. 
Categorical Variables: Expressed as frequency (percentage), and χ² test or 
Fisher's exact test (when expected frequency < 5) is used for inter-group comparison. 
3. 
Subgroup Analysis: Preset subgroup analyses by age (< 40 years and ≥ 40 
years), AMH level (< 0.5 ng/mL and 0.5-1.2 ng/mL), and number of oocytes retrieved 
(< 4 and ≥ 4). 
4. 
Statistical Software: SAS 9.4 and R 4.3.0 software are used, with P < 0.05 (two-
sided) considered statistically significant. 
X. Ethical Requirements and Informed Consent Form 

10.1 Ethics Committee Review 
This protocol, written informed consent form and materials directly related to subjects 
must be submitted to the Ethics Committee, and the research can be officially carried 
out only after obtaining the written approval of the Ethics Committee. The researcher 
must submit an annual research report to the Ethics Committee at least once a year 
(if applicable). When the research is terminated and/or completed, the researcher 
must notify the Ethics Committee in writing; the researcher must promptly report all 
changes that occur during the research work (such as revisions to the protocol and/or 
informed consent form) to the Ethics Committee, and these changes shall not be 
implemented until approved by the Ethics Committee, unless the changes are made 
to eliminate obvious and direct risks to subjects. In such cases, the Ethics Committee 
will be notified. 
10.2 Informed Consent 
The researcher must provide the subject or his/her legal representative with an easy-
to-understand informed consent form approved by the Ethics Committee, and give 
the subject or his/her legal representative sufficient time to consider this study. The 
subject shall not be enrolled until a signed written informed consent form is obtained 
from the subject. During the subject's participation, all updated versions of the 
informed consent form and written information will be provided to the subject. The 
informed consent form shall be retained as an important document of the clinical trial 
for inspection. 
XI. Insurance 
No insurance is required. 
XII. References 
[1] The POSEIDON Group. The POSEIDON criteria: a framework for identifying and 
managing low prognosis patients in IVF. Hum Reprod Open. 2016;2016(1):hrov008. 
[2] Zhang Y, Li J, Wang H, et al. Cumulative live birth rate after a single oocyte 
retrieval in patients with POSEIDON Category 4: a real-world study. J Assist Reprod 
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