Optimal Allogenic Platelet-rich Plasma Concentration to Improves in Vitro Maturation of Germinal Vesicle Oocytes From Women With PCOS: A Prospective Cohort Study
Phase: NA
Conditions: PCOS (Polycystic Ovary Syndrome)
Sponsor: Gadjah Mada University

STUDY PROTOCOL 
Official Title: Optimal Allogenic Platelet-rich Plasma Concentration for Oocyte In Vitro 
Maturation in Women With PCOS  
Unique Protocol ID: PRP-PCOS-COHORT-UGM  
NCT ID: Not yet assigned  
Document Date: January 5, 2023 
Sponsor: Universitas Gadjah Mada 
 
This study was approved by the Medical and Health Research Ethics Committee, Faculty of 
Medicine, Public Health and Nursing, Universitas Gadjah Mada (protocol number: 
KE/FK/0022/EC/2023, released January 5, 2023). A total of 34 germinal vesicle (GV) stage 
oocytes exhibiting normal morphology were retrieved via the ovum pick-up (OPU) procedure from 
15 women diagnosed with PCOS undergoing IVF treatment at Permata Hati Infertility Clinic 
between 2023 and 2024. An initial pilot experiment tested three different concentrations of PRP 
supplementation (5%, 25%, and 50%) using three GV-stage oocytes per group. The concentration 
that provided the highest maturation rate while maintaining the quality and stability of the culture 
medium was selected as optimal. 
Following this, the remaining 28 GV-stage oocytes were randomly allocated into two groups: a 
control group receiving no PRP (0%, n=14) and a treatment group supplemented with PRP at the 
optimal concentration (5%, n=14). Prior to evaluation, cumulus-oocyte complexes (COCs) were 
enzymatically denuded with hyaluronidase (Vitrolife, Sweden) and mechanically cleaned using 
micropipettes (170 µm and 140 µm) to remove cumulus cells, enabling direct assessment of PRP 
effects on oocytes. Each oocyte was then cultured for 24 hours at 37°C in either control media (G-
IVF™) or treatment media containing PRP at one of the three tested concentrations. 
The primary outcome was oocyte maturation, defined by the transition from GV to MII stage, 
determined by the presence of the first polar body after 24 hours. Secondary outcomes included 
total oocyte score (TOS) and fertilization outcomes. TOS is a semi-quantitative morphological 
assessment of oocyte quality, based on six parameters: oocyte shape, size, ooplasm characteristics, 
perivitelline space, zona pellucida thickness, and polar body morphology. Each parameter was 
scored on a standardized three-point scale (+1, 0, −1) according to the system by Lazzaroni-Tealdi 
[14]. Raw TOS data and scoring criteria are available in Mendeley Data. Fertilization was 
evaluated approximately 17 ± 1 hours post-injection, consistent with standard pronuclear 
assessment for ICSI-derived zygotes, with fertilization defined by the presence of two pronuclei 
(2PN) and two polar bodies under microscopy. 
 
 
 

PRP and Culture Media Preparation 
To reduce confounding factors, allogenic PRP was utilized. A healthy female donor aged 20–30 
years, with a history of spontaneous pregnancy and no reproductive disorders, was selected. Blood 
(35 mL) was collected and centrifuged sequentially at 103 × g for 10 minutes and 230 × g for 15 
minutes, yielding approximately 12 mL of PRP, which was aliquoted into 1.5 mL microtubes and 
stored at −80°C until use. PRP preparation followed routine clinical procedures; however, details 
such as platelet and leukocyte counts or growth factor quantification were not recorded. 
Oocyte maturation was conducted in G-IVF PLUS medium (Vitrolife, Gothenburg, Sweden). PRP 
stock solutions were prepared at 5%, 25%, and 50% concentrations. Frozen aliquots were thawed 
at room temperature for 10 minutes prior to mixing with the culture medium, and remaining stocks 
were kept frozen until required. 
Statistical Analysis 
Maturation outcomes were analyzed using Fisher’s exact test in IBM SPSS Statistics version 25 
(IBM Corp., Armonk, NY, USA), with statistical significance defined as p < 0.05. This method 
was chosen due to the small sample size and comparable variance among groups. Because of 
unequal MII oocyte counts across groups, TOS analysis was not performed statistically to avoid 
bias from sample size imbalance; TOS results are presented descriptively.