In vitro maturation medium supplementation: utilization of repaglinide, L-carnitine, and mesenchymal stem cell-conditioned medium to improve developmental competence of oocytes derived from endometriosis mouse models

Abstract

Endometriosis (EMS) is one of the most prevalent causes for female infertility. Herein, we investigated the effect of the repaglinide (RG), L-carnitine (LC), and bone marrow mesenchymal stem cell-conditioned medium (BMSC-CM) supplementation during in vitro maturation (IVM) on the quality, maturation, and fertilization rates, as well as embryonic quality and development of oocytes derived from normal and EMS mouse model. Immature oocytes were collected from two groups of normal and EMS-induced female NMRI mice at 6-8 weeks of age. Oocytes were cultured in IVM medium unsupplemented (control group), or supplemented with 1 M RG, 0.3 and 0.6 mg/mL LC, and 25 and 50% BMSC-CM. After 24 h of oocyte incubation, IVM rate and antioxidant status were assessed. Subsequently, the rates of fertilization, cleavage, blastulation, and embryonic development were assessed. Our results demonstrated that supplementation of IVM medium with LC and BMSC-CM, especially 50% BMSC-CM, significantly enhanced IVM and fertilization rates, and markedly improved blastocyst development and total blastocyst cell numbers in EMS-induced mice compared to the control group (53.28±0.24 vs 18.09±0.10%). Additionally, LC and BMSC-CM were able to significantly modulate EMS-induced nitro-oxidative stress by boosting total antioxidant capacity (TAC) and mitigating nitric oxide (NO) levels. Collectively, LC and BMSC-CM supplementation improved oocyte quality and IVM rates, pre-implantation developmental competence of oocytes after in vitro fertilization, and enhanced total blastocyst cell numbers probably by attenuating nitro-oxidative stress and accelerating nuclear maturation of oocytes. These outcomes may provide novel approaches to refining the IVM conditions that can advance the efficiency of assisted reproductive technologies in infertile couples.

Figure 1. Histological comparison of normal (A) and endometriosis-induced (B) ovaries of mice (scale bar: 100 μm). A, boxed area shows healthy growing follicle. B, boxed area shows atretic follicles.

Figure 2. Different stages of in vitro maturation of mice oocytes (scale bar: 100 μm). GV: germinal vesicle; GVBD: germinal vesicle breakdown; MII: metaphase II.

Figure 3. Different stages of mouse embryo cleavage at different times of in vitro culture [scale bar: 50 μm (A, B, D), 20 μm (C, E)]. A, 2 cells; B, 4 cells; C, Morula; D, expanded blastocysts; E, hatching blastocysts.

Figure 4. Differential staining of mice blastocyst after 5 days of in vitro culture (scale bar: 20 μm). ICM: inner cell mass, stained with propidium iodide would appear red. TE: trophectoderm, nuclei labeled with Hoechst 33258 would appear blue. Red and blue do not appear because these images are in black and white.