Advanced maternal age (AMA) has become prevalent globally. With aging, weakened antioxidant defense causes loss of normal function in the ovary and uterus due to oxidative stress. Here, we aimed to improve embryo development in AMA mice by intravenous injection (IV) of human adipose stem cell conditioned medium (ASC-CM) at various frequencies and intervals as an antioxidant intervention. Four- and six-month-old female ICR (Institute of Cancer Research) mice were randomly divided into groups IV treated with human ASC-CM under different conditions, and in vitro and in vivo embryo development were evaluated. Consequently, compared to the control group, blastocyst formation rate of parthenotes was significantly promoted in 4-month-old mice and the mean number of implanted fetuses after natural mating was significantly increased by approximately two-fold in 6-month-old mice. Through gene analysis, the anti-apoptotic and anti-oxidative effects of human ASC-CMs were confirmed in the ovaries and uterus of pregnant mice at both ages. In particular, ovarian expression of gpx1 and catalase drastically increased in 6-month-old mice. Furthermore, the levels of gpx1 and catalase were further increased, with a high frequency of injection regardless of age. Thus, we demonstrated for the first time the anti-oxidative effect of human ASC-CM administration against ovarian aging and the optimal injection condition.

The quality of embryos produced by assisted reproductive techniques should be advanced by the improvement of in vitro culture conditions for successful implantation and pregnancy maintenance. We investigated the anti-oxidative effect of human adipose stem cell (ASC) conditioned medium with its optimal basal medium, Dulbecco′s modified Eagle′s medium (DMEM-CM), or keratinocyte serum-free medium (KSFM-CM) as supplements during in vitro culture (IVC) of in vitro fertilized mouse embryo. At first, preimplantation embryo development was evaluated in KSFM-CM and DMEM-CM supplemented cultures at various concentrations. The blastocyst (BL) and hatched BL formation rates were significantly increased in 5% DMEM-CM, while no difference was observed from KSFM-CM. Next, comparing the efficacy of KSFM-CM and DMEM-CM at the same concentration, DMEM-CM enhanced the developmental rate of 16 cells, morula, BL, and hatched BL. The expression level of reactive oxygen species decreased and that of glutathione increased in BL cultured with DMEM-CM, which confirms its anti-oxidative effect. Furthermore, apoptosis in BL cultured with DMEM-CM was reduced compared with that in KSFM-CM. This study demonstrated that the comparative effect of human ASC-CM made of two different basal media during mouse embryo IVC and anti-oxidative effect of 5% DMEM-CM was optimal to improve preimplantation embryo development.

Oxidative stress is a major cause of damage to the quantity and quality of embryos produced in vitro. Antioxidants are usually supplemented to protect embryos from the suboptimal in vitro culture (IVC) environment. Amniotic membrane-derived mesenchymal stem cells (AMSC) have emerged as a promising regenerative therapy, and their paracrine factors with anti-oxidative effects are present in AMSC conditioned medium (CM). We examined the anti-oxidative potential of human AMSC-CM treatment during IVC on mouse preimplantation embryo development and antioxidant gene expression in the forkhead box O (FoxO) pathway. AMSC-CM (10%) was optimal for overall preimplantation embryo developmental processes and upregulated the expression of FoxOs and their downstream antioxidants in blastocysts (BL). Subsequently, compared to adipose-derived mesenchymal stem cell (ASC)-CM, AMSC-CM enhanced antioxidant gene expression and intracellular GSH levels in the BL. Total antioxidant capacity and SOD activity were greater in AMSC-CM than in ASC-CM. Furthermore, SOD and catalase were more active in culture medium supplemented with AMSC-CM than in ASC-CM. Lastly, the anti-apoptotic effect of AMSC-CM was observed with the regulation of apoptosis-related genes and mitochondrial membrane potential in BL. In conclusion, the present study established AMSC-CM treatment at an optimal concentration as a novel antioxidant intervention for assisted reproduction.

Background/Objectives: Oxidative stress is a major cause of impaired oocyte quality and early embryo development, a challenge that still needs to be addressed in assisted reproduction. Mesenchymal stem cell secretomes have been investigated as cell-free therapeutics with antioxidant activity and relevant anti-apoptotic effects. This study aimed to evaluate the effects of human amniotic membrane-derived mesenchymal stem cell-conditioned saline (AMSC-CS) as an injectable formulation on oxidative stress–related markers in ovarian tissue and preimplantation developmental outcomes. Methods: AMSC-CS was administered intravenously to female mice in a dose-dependent manner. Safety assessments were conducted to evaluate systemic and target organ toxicity within the dosage range. In vitro fertilization (IVF) outcomes and oxidative status in ovaries, oocytes, and embryos were evaluated following treatment with low, medium, and high doses of AMSC-CS (1, 3, and 5 μL/g). Results: As an injectable formulation, the safety assessments did not reveal systemic or target organ toxicity of AMSC-CS within the dosage range. Medium-to-high doses of AMSC-CS improved the expression of folliculogenesis-related genes and decreased oxidative stress and apoptosis signaling in ovarian tissue. At the high dose, AMSC-CS promoted preimplantation embryo development to the blastocyst and hatched blastocyst stages, along with improved blastocyst quality and reduced oxidative stress in oocytes and blastocysts. Conclusions: These findings suggest that AMSC-CS at medium-to-high doses, as an injectable formulation with antioxidant activity, may be a promising adjunct for assisted reproductive technologies.

In vitro maturation (IVM) of oocytes remains less efficient than in vivo maturation, partly because conventional culture media cannot fully reproduce the follicular microenvironment. This study evaluated whether human amniotic membrane stem cell-conditioned medium (hAMSC-CM) improves IVM efficiency of mouse cumulus–oocyte complexes (COCs) recovered from ICR females primed with 0, 5, or 10 IU pregnant mare serum gonadotropin (PMSG). hAMSC-CM was added at 10% or 20% (v/v), and maturation, cleavage, blastocyst formation, and gene expression in MII oocytes, cumulus cells, and blastocysts were assessed. Supplementation with 20% hAMSC-CM significantly increased maturation rates across all PMSG groups, reaching 89.4 ± 1.4% versus 59.5 ± 1.3% in the 5 IU PMSG control (p < 0.05). With PMSG priming, 20% hAMSC-CM also enhanced cleavage and blastocyst formation. Molecular analyses showed suppression of CASPASE9 in MII oocytes, upregulation of AMH, BMP15, and GDF9 with downregulation of AMHR in cumulus cells, and increased IGF1 and BCL2 expression in blastocysts. These findings indicate that 20% hAMSC-CM, together with optimized PMSG priming, improves mouse oocyte IVM efficiency and embryo developmental competence.