A Pilot Study of the Effect of RECO-18 Containing Natural Plant Extracts on Infertile Women Undergoing in Vitro Fertilization-embryo Transfer
Phase: NA
Conditions: Fertility Disorders, Infertility, Female
Sponsor: Sun Yat-Sen Memorial Hospital of Sun Yat-Sen University

A Pilot Study of the Effect of RECO-18 Containing Natural Plant 
Extracts on Infertile Women Undergoing in Vitro 
Fertilization-embryo Transfer 
 Clinical Study Protocol 
Sponsor: Sun Yat-sen Memorial Hospital of Sun Yat-sen University  
Version number: 01 
Version date: 2022-3-20 
 
Objectives: 
To investigate the effect of RECO-18 pretreatment on pregnancy outcomes of IVF/ICSI in 
infertile women when compared with oral administration of multi-vitamins. 
Design: 
This study is a prospective, randomized, controlled and superiority clinical trial. 
Participants: 
Eighty infertile patients undergoing IVF/ICSI are randomized to either RECO-18 treatment or 
multi-vitamins with the ratio of 1:1.  
Inclusion criteria 
• female, 20 to 40 years old;  
• the 1st or 2nd cycle of IVF/ICSI treatment;  
• BMI≤30Kg/m2, 4) with bilateral ovaries;  
• be eligible for IVF/ICSI treatment.  
Exclusion criteria  
• repeated implantation failures (with previous 3 or more IVF/ICSI failures);  
• moderate to severe endometriosis;  
• untreated hydrosalpinx;  
• untreated endometrial disease;  
• contraindications for assisted reproductive techniques or gestation;  
• a history of ovarian surgery;  
• expected poor ovarian response (POR) or previous POR;  
• polycystic ovarian syndrome;  
• participants in clinical trials of other drugs within one month prior to enrollment;  
• hypersensitivity to follicle-stimulating hormone-α, FSH, human menopausal gonadotropin, 
LH or excipients;  
• uncontrolled endocrine diseases (such as hyperthyroidism, hypothyroidism, adrenal gland 
disease, obesity, etc.);  

• percutaneous epididymal sperm aspiration or testicular sperm aspiration. 
Research procedure: 
Eighty infertile patients undergoing IVF/ICSI are randomized to either RECO-18 treatment 
(treatment group, n=40) or multi-vitamins (control group, n=40). 
The treatment group begins to take Reco-18 on the 1st to 5th day of menstruation with a dosage 
of 4 pills per day for the whole menstrual cycle, then perform ovulation induction on the second 
menstrual cycle, and continue to take Reco-18 until the day of oocyte retrieval.  
The control group takes the multi-vitamins (Elevit, Bayer S.A.) with the dosage of one tablet 
per day as the same period in the treatment group. 
Primary and secondary outcomes 
The primary outcome is the ongoing pregnancy rate at 12 weeks' gestation;  
The secondary indicators are the number of oocytes retrieved, normal fertilization rate, the high 
quality embryos rate, implantation rate, clinical pregnancy rate, and early miscarriage rate. 
Statistical analysis: 
Data are analyzed using SPSS 19.0 statistical software. Measurement data are expressed as 
mean ± standard deviation, and categorical variables are described as percentages. Data are 
compared using the independent sample t-test or chi-squared test. P<0.05 is considered statistical 
significance. 
Plan of follow-ups: 
 
Screening and 
randomization 
IVF/ICSI treatment 
Follow-up 
V1 
V2 
V3 
V4 
V5 
V6 
 
Before 
IVF/ICSI 
treatment 
IVF/ICSI 
treatment 
cycle 
Thawed 
ET cycle 
2 weeks 
after ET 
4-5 weeks 
after ET 
12 weeks of 
gestation 
Informed consent 
√ 
 
 
 
 
 
Eligibility Criteria 
√ 
 
 
 
 
 
Personal information 
√ 
 
 
 
 
 
History 
√ 
 
 
 
 
 
Companied medication 
√ 
√ 
√ 
√ 
√ 
√ 
Physical examination 
√ 
 
 
 
 
 
Vital signs 
√ 
√ 
√ 
 
 
 
Sex hormones 
√ 
√ 
√ 
 
 
 
Ultrasound 
√ 
√ 
√ 
 
√ 
√ 
Complication and 
adverse events 
 
√ 
√ 
√ 
√ 
√ 
 
 

1. Study protocol summary 
How to improve the fertility of infertile women has become a hot topic in the field of 
reproductive medicine. Animal experiment has shown that RECO-18 significantly improved the 
female fertility in mice, and the specific mechanism was related to reducing follicular atresia, 
promoting follicle development and improving oocyte quality. Therefore we aim to conduct a pilot 
study to explore the effect of RECO-18 in infertile women undergoing in vitro 
fertilization-embryo transfer (IVF-ET). This study is a prospective, randomized, controlled 
clinical study. The treatment group takes RECO-18 while the control group takes the 
multi-vitamins. The primary indicator is the ongoing pregnancy rate at 12 weeks' gestation; the 
secondary indicators are the number of oocytes retrieved, normal fertilization rate, the high quality 
embryos rate, implantation rate, clinical pregnancy rate, and early miscarriage rate. 
2. Research background 
In recent years, the group of infertile women has gradually expanded with the effect of 
environmental pollution, diet and sleeping habits. The decline of female fertility is manifested 
with decreased ovarian reserve and poor oocyte quality. With the increasing education and social 
status improvement in female, more and more women have postponed their childbearing plans. It 
is reported that the average childbearing age of women is nearly 30 years old in our country. 
Moreover, the liberalization of two-child and three-child policy maybe further delays female 
reproductive age. Therefore, how to improve the female fertility has become a social and scientific 
problem in China, and it is urgent to develop new technologies and products to improve the 
fertility of infertile women. 
In female mammals, a follicle is the basic functional unit of the ovary, consisting of an oocyte 
and single or multiple layers of granule cells. According to the different stages of the oocytes, 
there are primordial follicle, primary follicle, secondary follicle, antral follicle, and preovulatory 
follicle. Primordial follicles locate in the resting follicle pool of the ovary with a certain number. 
The number of human primordial follicles in the ovaries at birth is around 400,000, and decreases 
with age. When the number of primordial follicles decreases to less than 1000, a female enters the 
menopause at about 51 years old with disabled fecundity [1, 2]. The optimal age for childbearing 
is around 25 years old, and ovarian function begins to decline in 30 years old. After the age of 35, 
ovarian reserve decreases dramatically with the acceleration of primordial follicle activation, 
meanwhile oocyte quality is declining with the manifestation of high proportion of mature oocyte 
aneuploidy, low fertilization rate and embryo implantation rate, and high miscarriage rate. Ageing 
woman is suggested to consult in assisted reproductive center if she has prepared for pregnancy 
over 6 months but fails in conception. However, it is noteworthy that the success probability of 
assisted reproduction technology (ART) in aging woman is relative low, because traditional 
ovarian stimulation is not very effective. Oocyte donation is the effective treatment for aging 
woman with low fecundity or even infertility, but most women want to have their own babies, and 
hardly accept it in the mind. Personalized ovarian stimulation protocol and preimplantation 

genetic testing are used as clinical intervention, but the treatments increase the costs, and low 
chance of pregnancy still exists. It is closely related to poor oocyte quality caused by ovarian 
aging. Therefore, how to protect ovarian reserve and improve oocyte quality is the key to improve 
female fertility [3-5]. 
Granulosa cells differentiate into mural granulosa cells and cumulus cells at the stage of the 
secondary follicle. The cumulus cells are connected to the oocyte by gap junctions, and gradually 
separate from the mural granulosa cells to form follicular cavity. The follicular cavity is filled with 
follicular fluid composed of plasma exudate and local secretions, which constitutes the internal 
environment of oocyte development and plays an important regulatory role in follicular 
development, maturation, ovulation and atresia. In the process of ovarian aging, the ovarian 
chronic inflammatory environment and angiogenesis abnormality around the follicle increase the 
oxidative stress level, decrease mitochondrial vitality, and increase cell apoptosis level, which lead 
to abnormal granulosa cell function and follicular microenvironment changes, and finally oocyte 
is damaged with poor quality. Human preovulatory follicular fluid contains a large number of 
antioxidant enzymes, such as CAT, GSSPx, GST, etc, which mainly prevent the damage of 
oxidative stress to oocytes. The decreased level of GST and CAT and increased SOD activity are 
observed in the follicular fluid of aging woman. The decrease in the CAT / SOD ratio indicates a 
diminished scavenging ability of activating oxide in the follicle [6, 7]. Due to the important role of 
oxidative stress in ovarian aging, a large number of studies have shown that the application of 
antioxidants such as Vitamin C, Vitamin E and CoQ 10 effectively improve oxidative stress levels 
in oocytes, restore mitochondrial function and improve oocyte quality [8-10]. In 2021, consensus 
on reproductive health and multiple micronutrient in China recommends that infertile woman 
takes multiple micronutrient containing folic acid of 0.8mg before ART or on the day of embryo 
transfer to reduce the risk of neural tube defects. However, the adjuvant drugs have not been fully 
confirmed in evidence-based medicine clinical research, and have not been routinely 
recommended to aging woman in the guideline. 
Mitochondrial function is closely related to oocyte quality, and the effects may be more 
pronounced in infertile woman. The lack of multiple micronutrients is more common in infertile 
woman, and the supplementation of multiple micronutrients has an antioxidant effect, which 
reduce the damage of oxidative stress to fertility and help to improve the outcomes of ART. 
RECO-18 is a functional food containing a variety of plant extracts (sophora flower bud, clove, 
yam) and enzymatic soybean phospholipids, and its specific nutrients are list in Table 1. In 
previous in vivo and in vitro senescent models, RECO-18 was found to improve oocyte quality 
mainly by regulating the mitochondrial apoptosis pathway. Animal experiment has shown that 
RECO-18 significantly improved the female fertility in mice (Figure 1, Figure2, Figure 3), and the 
specific mechanism was related to reducing follicular atresia, promoting follicle development and 
improving oocyte quality. Therefore we aim to conduct a pilot study to explore whether RECO-18 

plays a role in improving oocyte and embryo quality and pregnancy outcomes in infertile women 
undergoing IVF-ET. 
Table 1. Nutrients of RECO-18  
Project 
Per 100g  
Energy 
1860k.J 
Protein 
20.2g 
Fat 
14.9g 
Carbohydrate 
56.8g 
Sodium 
334mg 
Vitamin A 
14072μgRE 
Vitamin D 
245.0μg 
Vitamin B1 
57.00mg 
Vitamin B2 
35.70mg 
Vitamin B6 
47.00mg 
Vitamin B12 
64.20μg 
Vitamin C 
2801.0mg 
Niacin 
347.00mg 
Folic acid 
9527μg 
Iron 
447.0mg 
Zinc 
202.00mg 
 

 
Figure 1: RECO-18 improved oocytes quality in in vitro and in vivo senescent models. A and C, 
RECO-18 improved mitochondrial distribution in in vitro aging COC; B and D, RECO-18 
reduced ROS level in in vitro aging COC; E and G, RECO-18 reduced TUNEL fluorescence 
signal in in vivo aging COC ; F and H, RECO-18 reduced ROS level of oocytes in in vivo aging 
COC. 
 

 
 
Figure 2: RECO-18 improved the aging oocyte quality mainly by improve the mitochondrial 
apoptosis pathway of cumulus cells. A and B, Western blot showed that RECO-18 improved the 
apoptosis pathway in aging COC; C, RECO-18 improved the mitochondrial distribution in aging 
COC; and D and E, RECO-18 increased the cardiolipin level in aging COC; F and G, RECO-18 
reduced cytochrome C level in aging COC. 

 
Figure 3: RECO-18 improved oocyte quality and fertility in aging mice. A, RECO-18 increased 
pregnancy rate of aging mice; B, RECO-18 increased litter size of ageing mice; C, RECO-18 
increased the weaning weight of offspring in mice; D, RECO18 increased the ovulation of aging 
mice; E, RECO-18 reduced the fragmentation rate of oocyte in aging mice during ovarian 
stimulation; F and G, RECO-18 improved the mitochondrial membrane potential of oocyte in 
aging mice. 
3. Research purpose 
To investigate the effect of RECO-18 pretreatment on pregnancy outcomes of IVF-ET in 
infertile women when compared with oral administration of multi-vitamins. 
4. Research design 
This study is a prospective, randomized, controlled and superiority clinical trial. Subjects will 
be recruited from the infertile patients undergoing IVF/ICSI at the Reproductive Medicine Center 
of Sun Yat-sen Memorial Hospital, Sun Yat-sen University. 
The treatment group begins to take Reco-18 on the 1st to 5th day of menstruation with a dosage 
of 4 pills per day for the whole menstrual cycle, then perform ovulation induction on the second 
menstrual cycle, and continue to take Reco-18 until the day of oocyte retrieval. The control group 

takes the multi-vitamins (Elevit, Bayer S.A.) with the dosage of one tablet per day as the same 
period in the treatment group. 
5. Research protocol  
5.1 Sample size 
At first it is designed to include 80 infertile patients (40 cases in each group), then the sample 
size is calculated based on the preliminary study results.  
5.2 Inclusion criteria 
• female, 20 to 40 years old;  
• the 1st or 2nd cycle of IVF/ICSI treatment;  
• BMI≤30Kg/m2, 4) with bilateral ovaries;  
• be eligible for IVF/ICSI treatment.  
5.3 Exclusion criteria  
• repeated implantation failures (with previous 3 or more IVF/ICSI failures);  
• moderate to severe endometriosis;  
• untreated hydrosalpinx;  
• untreated endometrial disease;  
• contraindications for assisted reproductive techniques or gestation;  
• a history of ovarian surgery;  
• expected poor ovarian response (POR) or previous POR;  
• polycystic ovarian syndrome;  
• participants in clinical trials of other drugs within one month prior to enrollment;  
• hypersensitivity to follicle-stimulating hormone-α, FSH, human menopausal gonadotropin, 
LH or excipients;  
• uncontrolled endocrine diseases (such as hyperthyroidism, hypothyroidism, adrenal gland 
disease, obesity, etc.);  
• percutaneous epididymal sperm aspiration or testicular sperm aspiration. 
5.4 Exit criteria 
1) Patients request to drop out; 2) the cycle is cancelled due to low ovarian response or 
premature ovulation; 3) actual treatment deviates from the protocol; 4) patients lost follow-up. 
5.5 The measurement of research results 
Primary indicator: the ongoing pregnancy rate at 12 weeks' gestation.  
Secondary indicators: the number of oocytes retrieved, the normal fertilization rate and the rate 
of high quality embryos, implantation rate, clinical pregnancy rate, and early miscarriage rate. 
Randomization: randomized envelope method is used in the study. Random sequence is firstly 
generated by an independent statistician, secondly the grouping is randomized according to 

random number and put into an opaque sealed envelope by the independent personnel, at last the 
investigator divides the subjects in line with the sealed envelope. 
Random ratio: 1:1 
Grouping: the treatment group takes RECO-18 while the control group takes the 
multi-vitamins. 
5.6 Informed consent  
Before the initiation of study, the informed consent form (ICF) should be used to inform the 
potential subjects of benefits and risks in simple language. ICF should specify that the informed 
consent is signed voluntarily, and the potential benefits and risks associated with study are 
specified, and subjects may drop out from the study at any time. 
5.7 Ethics 
The study process and informed consent acquisition should comply with Helsinki Declaration. 
This study has been approved by the Medicine Ethics Committee of Sun Yat-sen Memorial 
Hospital of Sun Yat-sen University. Every subject should sign the informed consent. The 
informed consent copy, and contact information of the investigator and Medicine Ethics 
Committee must be provided to the subjects. 
5.8 Research method 
5.8.1 Collection of basal data: age, infertility legnth, weight, and body mass index (BMI). 
5.8.2 Collection of basal endocrine indexes: follicle stimulating hormone (FSH), luteinizing 
hormone (LH), estradiol, testosterone, dehydroepiandrosterone sulfate (DHEAS), sex hormone 
binding globulin (SHBG), and anti-müllerian  hormone (AMH). 
5.8.3 Collection of biochemical indexes: alanine aminotransferase (ALT), aspertate 
aminotransferase (AST), creatinine, total cholesterol (TC), triglycerides (TG) , high-density 
lipoprotein (HDL), low-density lipoprotein (LDL), apolipoproteins B (ApoB), high-sensitivity 
C-reactive protein (Hs-CRP), fasting glucose, and fasting insulin. 
5.8.4 Ultrasound examination: bilateral ovarian size, follicle number, and antral follicle count 
(AFC);  
5.8.5 Controlled ovarian stimulation and IVF/ICSI: 
a. Controlled ovarian stimulation 
Gonadotropin-releasing hormone (GnRH) antagonist protocol: gonadotropin (Gn) 100 IU to 
250 IU/day is individually administered to stimulate follicular growth on the 2nd to 4th day of 
menstruation. GnRH antagonist is added when dominant follicle reaches ≥14mm in diameter and 
LH level is ≥10IU/L, and is stopped until the day of human chorionic gonadotropin (HCG) 
administration.   
GnRH agonist long protocol: in the midluteal phase of the menstrual cycle confirmed by 
ultrasound or hormone test or after 2 weeks of oral contraceptive use, pituitary downregulation is 
achieved by injection of 0.93-1.87mg long-acting GnRH agonist. 14 to 21 days later, vaginal 
ultrasound and blood hormone tests are performed to evaluate the complete downregulation of the 

pituitary gland. Ovarian stimulation is initiated with Gn 100U-250U/day, and adjustment of Gn 
dosage is performed according to serum hormone level and follicle growth.  
b. Trigger: recombinant HCG of 250ug is administered to induce oocyte maturation when at 
least two follicle ≥18mm or three follicles ≥17mm or four follicles ≥16mm in diameter.  
c. Oocyte retrieval, luteal support and embryo transfer: oocyte recovery is performed 34 to 38 
hour after the trigger. Luteal support is provided on the day of oocyte retrieval, with vaginal 
micronized progesterone capsules (600mg/day) combined with oral dydrogesterone (30mg/day) 
until the pregnancy test. With the use of conventional IVF or ICSI fertilization, 1 to 2 cleavage 
stage embryos or day 5 blastocysts are transferred to the uterus, and the rest available embryos are 
cryopreserved. 
d. If fresh embryo transfer is cancelled due to medical reasons, pregnancy outcome of the first 
thawed embryo transfer is collected. The endometrial preparation, thawed embryo transfer and 
luteal support are based on the routine operation. 
5.8.6 Collection of discarded sample: 
a. Follicular fluid is collected during oocyte retrieval. 
b .The cumulus oocyte complex (COCs) is put into hyaluronidase by the digestion for 10 to 30 
seconds to remove the cumulus cells, and the oocytes are picked up and cumulus cells are 
collected. 
5.8.7 Follow-up: 
A total of three visits are followed up during the treatment procedure. 
a. At the 14th day after embryo transfer: to record urinary pregnancy test, serum HCG and 
progesterone level. 
If pregnancy test is positive, the following follow-up continues, otherwise it discontinues. 
b. Four to five weeks after embryo transfer: to perform transvaginal ultrasound. Clinical 
pregnancy is defined as a gestational sac and fetal heart beat detected on ultrasound. 
c. At 12 weeks' gestation: to perform ultrasound. Ongoing pregnancy is defined as live fetuses 
detected on ultrasound. 
6. Safety evaluation 
6.1 Adverse events: Any adverse medical events occurring during the study period are defined 
as adverse events, such as drug reactions, adverse reactions associated with ovarian stimulation, 
complications associated with pregnancy, and other discomfort complaints 
6.2 Adverse events report: 
Mild adverse events are recorded in the progress note. 
Serious adverse events are submitted to Medicine Ethics Committee within 48 hours of 
occurrence. 
6.3 Safety indexes are adverse events occurring in the treatment group and the control group, 
such as gastrointestinal reactions and allergies. 
7. Data collection and management 

The data are collected and managed by the study team members, and personal information 
involving subject privacy will not appear in the results except for necessary. It should be applied 
to view subject information in accordance with the rules and regulations of department. The 
information is only open to the team members, and strictly forbidden to share with irrelevant 
personnel. 
Data are analyzed using SPSS 19.0 statistical software. Measurement data are expressed as 
mean ± standard deviation, and categorical variables are described as percentages. Data are 
compared using the independent sample t-test or chi-squared test. P<0.05 is considered statistical 
significance. 
7.1 Data Analysis Set: 
Full Analysis Set (FAS): the FAS include all randomized subjects receiving treatment at least 
once. According to the intent-to-treat population (ITT) principle, all randomized cases accepting 
intervention at least once should be analyzed. 
Safety Set (SS): all randomized subjects accepting intervention at least once and receiving 
post-treatment safety evaluation at least once should be included in the SS. SS is the safety 
evaluation population for this trial. 
Per-protocol population set (PPS): all subjects in the FAS without major protocol violation will 
be included. Major protocol violation should be defined in the statistical analysis plan before 
database locking and study unblinding. Subjects will be analyzed based on the treatment. 
7.2 Analysis of primary indicator and secondary indicators: 
Analysis of primary indicator: the two-sample rate test is used to compare the ongoing 
pregnancy rate at 12 weeks' gestation in the treatment group and the control group. Categorical 
variable is compared between groups using Pearson chi-squared test or Fisher's exact probabilistic 
method. Based on the FAS, the primary endpoint calculates the ongoing pregnancy rate at 12 
weeks' gestation and its 95% confidence interval. Logistic regression is used to analyze the 
baseline effects, and the adjusted factors included age, infertility length, BMI, AMH, AFC, 
ovulation stimulation protocol, etc. 
Analysis of secondary indicators: measurement data are compared using independent sample 
test or nonparametric analysis, and categorical variables are compared using Pearson chi-squared 
test or Fisher's exact probabilistic method between groups. Measurement data are analyzed by 
covariance analysis, and qualitative indicators are analyzed by Logistic regression to evaluate the 
baseline effects. Adjusted factors refer to the analysis of primary indicators. 
7.3 Safety indicators 
Use descriptive statistics to summarize laboratory examination and vital signs as safety 
evaluation indicators, and summarize the changes of each index relative to the baseline. The 
indexes are classified as normal or abnormal (without clinical significance), and calculated the 
case number and percentage in each classification. Other safety indicators are summarized or 
listed as appropriate. 

7.4 Data management and quality control 
7.4.1 Database establishment  
The Epidata electronic database is established based on the case report form before the 
initiation of study. 
7.4.2 Data filling and review 
Case report forms should be filled by designated, trained researchers with uniform regulation. 
Project principal assigns the researcher to review the completeness and accuracy of the case report   
form, and guide the researcher to make any necessary corrections or additions. When the data are 
questionable, fill in the data query form, and the researcher should respond the question and 
confirm the correction. Keep the signed data query form. 
7.4.3 Data entry and verification 
In order to ensure the accuracy of the data, two data administrators check the data 
independently. For any questions in the case report form, the data administrators generate the 
question answer form (DRQ) and send it to the researcher through clinical research associate. The 
researchers recheck the data and reply in time, and the data administrators revise and input the 
data accordingly. 
7.4.4 Data audit and database locking 
After all questions of the data in the system are resolved, the principal investigator, statistician 
and data administrator first review the data and determine the association of adverse events and 
study, then determine the analyzed data set, last lock the audited data without any change. If there 
is any question after the database lock, it should be corrected in the statistical analysis process, 
and be recorded in detail. 
8. Quality Management  
8.1 The research team has rich clinical experience and has received GCP training in order to 
conform to the protocol and ethical requirements 
8.2 Animal experiments have shown that RECO18 significantly improved the fertility of mice, 
which sets the foundation for clinical research. 
8.3 Reco-18 is a food for special dietary use based upon "National Food Safety Standard and 
Nutritional Supplementary Food for Pregnant and Lactating Women” (GB31601-2015). It 
contains multiple micronutrients (vitamins and minerals) and active constituent of Reco-18, and 
has passed through food inspection by a third party. Production license number is 
SC11337082906741. 
9. Pre-assessment of risks and benefits and risk control preplan  
9.1 Potential benefits 
9.1.1 Subjects may get more opportunities for pregnancy with the participation in the study. 
Meanwhile the study may provide indirect benefit for other patients and generate social benefits 
9.1.2 Researchers may develop an adjuvant therapy to improve the fertility of infertility patients, 
so as to benefit the majority of patients. 

9.1.3 Medical institution may provide efficient service for patients, and raise the visibility. 
9.2 Potential risks 
9.2.1 For researchers and medical institution, the study may not meet the expectation and 
achieve the research purpose, resulting in the waste of medical resources. 
9.2.2 For the patients, the fertility of the subjects may not be ultimately improved.  
9.3 Risk control measures 
9.3.1 Subjects are fully informed and sign informed consent before participation. 
9.3.2 If serious adverse events occur, stop the study immediately and report to the department 
and Medicine Ethics Committee in the first time, and control the impact of adverse events to a 
minimum. 
10. References 
1. Li J, Kawamura K, Cheng Y, Liu S, Klein C, Liu S, Duan EK, Hsueh AJ. (2010) Activation 
of dormant ovarian follicles to generate mature eggs. Proc Natl Acad Sci U S A. 107, 
10280-10284. 
2. Dou X, Sun Y, Li J, Zhang J, Hao D, Liu W, Wu R, Kong F, Peng X,Li J. (2017) Short-term 
rapamycin treatment increases ovarian lifespan in young and middle-aged female mice. Aging 
Cell. 16, 825-836. 
3. Li Q, Geng X, Zheng W, Tang J, Xu B, Shi Q. (2012) Current understanding of ovarian 
aging. Sci China Life Sci, 55, 659-669. 
4. Broekmans FJ, Soules MR, Fauser BC. (2009) Ovarian aging: mechanisms and clinical 
consequences. Endocr Rev. 30, 465-493. 
5. Tatone C, Amicarelli F, Carbone MC, Monteleone P, Caserta D, Marci R, Artini PG, 
Piomboni P, Focarelli R. (2008) Cellular and molecular aspects of ovarian follicle ageing. Hum 
Reprod Update. 14, 131-142. 
6. Tarin JJ, Perez-Albala S, Cano A. (2002) Oral antioxidants counteract the negative effects of 
female aging on oocyte quantity and quality in the mouse. Mol Reprod Dev.61, 385-397.  
7. Wang L, Tang J, Wang L, Tan F, Song H, Zhou J, Li F. (2021) Oxidative stress in oocyte 
aging and female reproduction. J Cell Physiol. 236, 7966-7983. 
8. Özcan P, Fıçıcıoğlu C, Kizilkale O, Yesiladali M, Tok OE, Ozkan F, Esrefoglu M. (2016) 
Can Coenzyme Q10 supplementation protect the ovarian reserve against oxidative damage? J 
Assist Reprod Genet. 33, 1223-1230. 
9. Homer HA. (2021) Understanding oocyte ageing: can we influence the process as clinicians? 
Curr Opin Obstet Gynecol. 33, 218-224. 
10. Gennari Verruma C, Credendio Eiras M, Fernandes A, Vila RA, Libardi Miranda Furtado C, 
Silveira Ramos E, Barbosa Lôbo R. (2021) Folic acid supplementa tion during oocytes maturation 
influences in vitro production and gene expression of bovine embryos. Zygote. 29, 342-349. 

11. Reproductive Endocrinology Committee of China Medical Education Association. (2021) 
Consensus on Reproductive health and multiple micronutrient in China. Chinese Journal of 
Practical Gynecology and Obstetrics.37,453-456.