Conditioned Media and Extracellular Vesicles Derived from Human Wharton's Jelly Mesenchymal Stem Cells Improve the in vitro Maturation of Immature Oocytes in Normal and PCOS Mouse Model

doi:10.21203/rs.3.rs-7410176/v1

Download PDF

Research Article

Conditioned Media and Extracellular Vesicles Derived from Human Wharton's Jelly Mesenchymal Stem Cells Improve the in vitro Maturation of Immature Oocytes in Normal and PCOS Mouse Model

https://doi.org/10.21203/rs.3.rs-7410176/v1

This work is licensed under a CC BY 4.0 License

Journal Publication

published 27 Dec, 2025

Read the published version in BioMedical Engineering OnLine →

You are reading this latest preprint version

Background

The effects of conditioned medium (CM) and extracellular Vesicles (EVs) derived from human Wharton’s jelly mesenchymal stem cells (hWJMSCs) on in vitro maturation (IVM) of immature oocytes in both normal and polycystic ovary syndrome (PCOS)-induced mice were investigated. PCOS was induced in adult female NMRI mice by administering letrozole (90 µg/kg/day) via gavage for one week. Germinal vesicle (GV) oocytes were collected from both PCOS-induced and normal mice, while mature oocytes (MII) were obtained from superovulated normal mice to serve as controls. The experimental groups included 7 groups: Control (MII oocytes), 3 IVM groups (In vitro maturation of GV oocytes): IVM (with simple IVM media), IVM + CM and IVM + EVs (IVM media supplemented with CM and EVs, respectively), and three PCOS groups (In vitro maturation of GV oocytes from PCOS-induced mice): PCOS IVM (with simple IVM media), PCOS IVM + CM and PCOS IVM + EVs (IVM media supplemented with CM and EVs, respectively). After IVM was conducted in all groups, mature oocytes were harvested and assessed for maturation rate, morphology, viability, and gene expression profiles of key regulators (CDK1, CCNB1, MAP2K). Developmentally competent oocytes were selected using Brilliant Cresyl Blue staining and then subjected to in vitro maturation with or without CM or EVs supplementation. Nuclear maturation was evaluated via orcein staining, while viability was assessed using Trypan Blue. Morphometric parameters were measured using ImageJ software. Real-time PCR was utilized for the evaluation of gene expression of targeted genes.

Results

Results demonstrated that in BCB + oocytes, CM and EVs significantly improved mature oocytes compared to IVM (P < 0.05). Oocytes from PCOS-induced mice exhibited reduced maturation and increased degeneration, which were rescued by CM and EV treatment. Gene expression analysis revealed downregulation of MAP2K, CCNB1, and CDK1 in IVM and PCOS IVM groups compared to the control group, while CM supplementation restored their expression. Oocyte diameter and viability were significantly enhanced in IVM + CM compared to IVM (P < 0.05).

Conclusions

These findings suggest that hWJMSC-derived secretomes, particularly CM, enhance oocyte maturation and quality, offering potential therapeutic benefits for IVM in both normal and PCOS conditions.

Extracellular Vesicles

PCOS

IVM

Conditioned media

Oocyte

Polycystic ovary syndrome is a common hormone disorder that affects women during their childbearing years. The number of women diagnosed with PCOS varies depending on the set of diagnostic rules used (1). During fertility treatments, women with PCOS typically produce more eggs when given ovary-stimulating hormones compared to women without PCOS (2). However, because of alterations in the follicular fluid, hormonal imbalance, and oocyte microenvironment, a significant proportion of these oocytes remain immature, compromising their developmental potential (3, 4).

Currently, in vitro maturation (IVM) serves as an effective approach to rescue oocytes that fail to mature in vivo following ovarian stimulation, enabling their progression to mature oocytes at the stage of metaphase II (MII) (5). Consequently, IVM has been proposed as a tailored approach for PCOS patients undergoing Assisted Reproductive Techniques (6). However, despite its potential, the clinical application of IVM remains limited due to low maturation and fertilization rates, ultimately resulting in lower pregnancy success even in non-PCOS patients (7, 8).

A key challenge in IVM lies in replicating the physiological follicular environment in vitro, as current culture conditions fail to support oocyte maturation fully (9).

The process of oocyte maturation involves coordinated signaling pathways and cell cycle regulators, including CDC2 (also known as CDK1), CCNB1 (cyclin B1), and MAP2K (MEK1/2)(10). CDC2/CDK1, in complex with cyclin B1, forms the maturation-promoting factor (MPF), which is crucial for the germinal vesicle breakdown, spindle formation, chromosome condensation, and consequently, oocyte progression of the maturation oocyte (11).

The levels and activity of CCNB1 are tightly regulated, ensuring the timely activation and inactivation of MPF during maturation. Additionally, MAP2K (MEK1/2), part of the MAPK/ERK signaling pathway, modulates cytoplasmic and nuclear maturation processes by regulating the activity of cyclin-dependent kinases and other downstream effectors (10). Activation of the MAPK pathway is essential for spindle assembly, oocyte cytoplasmic maturation, and successful completion of meiosis II (12). Together, CDC2, CCNB1, and MAP2K orchestrate the intricate signaling networks that underpin oocyte maturation, ensuring the oocyte reaches developmental competence for fertilization (12).

Recent advances in IVM research have focused on optimizing culture conditions to enhance meiotic resumption and cytoplasmic maturation- two critical determinants of oocyte quality (13, 14). In this context, novel culture medium additives have emerged as potential tools to improve oocyte maturation efficiency and developmental outcomes (15, 16).

Nowadays, mesenchymal stem cells (MSCs) are recognized as potential therapeutic agents for treating a wide range of diseases largely attributed to their capacity to secrete bioactive molecules (17). Conditioned medium (CM) derived from MSCs has shown significant clinical relevance, as it contains a complex mixture of bioactive factors capable of exerting therapeutic effects (18). MSC-derived CM are rich in growth factors, cytokines, and tissue regeneration mediators that can modulate in vitro maturation (13).

Extracellular Vesicles (EVs) are membrane-enclosed particles present in various biological fluids, including plasma, urine, follicular fluid, and culture supernatants of MSCs (19). The cells secrete these lipid bilayer vesicles into the extracellular environment, where they coordinate communication between cells by transporting diverse cargoes such as cytokines, proteins, microRNAs, and lipids (19). Many of the regenerative effects associated with MSC therapy are mediated through EVs (20). In the process of reproduction, EVs influence key reproductive processes, including oocyte maturation, sperm capacitation, fertilization, and the development and implantation of the embryo (21).

Therefore, the IVM medium was supplemented with CM or EVs of human Wharton’s jelly mesenchymal stem cells (hWJMSCs) in a mouse model of PCOS. After conducting the IVM, the maturation rates (GV, MI, MII stages), oocyte quality parameters such as zona pellucida thickness, perivitelline space size, and oocyte diameter, oocyte morphology, viability, nuclear maturation, and the expression levels of key maturation-related genes, including CDK1, CCNB1, and MAP2K, were evaluated in mature oocytes.

Ethics statement

This study was conducted with approval from the Shiraz University of Medical Sciences Animal Ethics Committee (IR.SUMS.REC.1400.566) and also adhered to the guidelines set forth by the ARRIVE (Animal Research: Reporting of In Vivo Experiments) to ensure rigorous reporting, transparency, and reproducibility.

Isolation and cultivation of hWJMSCs and production of hWJMSCs

Umbilical cords were collected from full-term infants born by cesarean section at Hafez Hospital, after obtaining written informed consent from their parents following the protocols approved by the Ethics Committee of Shiraz University of Medical Sciences (IR.SUMS.REC.1400.566). All the process related to isolation and cultivation of hWJMSCs, characterization, differentiation od the cells, CM and EVs extraction was done according to a previous study (17). After collection, the umbilical cords were immediately placed in ice-cold phosphate-buffered saline (PBS) (Shellmax, USA). The solution was supplemented with antibiotics (100 U/mL streptomycin and 100 µg/mL penicillin (Gibco, UK)). Initially, the umbilical vein was incised, followed by scraping away the endothelial lining of the vein and the amniotic epithelium, after which both arteries were removed. Later, tiny explant fragments, approximately 4 by 5 mm cultured in α-Minimal Essential Medium (α-MEM) (Shellmax, USA), 10% fetal bovine serum (FBS) (Gibco, UK), 1% L-glutamine (Gibco, UK), 100 U/mL penicillin (Gibco, UK), and 100 µg/mL streptomycin (Gibco, UK) in a culture dish (SPL Lifesciences, Korea). The MSCs started migrating from the explant roughly 15 days later. For the subsequent experiments, the hWJMSC from passages 3 to 6 was employed.

Characterization of hWJMSCs

Following the growth expansion phase, the isolated MSCs were examined under a light microscope to evaluate cell morphology.

Flow cytometry-based assay

To examine cell surface markers, hWJMSCs at passage three were subjected to flow cytometry. Using the 0.25% Trypsin-EDTA (Dacell, Iran), the cells were detached from the flask (NEST, China) and were subsequently centrifuged at 1200 rpm for 5 minutes. The cell suspension was adjusted to a final concentration of 10⁶ cells per mL in 100 µL PBS (Shell max, USA) and incubated at 4°C with the following anti-human antibodies: CD73-PE, CD144, phycoerythrin-conjugated CD34, and CD90 for 30 minutes (all purchased from Abcam, UK). Cell surface staining was carried out using fluorescein isothiocyanate or phycoerythrin-conjugated isotype antibodies. Cell analysis was performed using flow cytometry and FlowJo™ software (TreeStar, Ashland, OR, USA).

Differentiation into adipocytes and osteocytes

Adipogenic and osteogenic differentiation media were applied to passage 3 cells for 3 and 4 weeks, respectively. A fresh medium was provided every 72 hours. The adipogenic differentiation medium was composed of α-MEM (Shellmax, USA) supplemented with 100 nM dexamethasone (Sigma, USA), 50 µg/mL ascorbic acid-2 phosphate (Merck, Germany), 10% FBS (Gibco, UK), and 50 µg/mL indomethacin (Sigma, USA). The differentiation of adipocytes was examined through oil-red O staining. Initially, the cells were fixed using 4% paraformaldehyde (Merck, Germany). In the next step, staining was performed using 0.5% oil-red O (Sigma Aldrich, USA) in isopropyl alcohol (Merck, Germany). The α-MEM supplemented with 10% FBS (Kiazist, Iran), 10 nM dexamethasone (Sigma, USA), 50 µg/mL ascorbic acid-2 phosphate (Merck, Germany), 2.1604 g/L 6-glycerol phosphate, and 10 mM β-glycerophosphate (Sigma, USA), was used as the osteogenic differentiation medium. Methanol (Merck, Germany) was used to fix the cells, and stained with Alizarin-red S (Sigma Aldrich, USA) to determine the MSCs' potential to differentiate into osteoblasts and observed with an inverted phase-contrast microscope.

Preparation of conditioned media

Conditioned medium (CM) was prepared from human Wharton's jelly mesenchymal stem cells (hWJMSCs). When the cells reached 70–75% confluence, the complete culture medium was removed. The cell monolayer was washed twice with phosphate-buffered saline (PBS), and serum-free medium was added. After 48 hours of culture, the medium was collected and centrifuged (12,000 × g, 10 min, 4°C). The supernatant was then processed: a fresh portion was used for extracellular vesicle (EV) isolation, and the remainder was kept in -80°C to be lyophilized to prepare the CM.

Extraction of EVs

The Exocib kit (Cib Biotech, Iran) was employed to isolate EVs from MSCs-CM. In short, the MSCs-CM were gathered and centrifuged at 3000 r/min for 10 minutes to remove cell fragments. Afterward, the supernatant and Exocib solution were mixed at a 5:1 ratio and incubated at 4°C for 12 hours at 4°C followed by centrifugation at 3000 r/min for 40 min. Then, after discarding the supernatant, the pellet was mixed again in 100 µL of PBS. After isolation, the EVs were preserved at -20°C.

Transmission electron microscopy (TEM)

The size and shape of EVs were assessed by transmission electron microscopy (LEO 906E, Zeiss, Germany). After a 1:10 dilution in PBS (Shell max, USA), the EVs were placed on copper grids, dried at room temperature, and observed via TEM without the use of staining. ImageJ software was used to determine the size distribution of EVs (Java 1.8.0_112).

Dynamic light scattering (DLS)

The DLS approach allows for the measurement of particle size distributions spanning from 1 nm up to 6 µm. Particles such as EVs disperse light upon being hit by the laser beam.

By analyzing changes in scattered light patterns, a mathematical model based on light scattering principles and Brownian motion was developed. Seven EV samples were assessed to calculate the average size distribution.

Experiments

A total of 84 NMRI (12 in each group) female mice, aged 6–8 weeks and weighing between 30 and 35 grams, were procured from the Comparative and Experimental Medicine Center at Shiraz University of Medical Sciences. The animals were housed in a controlled environment with a 12-hour light/dark cycle and ambient temperatures maintained between 20°C and 24°C. Food and water were provided ad libitum throughout the experimental period.

Monitoring the estrous cycle

Before experiments, vaginal smears were collected daily between 9:00 and 10:00 AM for 2 weeks to make sure that the animals were mature and had a regular sexual cycle. Vaginal cells were collected by lavage of distilled water, fixed on a slide, and stained by Giemsa to assess the estrous cycle stages, including proestrus, estrus, metestrus, and diestrus. A duration of 4 to 5 days was considered the definition of regular cycles (22).

Establishment of the PCOS mouse model

To induce PCOS, adult female NMRI mice received letrozole (Letrax 2.5, Abu Raihan Pharmaceutical Co, Tehran, Iran) at a dose of 90 µg/kg daily for one week by gavage (23, 24).

PCOS induction confirmation

To confirm the establishment of the PCOS model, the blood testosterone levels and the histology of the ovarian tissues were determined.

Serum testosterone assay

To compare the testosterone levels between control and PCOS mice, the animals were anesthetized using a CO2 chamber. Blood samples were then obtained through cardiac puncture and immediately centrifuged at 3000 rpm for 10 minutes. The resulting serum samples were collected and stored at -80°C. Later, the testosterone concentration was determined using an enzyme-linked immunosorbent assay (ELISA) with commercially available kits (Monobind Inc., USA).

Evaluation of the ovarian tissue

Following blood collection, the ovaries were excised and fixed in 10% buffered formalin. They were then embedded in paraffin and sectioned serially at a thickness of 5 µm. The tissue sections mounted on slides were stained with hematoxylin and eosin. The different types of ovarian follicles, including primordial, primary, secondary, Graafian, atretic follicles, corpus luteum, and ovarian cysts, were counted in the prepared sections using a light microscope (Olympus, Japan)(25).

Experimental design and groups

Both pregnant mare serum gonadotropin (PMSG, GONASER®, HIPRA, Amer, Spain) and human chorionic gonadotropin (hCG, Organon, Oss, The Netherlands) were administered at doses of 10 IU via intraperitoneal injection.

The animals were divided into seven groups as follows:

Control: Mice were administered PMSG followed by hCG 48 hours later. MII oocytes were collected 14 hours post-hCG administration.

IVM Group: Mice were administered PMSG. Forty-eight hours later, oocytes at the GV stage were collected and cultured for 24 hours in IVM media (26).

IVM + CM Group: Mice were administered PMSG. Forty-eight hours later, oocytes at the GV stage were collected and cultured for 24 hours in IVM media supplemented with hWJMSCs-derived conditioned media.

IVM + EVs Group: Mice were administered PMSG. Forty-eight hours later, oocytes at the GV stage were collected and cultured for 24 hours in IVM media supplemented with hWJMSCs-derived EVs.

PCOS IVM Group: PCOS-induced mice were administered PMSG. Forty-eight hours later, oocytes at the GV stage were collected and cultured for 24 hours in IVM media.

PCOS IVM + CM Group: PCOS-induced mice were administered PMSG. Forty-eight hours later, oocytes at the GV stage were collected and cultured for 24 hours in IVM media supplemented with hWJMSCs-derived conditioned media.

PCOS IVM + EVs Group: PCOS-induced mice were administered PMSG. Forty-eight hours later, oocytes at the GV stage were collected and cultured for 24 hours in IVM media supplemented with hWJMSCs-EVs.

Determining the optimal concentration of CM and EVs

To determine the optimal concentrations of CM and EVs for IVM, the maturation rate of GV-stage oocytes was assessed in IVM medium supplemented with various doses of these supplements. Initially, the protein concentrations of hWJMSCs-derived CM or EVs were measured using the Bradford Protein Assay Kit (ProtoCib, Cib Biotech, Iran) following the manufacturer's instructions. The protein concentrations were found to be 500 µg/ml for CM and 15 µg/ml for EVs.

Following this, GV-stage oocytes were randomly assigned to seven groups and cultured for 24 hours in IVM medium supplemented with different concentrations of hWJMSCs-derived CM or EVs. The optimal concentration was determined based on the oocyte maturation rate. The study identified 50 µg/ml for CM and 5 µg/ml for EVs as the most effective concentrations.

COCs collection and IVM procedure

In the control group, mature (MII) oocytes were harvested to serve as in vivo-matured of normal oocytes for comparison with those matured in vitro. Maturity was defined by the extrusion of the first polar body. Mice were superovulated via an IP injection of PMSG, followed 48 hours later by an IP injection of hCG. Fourteen hours after hCG administration, the mice were anesthetized using a CO₂ chamber. The oviducts were then transferred into handling medium (G-MOPS™, Vitrolife, Göteborg, Sweden), which was pre-incubated for 24 hours at 37°C. Under a stereomicroscope (Nikon, Tokyo, Japan), the oviducts were dissected using two insulin syringes (Helma Teb, Baspar Sanat Fakher, Saveh, Iran). The cumulus-oocyte complexes were released and collected with flame-polished Pasteur pipettes, and the complexes were placed into a few drops of hyaluronidase (80 IU/mL; Vitrolife) to separate the cumulus cells and harvest MII oocytes.

In all six IVM groups, 48 hours after PMSG injection, COCs were collected from ovarian dissection. The COCs were washed in handling medium and then cultured for 24 hours in 50 µl droplets covered with mineral oil, under a controlled environment of 5% CO₂ at 37°C. The culture medium consisted of basal IVM medium—Minimum Essential Medium α (α-MEM) supplemented with 0.1 IU/mL FSH (Follitropin alfa 75 IU, CinnaGen, Iran), 5 mIU/mL hCG (Folignan, DarouPakhsh, Iran), and 75 µg/mL Penicillin, 50 µg/mL streptomycin, and 5% fetal bovine serum (Gibco, UK) either alone or supplemented with hWJMSCs-derived CM or EVs according to the respective experimental groups.

Both mature oocytes from the control group and those obtained after IVM were used for subsequent analyses, including assessment of maturation rate, measurement of oocyte diameter, perivitelline space PVS, and ZP thickness. Nuclear maturation was examined using Orcein staining of chromatin, while oocyte viability was assessed with Trypan Blue (TB) staining. Additionally, in each group, a proportion of mature oocytes were stored at -80°C for gene expression profiling of CDk1, CCNB1, and MAP2K using real-time PCR.

Brilliant Cresyl Blue (BCB) staining

To assess the effects of selecting developmentally competent oocytes, the collected COCs were first washed three times with PBS. They were then exposed to 13 µM BCB in PBS at 37°C under humidified air conditions for 90 minutes. After this incubation, the COCs were rinsed three times with PBS and subsequently classified as BCB-positive (BCB+, indicated by blue cytoplasm) or BCB-negative (BCB−, indicated by colorless cytoplasm) (27).

Evaluation of the oocyte maturation rate

Oocyte maturation was evaluated using an inverted microscope (Nikon, Japan), and the proportions of oocytes at various stages, GV, MI, MII, or degenerated, were recorded. Mature oocytes were identified by the presence of the first polar body. For further experiments, selected oocytes exhibited a spherical shape, a well-defined zona pellucida, a clear perivitelline space, and a faintly granular cytoplasm without inclusions (28).

Examining nuclear maturation via Orcein staining of chromatin

Nuclear maturation of the oocytes was assessed using aceto- orcein staining, categorizing them into GV, GVBD, anaphase-telophase, and MII stages. The oocytes were placed on a glass slide and fixed using a 3:1 acetic acid-ethanol solution for 24 hours. Next, the oocytes were stained with Aceto-Orcein and examined under an inverted microscope to assess nuclear maturation (29).

Trypan blue (TB) staining for evaluating oocyte survival

Trypan blue (TB) is a dye extensively applied to stain dead cells. The principle of TB staining is that TB is negatively charged and binds only to damaged membranes. Intact cells allow the passage of very few select compounds through the membrane, and therefore do not absorb TB. MII oocytes of control group and those from IVM groups, were stained with TB. Those which did not stained considered to be viable. In contrast, cells with damaged membranes were stained a distinctive blue color, as readily observed under a microscope (30).

Measurement of Oocyte Diameter, PVS and ZP thickness After IVM

The MII oocytes harvested from mice in the control group and those harvested from IVM groups, were imaged at 200x magnification using a digital camera (Nikon, Tokyo, Japan) mounted on an inverted microscope (Nikon, Japan). Using image analysis software (ImageJ, ver. 1.41o; National Institutes of Health, Bethesda, MD), the diameter of each oocyte was determined by averaging the two perpendicular measurements including the ZP (31). Also, the PVS and ZP thickness of each oocyte was measured at four different locations using ImageJ software, and the mean value of these measurements was calculated for each oocyte.

Real-time RT-PCR

To assess the expression of Cdk1, Ccnb1, and Map2k in the mature oocytes, total RNA was extracted from three pools, each containing 25 oocytes in their respective groups using the RNX-Plus kit (Cinnagen, Iran). The primers were initially designed using Primer3 software, and were subsequently verified using NCBI Primer-BLAST to confirm target specificity and produced by Pishgam Tehran Company. The primer sequences are presented in Table 1.. To synthesize the cDNA, the mixture consists of 1 µg of RNA (which includes 2 µg of total RNA), 1 µg of a 50-µM oligo (dT)18 primer, and 5 µL of RNase-free distilled water. After incubation at 70°C for 10 minutes, the mixture was allowed to cool for at least 2 minutes. Next, a new mixture was prepared by adding 2 µL of 5× M-MLV buffer, 0.5 µL of dNTP mixture (10 mM each), 0.25 µL of 40 U/µL RNase inhibitor, and 0.25 µL of 200 U/µL RTase M-MLV (RNase H⁻), resulting in a final volume of 10 µL. The mixture underwent incubation first at 42°C for 60 minutes, then at 70°C for 15 minutes. Real-time quantitative PCR was performed using the SYBR Green I fluorescent dye reagent (SYBR® Premix Ex Taq™ II, Ampliqon Biotechnology Co., Ltd.) as well as an ABI-Step one Sequence Detection System. The experiment will be conducted three times. The findings will be presented as mean ± standard deviation, and group differences will be assessed using the 2^-ΔΔCT method (32).

Table 1
**Primer sequences used for real-time PCR analysis**
Gene	Sequence forward (5′–3′)	Sequence reverse (5′–3′)	Product size (bp)
Cdk1	TGCAATTCGGGAAATCTCTCTAT	CCATGGACAGGAACTCAAAGA	116
Map2k	GGAGTGGTCTTCAAGGTCTC	CTCCCGGATGATCTGGTTC	105
Ccnb1	GCCTGAGCCTGAACCT	TTCTGCAGGCGCACATC	118
β-actin	TCCTGACCCTGAAGTACCC	CACACGCAGCTCATTGTAGA	98

Statistical analysis

The data were analyzed using GraphPad Prism 9 software. After confirming normal distribution and homogeneity of variances, an independent t-test was used to compare testosterone levels and the number of ovarian follicles between control and PCOS-induced mice. For other intergroup comparisons, one-way analysis of variance (ANOVA) followed by Tukey’s post-hoc test was applied. A p value ≤ 0.05 was considered statistically significant.

Characterization of hWJMSCs

Observations with light microscopy demonstrated that the monolayer MSCs were morphologically homogeneous and spindle-shaped. Using flow cytometry, it was confirmed that the isolated cells expressed MSC markers (CD73, CD90) at high levels and hematopoietic markers (CD34, CD144) at low levels (Fig. 1. A). Additionally, Oil-Red O staining revealed the presence of lipid droplets in differentiating MSCs (Fig. 1. B), while Alizarin Red staining indicated calcium deposition, confirming osteoblast differentiation (Fig. 1. C).

Characterization of isolated EVs

Transmission electron microscopy

Using TEM microscopy, Round or disk-shaped particles were seen in groups (Fig. 2. A).

Dynamic light scattering

DLS is a method used to assess the dimensions of EVs. On average, EVs measured 17.82 nm in diameter.

Testosterone level

Letrozole-induced PCOS led to a significant rise in serum testosterone levels compared to the control group (P < 0.001) (Fig. 2. B).

Ovarian histopathology

In the ovaries of mice in the control follicles in different phases of development were observed; however, in the PCOS group cystic follicles were found. Also, the PCOS mice showed significantly lower corpus luteum (Fig. 3 and Table 2).

Yellow stars show Multiple ovarian cysts in the ovarian tissue of PCOS mice

Table 2
The number of follicles, corpus Luteum and cysts in the ovarian tissue of the PCOS and control mice
Groups	Primordial follicles	Primary follicles	Secondary follicles	Graafian follicles	Atretic follicles	Corpus Luteum	Ovarian cysts
Control	9.75 ± 1.4	7.75 ± 0.85	5.80 ± 0.76	0.80 ± 0.41	0.75 ± .55	4.70 ± 0.92	0
PCOS	3.35 ± 1.08*	4.0 ± 0.91*	2.85 ± 1.03*	0.35 ± .48*	8.25 ± 1.11*	0.65 ± 0.67*	9.25 ± 1.16*

Data are shown as mean ± SD.

* Significant difference between the PCOS and control groups (P < 0.001).

The Outcomes of IVM after selecting oocytes with BCB staining

The results of BCB staining are summarized in Table 3. The IVM + CM and IVM + EVs groups exhibited a significantly higher rate of MII oocytes derived from BCB + oocytes compared to the IVM group (P = 0.007 and P = 0.01, respectively). In contrast, the PCOS IVM group showed a significant reduction in the percentage of MII oocytes compared to the IVM group (P = 0.0007). Notably, supplementation with CM or EVs in PCOS oocytes significantly increased the rate of MII oocytes compared to the PCOS IVM group (P < 0.0001 and P = 0.03, respectively).

No significant differences were observed in the rate of MI oocytes between the IVM + CM and IVM + EVs groups compared to the IVM group, nor between the PCOS IVM + CM and PCOS IVM + EVs groups relative to the PCOS IVM group (P > 0.05).

The percentage of GV oocytes was significantly lower in the IVM + CM and IVM + EVs groups than in the IVM group (P = 0.03 for both) and the PCOS IVM + CM group when compared to the PCOS IVM group (P = 0.03). Additionally, the PCOS IVM + CM and PCOS IVM + EVs groups had significantly fewer degenerated oocytes than the PCOS IVM group (P = 0.02 for both).

The percentage of both MI, GV, and degenerated oocytes was remarkably higher in PCOS IVM when compared to the IVM group (P = 0.03, P = 0.02, and P = 0.008, respectively).

Table 3
Maturation rate of mice GV oocytes selected with BCB
Groups	NO. of oocyte	MII (% ± SD) NO. of oocyte	MI (% ± SD) NO. of oocyte	GV (% ± SD) NO. of oocyte	Deg (% ± SD) NO. of oocyte
IVM	182	62.08 ± 2.84 (113)	12.08 ± 2.26 (22)	15.93 ± 0.95 (29)	9.89 ± 0.48 (18)
IVM + CM	196	73.46 ± 1.34 ^a (144)	10.20 ± 0.34 (20)	8.16 ± 1.29^a (16)	8.16 ± 0.38 (16)
IVM + EVs	201	69.65 ± 1.75 ^a (140)	10.94 ± 1.84 (22)	8.95 ± 1.32^a (18)	10.44 ± 0.35 (21)
PCOS IVM	181	43.09 ± 0.77 ^a (78)	17.67 ± 1.62 ^a (32)	19.88 ± 0.38^a (36)	19.33 ± 0.45 ^a (35)
PCOS IVM + CM	190	57.36 ± 0.82^b (109)	15.78 ± 1.25 (30)	12.63 ± 0.30^b (24)	14.21 ± 0.30 ^b (27)
PCOS IVM + EVs	202	49.5 ± 1.15 ^b,c (100)	17.82 ± 0.94 (36)	17.82 ± 0.37^c (36)	14.85 ± 2.33 ^b (30)
^a significant difference vs IVM group, ^b significant difference vs PCOS IVM group, ^c significant difference vs PCOS IVM + CM group

Oocyte maturation rate without BCB selection of immature oocytes

The study evaluated the maturation rates of oocytes across different experimental groups, with a focus on metaphase II as the primary outcome (Fig. 4. and Table 4). The IVM group showed significantly lower MII rates compared to the Control group (P < 0.0001). The IVM group also had higher rates of MI oocytes (P = 0.002) and degenerated oocytes (P = 0.001), indicating reduced maturation efficiency.

IVM + CM significantly improved MII rates compared to IVM alone (P = 0.003), while IVM + EVs showed no significant improvement (P = 0.82). Both IVM + CM (P = 0.02) and IVM + EVs (P = 0.04) reduced GV-stage oocytes compared to IVM alone.

The PCOS IVM group had significantly lower MII rates than both the Control (P < 0.0001) and standard IVM group (P < 0.0001). PCOS IVM also exhibited higher GV oocytes (P < 0.0001) and degeneration rates (P < 0.0001) compared to Control.

PCOS IVM + CM restored MII rates compared to PCOS IVM (P < 0.0001). It also reduced GV oocytes (P = 0.006) and degeneration rates (P = 0.001). PCOS IVM + EVs showed no significant improvement in MII rates (P = 0.12). However, it marginally reduced degeneration (P = 0.03), with no effect on GV retention (P = 0.89).

Table 4
Percentage of Mature (MII), MI, GV, and degenerated oocytes in the immature oocytes of experimental groups selected without BCB
Groups	NO. of oocyte	MII (% ± SD) NO. of oocyte	MI (% ± SD) NO. of oocyte	GV (% ± SD) NO. of oocyte	Deg (% ± SD) NO. of oocyte
Control	148	71.62 ± 0.71 (106)	9.45 ± 1.1 (14)	10.81 ± 1.50 (16)	8.10 ± 2.18 (12)
IVM	191	54.97 ± 1.07^a (105)	15.70 ± 1.43^a (30)	14.65 ± 1.92 (28)	14.65 ± 0.93^a (28)
IVM + CM	216	63.88 ± 1.22^b (138)	15.27 ± 1.17 (33)	9.72 ± 1.07^b (21)	11.11 ± 0.74 (24)
IVM + EVs	179	56.42 ± 1.79^c (101)	13.40 ± 1.49 (24)	12.84 ± 1.02^c (23)	11.73 ± 0.62 (21)
PCOS IVM	187	37.96 ± 1.68^d (71)	15.50 ± 1.85 (29)	20.85 ± 1.15^d (39)	25.66 ± 1.99 (48)
PCOS IVM + CM	175	56 ± 1.55^e (98)	12.57 ± 1.5 (22)	16 ± 0.91 (28)	15.42 ± 1.26^e (27)
PCOS IVM + EVs	171	40.93 ± 0.98^f (70)	18.71 ± 0.95^f (32)	20.46 ± 1.19^f (35)	19.88 ± 1.15 ^e,f (34)

^a Significant difference with the control group, ^b Significant difference with the IVM group, c Significant difference with IVM + CM group, d Significant difference with IVM, ^e Significant difference with PCOS IVM group, f Significant difference with PCOS IVM + CM group (P < 0.05).

Effects of secretome (CM/EVs) from hWJMSCs on nuclear maturation of oocytes

Following orcein staining, it was observed that the proportion of MII oocytes was significantly lower in the IVM group compared to the control group (P < 0.0001). However, this rate increased in the IVM + CM group compared to the IVM group (P = 0.0014). Additionally, the rate was significantly higher in the IVM + CM group than in the IVM + EVs group (P = 0.04).

In the PCOS group, the proportion of MII oocytes was lower compared to the non-PCOS IVM group (P = 0.0246). Among PCOS samples, the IVM + CM group showed a higher rate than the PCOS IVM group (P = 0.002).

The percentage of degenerated oocytes was significantly higher in the IVM group compared to the control (P = 0.004), but it was significantly reduced in the IVM + EVs group relative to the IVM group (P = 0.0003). These findings are illustrated in Figs. 5 and 6.

Effects of hWJMSCs-derived CM or EVs on the survival rate of MII oocytes

Mature oocyte viability was evaluated across experimental groups (Figure. 7A). The proportion of viable metaphase II (MII) oocytes in the IVM group was significantly lower than in the control group (P < 0.0001). However, supplementation with conditioned medium (IVM + CM) or extracellular vesicles (IVM + EVs) significantly improved oocyte viability compared to IVM alone (P < 0.01 and P < 0.001, respectively).

In the PCOS model, IVM + CM resulted in fewer viable MII oocytes than standard IVM (P < 0.0001). Conversely, both PCOS IVM + CM and PCOS IVM + EVs exhibited significantly higher oocyte viability compared to PCOS IVM (P < 0.0001 and P < 0.001, respectively).

Effects of secretome (CM/EVs) from hWJMSCs on Morphometric parameters of oocytes

Comparative analysis of oocyte diameter across experimental groups revealed a significant reduction in the IVM group compared to the control group (P < 0.0001). However, oocyte diameter was significantly higher in the IVM + CM group than in both the IVM (P < 0.0001) and IVM + EVs (P = 0.0057) groups. Furthermore, both CM and EVs significantly increased oocyte diameter compared to the PCOS group (P < 0.0001 for both).

ZP thickness in PCOS IVM + CM increased significantly compared to PCOS IVM group (P = 0.0036).

There was no significant difference in PVS size among the studied groups (P > 0.05) (Figs. 7B–7D).

Effects of secretome (CM/EVs) from hWJMSCs on expression of Cdk1, Ccnb1, and Map2k

Map2k transcript levels were significantly downregulated in the IVM group relative to controls (P = 0.011) and further diminished in the PCOS IVM group compared to IVM alone (P = 0.012). In contrast, supplementation with CM (conditioned medium) in the IVM group led to a marked increase in Map2k expression (P = 0.003). Similarly, the PCOS IVM + CM group exhibited elevated Map2k levels compared to both PCOS IVM and PCOS IVM + EVs groups (P = 0.005 and P = 0.0485, respectively).

For Ccnb1, expression was significantly reduced in the IVM group versus controls (P = 0.007), while no significant difference was observed between the PCOS IVM and IVM groups (P > 0.05). Although Ccnb1 levels showed an upward trend in IVM + CM and IVM + EVs groups compared to IVM alone, this increase did not reach statistical significance (P > 0.05). However, in the PCOS IVM + CM group, Ccnb1 expression was significantly higher than in PCOS IVM (P = 0.027).

Cdk1 expression was significantly lower in the IVM group than in controls (P = 0.006) and exhibited a non-significant increase in the IVM + CM group (P = 0.022). Notably, Cdk1 levels were further reduced in PCOS IVM compared to IVM (P = 0.004). Conversely, the PCOS IVM + CM group displayed a significant upregulation relative to PCOS IVM (P = 0.021) (Fig. 8).

This study demonstrates that in vitro maturation efficiency is significantly compromised in both standard and PCOS models, as evidenced by reduced metaphase II oocyte yields, increased GV and degenerated oocytes, and downregulation of key maturation regulators (Cdk1, Ccnb1, and Map2k). However, supplementation with human Wharton’s jelly mesenchymal stem cell-derived conditioned medium consistently rescued oocyte competence, enhancing nuclear maturation rates, oocyte viability, and morphometric parameters (oocyte diameter and ZP thickness). Notably, BCB + oocytes treated with CM exhibited significantly higher MII rates compared to IVM alone, while PCOS IVM oocytes showed severe maturation defects, which were partially reversed by CM. EVs also improved outcomes but were less effective than CM, particularly in PCOS, where CM restored Map2k and Cdk1 expression—critical for meiotic progression. These findings highlight CM’s superior potential in optimizing IVM, especially in PCOS-associated oocyte dysfunction, likely due to its rich secretome of growth factors and regulatory molecules.

The developmental potential of oocytes refers to their capacity to resume meiosis, undergo fertilization, form a blastocyst, and ultimately give rise to healthy offspring (33). Although efforts have been made, the chances of implantation and live births remain very low for embryos developed from matured oocytes in IVM. On the other hand, research indicates that the cells in these embryos often have chromosomal abnormalities. Therefore, oocyte quality is highly important and studies are looking for the best maturation of immature oocytes and reducing their subsequent complications (34).

To enhance the efficiency of in vitro embryo production, the initial step involves choosing high-quality, suitable immature oocytes that produce the highest quality mature oocytes when transferred to the IVM environment (35, 36). The BCB staining method, a non-invasive technique that reveals glucose-6-phosphate dehydrogenase (G6PD) levels, is the optimal way to select immature oocytes. This enzyme is abundant in fresh, high-quality oocytes but declines as the oocyte ages (37). Research indicates that BCB + oocytes exhibit higher rate of cytoplasmic maturation, improved fertilization rates, and enhanced embryo development to the blastocyst stage compared to BCB- oocytes (38). Due to the benefits of this staining, we used this staining in this study to select high-quality immature oocytes and examine the effect of CM and EVs in both BCB + and BCB- groups.

The current study revealed significant differences in maturation outcomes between BCB-selected and non-selected oocytes during IVM. BCB + oocytes demonstrated superior developmental competence, with significantly higher MII rates in both standard IVM and PCOS models compared to their non-selected counterparts. This enhanced performance was accompanied by reduced rates of degeneration and GV arrest in BCB-selected oocytes, particularly when supplemented with CM. In contrast, non-selected oocytes exhibited poorer outcomes across all parameters, including lower MII rates, higher degeneration, and increased meiotic arrest. These results collectively demonstrate that BCB selection serves as an effective screening tool that not only identifies oocytes with greater developmental potential but also enhances their responsiveness to IVM optimization strategies, particularly in challenging conditions like PCOS. The stark contrast between BCB-selected and non-selected oocytes underscores the importance of pre-IVM competence assessment for improving ART outcomes.

This finding agrees with prior studies by Yan-Guang that employed the same staining technique for selecting viable immature mouse oocytes (38). Besides, in support of our findings of better developmental potential of BCB + oocytes, Opiela et al. (2008) found that BCB + bovine oocytes matured in vitro and then underwent in vitro fertilization (IVM/IVF) had significantly more ability to reach the 2-cell stage and blastocyst stage than BCB- oocytes in their ability (39). Research indicates that BCB + oocytes exhibit significantly elevated mRNA levels of genes related to mitochondrial biogenesis, suggesting this could contribute to their enhanced developmental potential (40).

Overall, the study demonstrates that both CM and EVs groups showed significantly higher MII oocyte yields compared to standard IVM for normal and PCOS mice, with CM proving more effective than EVs, likely due to its broader spectrum of soluble factors.

During maturation, oocytes experience nuclear transformations, exiting the diplotene stage of the first meiotic prophase and advancing to metaphase II, while releasing the first polar body. Observing the chromosomes of oocytes at this stage provides a more dependable method for assessing the progress of in vitro maturation (41). In this research, we conducted orcein staining for the initial time to assess nuclear maturation in mouse oocytes.

Orcein staining confirmed CM's superiority in restoring MII rates even in PCOS oocytes, while EVs were more effective at reducing degeneration, indicating distinct but complementary roles. PCOS oocytes exhibited significantly lower MII rates and higher degeneration, but CM supplementation restored maturation efficiency, highlighting its potential clinical relevance for PCOS patients undergoing IVM.

The viability and morphometric assessments collectively demonstrate that CM significantly enhances both oocyte survival rates and morphological parameters, key indicators of developmental competence, particularly in PCOS-derived oocytes, while EVs show more modest improvements. This consistent pattern suggests that although EVs contain essential bioactive factors, CM's richer composition of regulatory components provides more comprehensive support for oocyte maturation by simultaneously improving viability, restoring normal diameter, and maintaining proper ZP thickness. The superior efficacy of CM under challenging conditions like PCOS highlights its multifaceted protective and maturation-promoting effects, implying that while EVs offer therapeutic potential, CM's broader array of factors may be necessary for optimal IVM outcomes, especially in compromised oocytes.

Although existing commercial IVM media are costly and show limited effectiveness, MSC-CM holds promise due to its likely paracrine effects, justifying its exploration in this research. Recent decades have seen multiple studies attempting to overcome the absence of a follicular microenvironment in IVM through the addition of exogenous growth factors, follicular fluid, and co-culture systems that use fresh oocytes, denuded oocytes, and oviduct cells to simulate an in vivo setting (14, 42, 43).

Similar to our results, Ling et al. demonstrated that MSC-conditioned medium resulted in a greater rate of mouse oocyte maturation than α-MEM. Their findings might vary from ours because of differences in MSC origin, oocyte type, and culture techniques. Highlighting the importance of standardized protocols when comparing IVM outcomes across experimental models (44).

Adib et al. (2020) reported that both human testicular cell-conditioned medium and cumulus cell-conditioned medium (hCCCM) significantly improved oocyte maturation rates. However, these conditioned media were also associated with morphological alterations, including perivitelline space (PVS) enlargement and a higher incidence of irregular oocyte shape compared to control groups (45, 46).

The human umbilical cord mesenchymal stem cells significantly enhanced the maturation rates of both fresh and vitrified human oocytes. Additionally, it improved oocyte ultrastructures such as increased mitochondria-vesicle complexes, optimized cortical granule distribution, and enhanced mitochondrial-smooth endoplasmic reticulum aggregates. (47)

Several studies have confirmed that oocyte morphology significantly influences embryo development, and high-quality embryos are more likely to be obtained after IVM when morphologically normal oocytes are used (48, 49). Among the key abnormalities in the extracytoplasmic components, anomalies in the perivitelline space (PVS) are particularly notable. Some evidence suggests that an enlarged PVS may contribute to higher oocyte degeneration (49) and reduced fertilization success (50). Interestingly, however, one study found that oocytes with PVS abnormalities exhibited a significantly higher embryo development rate compared to normal oocytes, which contrasts with earlier findings (51).

Bahrami et al. (2022) reported that granulosa cell-conditioned medium markedly enhanced IVM outcomes by increasing the proportion of oocytes reaching the MII stage, improving mitochondrial membrane potential and oocyte viability, boosting fertilization in vitrified-warmed and IVM oocytes, and regulating the expression of key maturation-related genes in MII oocytes of the mouse (52).

Another study has shown that conditioned media from equine amniotic fluid mesenchymal stem cells promote maturation of porcine oocytes by modulating the expression of critical genes involved in cumulus cell-mediated maturation, improving mitochondrial distribution, enhancing cortical granule positioning, and ultimately enhancing blastocyst quality (53). Also, exosomes derived from human umbilical cord mesenchymal stem cells improved oocyte quality by increasing maturation rates, optimizing spindle formation, enhancing mitochondrial function, and boosting developmental potential (54).

Recent studies across multiple species demonstrate the critical role of follicular fluid-derived EVs (ffEVs) in enhancing oocyte maturation and developmental competence. In mares and porcines, follicular fluid-derived EVs significantly increased maturation rates (55, 56). Bovine studies revealed that ffEVs from preovulatory follicles and ampullary fluid upregulated maturation-related genes and improved fertilization rates, alongside metabolic quality markers (57). Further, equine ffEVs promoted cumulus expansion, viability, and MAPK pathway activation during maturation (58). Additional evidence shows that ffEVs enhance embryo development and oocyte resistance to aging (59). Collectively, these findings highlight ffEVs as conserved regulators of oocyte competence, with species-specific roles in altering gene expression.

In human ffEVs isolated from mature follicles, enhanced in vitro maturation of immature oocytes (60). Conversely, a study showed that ffEV supplementation does not improve nuclear maturation of cat oocytes in vitro (61).

The findings from our study, which demonstrated lower in vitro maturation IVM rates in oocytes from PCOS mice compared to controls, align with previous research highlighting the detrimental effects of PCOS-associated follicular microenvironment on oocyte quality. Liu et al. (2023) reported that EVs derived from the follicular fluid of PCOS women significantly impaired oocyte maturation, leading to increased mitochondrial mislocalization, spindle abnormalities, and upregulated antioxidant gene expression. These observations suggest that the altered composition of follicular fluid in PCOS may contribute to oxidative stress and dysfunctional oocyte maturation, potentially explaining the reduced IVM efficiency observed in our study (62).

Supporting this notion, Madkour et al. (2018) conducted a randomized controlled trial in which supplementation of IVM media with follicular fluid and cumulus-granulosa cell secretions from non-PCOS women significantly improved the maturation rates of immature oocytes from PCOS patients (63).

Consistent with the mentioned results of oocyte maturation and quality, gene expression analysis of maturation-related genes revealead significant alterations in the expression of key regulatory genes, Map2k, Ccnb1, and Cdk1, during IVM under different experimental conditions, particularly in the context of PCOS. The observed downregulation of Map2k in the IVM and PCOS IVM groups suggests impaired MAPK/ERK signaling, a critical pathway for oocyte maturation and metabolic homeostasis. The further reduction in PCOS IVM oocytes may reflect exacerbated dysfunction in PCOS, where aberrant folliculogenesis and oxidative stress are known to disrupt signaling cascades. The restoration of Map2k expression in IVM + CM and PCOS IVM + CM groups implies that conditioned medium (CM) contains factors—possibly growth factors or extracellular vesicles—that rescue MAPK pathway activity, potentially improving oocyte competence.

CCcnb1 and CDK1 suppression may contribute to maturation arrest in IVM. While the PCOS IVM group showed no further significant decline in Ccnb1, the marked reduction in Cdk1 suggests a PCOS-specific defect in the oocyte cell cycle. Notably, CM supplementation consistently upregulated both genes in PCOS IVM, reinforcing its potential to mitigate cell cycle disruptions.

Collectively, these results provide robust evidence that hWJMSC-derived CM and EVs enhance oocyte developmental competence, particularly in challenging conditions like PCOS, where conventional protocols often yield poorer results. By rescuing Map2k, Ccnb1, and Cdk1 expression, CM may promote nuclear and cytoplasmic maturation, ultimately improving embryo quality.

The superior performance of CM highlights its potential as an optimized supplement for IVM protocols, while EVs offer a more refined, cell-free alternative that may be advantageous for clinical translation.

Future research should employ proteomic/metabolomic profiling to identify key active components in CM/EVs for developing standardized IVM treatment. These findings should then be validated in translational models, with parallel investigation of synergistic effects when combined with adjuvants like antioxidants. Such efforts will optimize IVM protocols, enhancing oocyte quality and maturation rates in both normal and PCOS conditions for clinical application.

IVM

in vitro maturation

Conditioned Media

EVs

Extracellular Vesicles

PCOS

Poly Cystic Ovarian Syndrome

hWJMSCs

Human Wharton's Jelly Mesenchymal Stem Cells

BCB

Brilliant Cresyl Blue

Trypan Blue

PBS

Phosphate Buffer Saline

Acknowledgements

This study was supported by the Shiraz University of Medical Sciences, Shiraz, Iran (Grant No. 23144). This work is a part of the duties that were done by A Solati for phD program.

Author contributions

All authors contributed to the conceptualization and design of the study. Experiments were conducted by A.S., M.D., and S.B., Data analysis, interpretation, and preparation of figures and tables were performed by A.Z., S.A., F.Z., Z.KH., and SH. M. A.S., S.A., F.Z. supervised the study. Z. KH., and SH. M. provided methodological guidance. The first draft of the manuscript was written collaboratively by A.S. and S.A., with revisions and editing contributions from all authors. All authors critically reviewed earlier drafts and approved the final manuscript.

Funding

This study was supported by the Shiraz University of Medical Sciences, Shiraz, Iran (Grant No. 23144).

Data availability

No data sets were generated or analyzed during the current study.

Ethical statement

All procedures were approved by the Ethics Committee of Shiraz University of Medical Sciences (IR.SUMS.AEC.1400.566).

Consent for publication

Not applicable.

Declaration of conflicting interests

The authors declared no potential conflicts of interest.

Sengupta P, Dutta S. Polycystic Ovary Syndrome Beyond Reproduction: An Ethnic, Endocrine, and Lifestyle Enigma Demanding Holistic and Lifelong Solutions for the Modern Woman. Journal of Infertility and Reproductive Biology. 2025:1-3-1-3.
Agarwal S, Krishna D, Praneesh G, Rao KJJIRB. Tetrad of hormonal and biochemical manifestations in phenotypes of polycystic ovary syndrome. 2020;8(4):73–83.
Alaee S, Ekramzadeh M, Samare-Najaf M, Jahromi BN, Shokri S, Ghomashi10 F, et al. Nutritional Intake and Lifestyle in Infertile Women with and without Polycystic Ovary Syndrome: A Case-control Study. 2024;12(4):15-30-15-30.
Ward CP, Huddleston H, Masharani U, Mason AE, Frassetto L. Stringency in Polycystic Ovary Syndrome (PCOS) Criteria and Application in Clinical Study.
Jie H, Zhao M, Alqawasmeh OAM, Chan CPS, Lee TL, Li T, et al. In vitro rescue immature oocytes–a literature review. 2022;25(4):640–50.
Grynberg M, Sermondade N, Sellami I, Benoit A, Mayeur A, Sonigo CJF, et al. In vitro maturation of oocytes for fertility preservation: a comprehensive review. 2022;3(4):211–26.
Mackens S, Pareyn S, Drakopoulos P, Deckers T, Mostinckx L, Blockeel C, et al. Outcome of in-vitro oocyte maturation in patients with PCOS: does phenotype have an impact? 2020;35(10):2272-9.
Jayakrishnan K, Nambiar D, Fency JLJJIRB. Comparison of ovarian response between PCOS and Non-PCOS patients undergoing ICSI with antagonist protocol. 2015;3(3):199–207.
Nikmard F, Hosseini E, Bakhtiyari M, Ashrafi M, Amidi F, Aflatoonian R. The boosting effects of melatonin on the expression of related genes to oocyte maturation and antioxidant pathways: a polycystic ovary syndrome- mouse model. J Ovarian Res. 2022;15(1):11.
Filatov M, Khramova Y, Semenova M. Molecular Mechanisms of Prophase I Meiotic Arrest Maintenance and Meiotic Resumption in Mammalian Oocytes. Reprod Sci. 2019;26(11):1519–37.
Landim-Alvarenga F, Maziero RJAR. Control of oocyte maturation. 2018;11(3):150–8.
Jiang Y, He Y, Pan X, Wang P, Yuan X, Ma B. Advances in Oocyte Maturation In Vivo and In Vitro in Mammals. Int J Mol Sci. 2023;24(10):9059.
Akbari H. The effect of conditioned media on mouse oocytes ultrastructure following in vitro maturation. Gene Reports. 2020;20:100732.
Alaee S, Zal F, Razban V, Talaei-Khozani T, Shokri S, Khodabandeh Z. PRP Influences Maturation and Fertilisation of Immature Mouse Oocytes. Anat Histol Embryol. 2024;53(6):e13112.
Alaee S, Asadollahpour R, Hosseinzadeh Colagar A, Talaei-Khozani T. The decellularized ovary as a potential scaffold for maturation of preantral ovarian follicles of prepubertal mice. Systems Biology in Reproductive Medicine. 2021;67(6):413–27.
Walls ML, Hart RJ. In vitro maturation. Best Pract Res Clin Obstet Gynaecol. 2018;53:60–72.
Bahmyari S, Alaee S, Khodabandeh Z, Talaei-Khozani T, Dara M, Mehdinejadiani S, et al. The effects of Wharton’s jelly MSCs secretomes for restoring busulfan-induced reproductive toxicity in male mice. Human and Experimental Toxicology. 2024;43:09603271241269019.
Smolinska V, Bohac M, Danisovic L. Current status of the applications of conditioned media derived from mesenchymal stem cells for regenerative medicine. Physiol Res. 2023;72(S3):S233-S45.
Hasan MM. Characterization of follicular fluid-derived extracellular vesicles and their contribution to periconception environment. Tartu; 2022.
Worthington EN, Hagood JS. Therapeutic Use of Extracellular Vesicles for Acute and Chronic Lung Disease. Int J Mol Sci. 2020;21(7):2318.
Capra E, Lange-Consiglio AJB. The biological function of extracellular vesicles during fertilization, early embryo—maternal crosstalk and their involvement in reproduction: review and overview. 2020;10(11):1510.
Alaee S, Bagheri MJ, Ataabadi MS, Koohpeyma F. Capacity of Mentha spicata (spearmint) extracts in alleviating hormonal and folliculogenesis disturbances in a polycystic ovarian syndrome rat model. World J Vet. 2020(3):451–6.
Lohrasbi P, Karbalay-Doust S, Mohammad Bagher Tabei S, Azarpira N, Alaee S, Rafiee B, et al. The effects of melatonin and metformin on histological characteristics of the ovary and uterus in letrozole-induced polycystic ovarian syndrome mice: A stereological study. Int J Reprod Biomed. 2022;20(11):973–88.
Rafiee B, Karbalay-Doust S, Tabei SMB, Azarpira N, Alaee S, Lohrasbi P, et al. Effects of N-acetylcysteine and metformin treatment on the stereopathological characteristics of uterus and ovary. European journal of translational myology. 2022;32(2):10409.
Ataabadi MS, Bahmanpour S, Yousefinejad S, Alaee S. Blood volatile organic compounds as potential biomarkers for poly cystic ovarian syndrome (PCOS): An animal study in the PCOS rat model. The Journal of Steroid Biochemistry Molecular Biology. 2023;226:106215.
Zare Z, Abouhamzeh B, Masteri Farahani R, Salehi M, Mohammadi M. Supplementation of L-carnitine during in vitro maturation of mouse oocytes affects expression of genes involved in oocyte and embryo competence: An experimental study. Int J Reprod Biomed. 2017;15(12):779–86.
Murin M, Strejcek F, Bartkova A, Morovic M, Benc M, Prochazka R, et al. Intranuclear characteristics of pig oocytes stained with brilliant cresyl blue and nucleologenesis of resulting embryos. Zygote. 2019;27(4):232–40.
Jafarzadeh H, Nazarian H, Ghaffari Novin M, Shams Mofarahe Z, Eini F, Piryaei A. Improvement of oocyte in vitro maturation from mice with polycystic ovary syndrome by human mesenchymal stromal cell–conditioned media. Journal of cellular biochemistry. 2018;119(12):10365–75.
Fazelian-Dehkordi K, Talaei‐Khozani T. Three‐dimensional in vitro maturation of rabbit oocytes enriched with sheep decellularized greater omentum. Veterinary Medicine and Science. 2022;8(5):2092–103.
Zand E, Fathi R, Nasrabadi MH, Atrabi MJ, Spears N, Akbarinejad V. Maturational gene upregulation and mitochondrial activity enhancement in mouse in vitro matured oocytes and using granulosa cell conditioned medium. Zygote. 2018;26(5):366–71.
Kim J, You J, Hyun SH, Lee G, Lim J, Lee E. Developmental competence of morphologically poor oocytes in relation to follicular size and oocyte diameter in the pig. Mol Reprod Dev. 2010;77(4):330–9.
Liu J, Lu X, Wang W, Zhu J, Li Y, Luo L, et al. Activity of MPF and expression of its related genes in mouse MI oocytes exposed to cadmium. Food Chem Toxicol. 2018;112:332–41.
Sirard MA, Richard F, Blondin P, Robert C. Contribution of the oocyte to embryo quality. Theriogenology. 2006;65(1):126–36.
Anderiesz C, Fong CY, Bongso A, Trounson AO. Regulation of human and mouse oocyte maturation in vitro with 6-dimethylaminopurine. Hum Reprod. 2000;15(2):379–88.
Vernunft A, Alm H, Tuchscherer A, Kanitz W, Hinrichs K, Torner HJT. Chromatin and cytoplasmic characteristics of equine oocytes recovered by transvaginal ultrasound-guided follicle aspiration are influenced by the developmental stage of their follicle of origin. 2013;80(1):1–9.
Opiela J, Lipiński D, Słomski R, Kątska-Książkiewicz LJARS. Transcript expression of mitochondria related genes is correlated with bovine oocyte selection by BCB test. 2010;118(2–4):188–93.
Alm H, Torner H, Löhrke B, Viergutz T, Ghoneim I, Kanitz WJT. Bovine blastocyst development rate in vitro is influenced by selection of oocytes by brillant cresyl blue staining before IVM as indicator for glucose-6-phosphate dehydrogenase activity. 2005;63(8):2194–205.
Wu YG, Liu Y, Zhou P, Lan GC, Han D, Miao DQ, et al. Selection of oocytes for in vitro maturation by brilliant cresyl blue staining: a study using the mouse model. Cell Res. 2007;17(8):722–31.
Opiela J, Kątska-Książkiewicz L, Lipiński D, Słomski R, Bzowska M, Ryńska B. Interactions among activity of glucose-6-phosphate dehydrogenase in immature oocytes, expression of apoptosis-related genes Bcl-2 and Bax, and developmental competence following IVP in cattle. Theriogenology. 2008;69(5):546–55.
Opiela J, Lipiński D, Słomski R, Kątska-Książkiewicz L. Transcript expression of mitochondria related genes is correlated with bovine oocyte selection by BCB test. Animal Reproduction Science. 2010;118(2–4):188–93.
El-Sokary MM, Kandiel MM, Mahmoud KGM, Abouel-Roos ME, Sosa GA. Evaluation of viability and nuclear status in vitrified mature buffalo oocytes. Glob Vet. 2013;10:297–302.
Lee SH. Effects of Human Endothelial Progenitor Cell and Its Conditioned Medium on Oocyte Development and Subsequent Embryo Development. Int J Mol Sci. 2020;21(21):7983.
Bhardwaj R, Ansari MM, Parmar MS, Chandra V, Sharma GT. Stem Cell Conditioned Media Contains Important Growth Factors and Improves In Vitro Buffalo Embryo Production. Anim Biotechnol. 2016;27(2):118–25.
Ling B, Feng DQ, Zhou Y, Gao T, Wei HM, Tian ZG. Effect of conditioned medium of mesenchymal stem cells on the in vitro maturation and subsequent development of mouse oocyte. Braz J Med Biol Res. 2008;41(11):978–85.
Adib M, Seifati SM, Ashkezari MD, Akyash F, Khoradmehr A, Aflatoonian B. Effect of human testicular cells conditioned medium on in vitro maturation and morphology of mouse oocytes. International Journal of Fertility & Sterility. 2020;14(3):176.
Adib M, Seifati SM, Ashkezari MD, Khoradmehr A, Rezaee-Ranjbar-Sardari R, Tahajjodi SS, et al. The effect of the human cumulus cells-conditioned medium on in vitro maturation of mouse oocyte: An experimental study. International Journal of Reproductive BioMedicine. 2020;18(12):1019.
Akbari H, Vaghefi SHE, Shahedi A, Habibzadeh V, Mirshekari TR, Shekari MA, et al. Conditioned mediums and human oocytes in vitro maturation. Int J Pharm Phytopharm Res (eIJPPR). 2018;8(2):64–71.
Khalili MA, Mojibian M, Sultan A-M. Role of oocyte morphology on fertilization and embryo formation in assisted reproductive techniques. Cell Journal (Yakhteh). 2005.
Mikkelsen AL, Lindenberg S. Morphology of in-vitro matured oocytes: impact on fertility potential and embryo quality. Human Reproduction. 2001;16(8):1714–8.
Plachot M, Selva J, Wolf J, Bastit P, De Mouzon J. Consequences of oocyte dysmorphy on the fertilization rate and embryo development after intracytoplasmic sperm injection. A prospective multicenter study. Gynecologie, Obstetrique Fertilite. 2002;30(10):772–9.
Hassa H, Aydın Y, Taplamacıoğlu F. The role of perivitelline space abnormalities of oocytes in the developmental potential of embryos. Journal of the Turkish German Gynecological Association. 2014;15(3):161.
Bahrami Z, Hatamian N, Talkhabi M, Zand E, Mottershead DG, Fathi R. Granulosa Cell Conditioned Medium Enhances The Rate of Mouse Oocyte In Vitro Maturation and Embryo Formation. Cell Journal (Yakhteh). 2022;24(10):620.
Park A, Oh HJ, Ji K, Choi EM, Kim D, Kim E, et al. Effect of passage number of conditioned medium collected from equine amniotic fluid mesenchymal stem cells: porcine oocyte maturation and embryo development. International Journal of Molecular Sciences. 2022;23(12):6569.
Song J, Guo X, Zhang B, Zhang Q, Han Y, Cao D, et al. Human umbilical cord mesenchymal stem cells derived exosomes improved the aged mouse IVM oocytes quality. Reproductive Sciences. 2024;31(9):2808–19.
Gabryś J, Kij-Mitka B, Sawicki S, Kochan J, Nowak A, Łojko J, et al. Extracellular vesicles from follicular fluid may improve the nuclear maturation rate of in vitro matured mare oocytes. Theriogenology. 2022;188:116–24.
Kang H, Bang S, Kim H, Han A, Miura S, Park HS, et al. Follicular fluid-derived extracellular vesicles improve in vitro maturation and embryonic development of porcine oocytes. Korean Journal of Veterinary Research. 2023;63(4):40.1-.7.
Pakniyat Z, Azari M, Kafi M, Ghaemi M, Hashemipour SMA, Safaie A, et al. The effect of follicular and ampullary fluid extracellular vesicles on bovine oocyte competence and in vitro fertilization rates. Plos One. 2025;20(6):e0325268.
Gabryś J, Gurgul A, Szmatoła T, Kij-Mitka B, Andronowska A, Karnas E, et al. Follicular fluid-derived extracellular vesicles influence on in vitro maturation of equine oocyte: impact on cumulus cell viability, expansion and transcriptome. International Journal of Molecular Sciences. 2024;25(6):3262.
Shedova E, Singina G, Uzbekova S, Uzbekov R, Lukanina V, Tsyndrina E. Effect of extracellular vesicles of follicular origin during in vitro maturation and ageing of bovine oocytes on embryo development after in vitro fertilization. Sel'skokhozyaistvennaya Biologiya. 2022.
Makieva S, Saenz-de-Juano MD, Almiñana C, Bauersachs S, Bernal-Ulloa S, Xie M, et al. Treatment of human oocytes with extracellular vesicles from follicular fluid during rescue in vitro maturation enhances maturation rates and modulates oocyte proteome and ultrastructure. bioRxiv. 2025:2025.02. 05.636623.
Dahal R, Nagashima J, Songsasen N, Wood T. 150 The influence of follicular fluid extracellular vesicles on in vitro maturation of oocytes in the domestic cat. Reproduction, Fertility and Development. 2021;34(2):313-.
Liu C, Wang M, Yao H, Cui M, Gong X, Wang L, et al. Inhibition of oocyte maturation by follicular extracellular vesicles of nonhyperandrogenic PCOS patients requiring IVF. The Journal of Clinical Endocrinology & Metabolism. 2023;108(6):1394–404.
Madkour A, Bouamoud N, Kaarouch I, Louanjli N, Saadani B, Assou S, et al. Follicular fluid and supernatant from cultured cumulus-granulosa cells improve in vitro maturation in patients with polycystic ovarian syndrome. Fertility and sterility. 2018;110(4):710–9.

No competing interests reported.

Download PDF

Journal Publication

published 27 Dec, 2025

Read the published version in BioMedical Engineering OnLine →

Editorial decision: Revision requested
30 Sep, 2025
Reviews received at journal
30 Sep, 2025
Reviews received at journal
29 Sep, 2025
Reviews received at journal
26 Sep, 2025
Reviewers agreed at journal
02 Sep, 2025
Reviewers agreed at journal
02 Sep, 2025
Reviewers agreed at journal
29 Aug, 2025
Reviewers agreed at journal
28 Aug, 2025
Reviewers invited by journal
28 Aug, 2025
Editor assigned by journal
20 Aug, 2025
Submission checks completed at journal
20 Aug, 2025
First submitted to journal
19 Aug, 2025

You are reading this latest preprint version

Conditioned Media and Extracellular Vesicles Derived from Human Wharton's Jelly Mesenchymal Stem Cells Improve the in vitro Maturation of Immature Oocytes in Normal and PCOS Mouse Model

Status:

Journal Publication

Version 1

Abstract

Background

Results

Conclusions

Figures

Introduction

Materials and Methods

Ethics statement

Isolation and cultivation of hWJMSCs and production of hWJMSCs

Characterization of hWJMSCs

Flow cytometry-based assay

Differentiation into adipocytes and osteocytes

Preparation of conditioned media

Extraction of EVs

Dynamic light scattering (DLS)

Experiments

Monitoring the estrous cycle

Establishment of the PCOS mouse model

PCOS induction confirmation

Serum testosterone assay

Evaluation of the ovarian tissue

Experimental design and groups

Determining the optimal concentration of CM and EVs

COCs collection and IVM procedure

Brilliant Cresyl Blue (BCB) staining

Evaluation of the oocyte maturation rate

Examining nuclear maturation via Orcein staining of chromatin

Measurement of Oocyte Diameter, PVS and ZP thickness After IVM

Real-time RT-PCR

Statistical analysis

Results

Characterization of hWJMSCs

Characterization of isolated EVs

Transmission electron microscopy

Dynamic light scattering

Testosterone level

Ovarian histopathology

The Outcomes of IVM after selecting oocytes with BCB staining

Oocyte maturation rate without BCB selection of immature oocytes

Effects of hWJMSCs-derived CM or EVs on the survival rate of MII oocytes

Effects of secretome (CM/EVs) from hWJMSCs on Morphometric parameters of oocytes

Effects of secretome (CM/EVs) from hWJMSCs on expression of Cdk1, Ccnb1, and Map2k

Discussion

Conclusion

Abbreviations

Declarations

References

Additional Declarations

Status:

Journal Publication

Version 1