{"paper_id":"9f7141dd-d118-4662-92bd-d73b385b0558","body_text":"Endometriosis (EMS) is a detrimental condition of the female reproductive system in\nwhich the endometrium (uterine lining) grows outside the uterus, most commonly on\nthe ovary and peritoneum. The main symptoms of the disease are pelvic pain,\ndysmenorrhea, and dyspareunia ( 1 ). In\naddition to the fact that EMS impacts up to 15% of women of reproductive age, 25-40%\nof women with infertility have been estimated to suffer from EMS ( 2 ). However, the exact pathophysiology of EMS\nrelated to infertility is still unknown. It can be detrimental to fertility directly\nby distorting tubo-ovarian anatomy or indirectly by invoking inflammatory and\noxidative damage to the oocytes resulting in poorer quality oocytes ( 3 , 4 ). In\naddition, EMS is currently believed to be detrimental to the ovaries based on\nmolecular, histological, and morphological evidence ( 5 ).\nOver the past three decades, assisted reproductive technology (ARTs) has encompassed\nthe management of almost all types of infertility, including EMS. In this context,\nrecent studies have shown that oocytes retrieved from EMS-affected ovaries are more\nlikely to fail  in vitro  maturation (IVM) and to show altered\nmorphology and lower cytoplasmic mitochondrial content ( 3 ). Moreover, oocyte quality is reflected in the ability of the\ncell to complete maturation and undergo successful fertilization and plays a vital\nrole in embryonic development during fertilization ( 3 ). The available evidence suggests that a reduction in the quality of\noocytes retrieved is consistently associated with EMS, differently than other causes\nof infertility ( 5 ). In essence, EMS has a\nnegative impact not only on the receptivity of the endometrium but also on the\ndevelopment of oocytes and embryogenesis ( 3 ).\nHowever, human oocytes are relatively rare for research, and their use in invasive\ninvestigations is typically unviable because it prevents their use in ARTs. On this\npremise, animal models may be beneficial in elucidating the pathophysiology of\nEMS-associated infertility.\nNitro-oxidative stress is a condition that reflects an imbalance between the systemic\nmanifestation of reactive oxygen species (ROS) and reactive nitrogen species (NOS)\nand the ability of a biological system to readily detoxify the reactive\nintermediates or to repair the resulting damage ( 6 ). Oxidative stress may have detrimental effects on oocytes, the\nfertilization process, and subsequent embryo development ( 3 , 4 ). Recent studies\nhave demonstrated that free radicals play a critical role in the pathophysiology of\nEMS ( 7 ). Moreover, the follicular fluid of\npatients with EMS shows increased levels of reactive species and a reduction in\ntotal antioxidant capacity ( 4 , 7 ).\nRepaglinide (RG) is an oral anti-hyperglycemic medication used to treat\nnon-insulin-dependent diabetes mellitus. It belongs to the meglitinide class of\nshort-acting insulin secretagogues, which induce insulin secretion by attaching to\nthe β cells of the pancreas ( 8 ). RG achieves\nthis by inhibition of the K-ATP-sensitive channels in the membrane of the β cells\n( 9 ). This depolarizes the β cells,\nallowing voltage-gated calcium channels to open, and the subsequent calcium influx\nstimulates insulin release ( 10 ). It has been\nreported that RG could up-regulate glutathione reductase and glutathione levels,\nthereby enhancing the anti-oxidative defenses ( 11 ). While the potential of RG in treating diabetes has been\ninvestigated well, there is little information to support its effect on oocyte\nmaturation and subsequent developmental process.\nL-carnitine (LC; β-hydroxy-c-trimethylammonium-butyric acid) is a vital cofactor that\nmay be generated endogenously or received through dietary sources and plays an\nimportant role in cell metabolism ( 12 ). LC is\ncrucial for fatty acid metabolism because it facilitates the transport of long-chain\nfree fatty acids into the mitochondrial matrix, where they may be used for\nbeta-oxidation ( 12 , 13 ). Furthermore, LC transports acetyl groups from the inside\nto the outside of the mitochondrial membrane, regulating glucose metabolism and, as\na result, affecting cell ATP levels ( 13 ). LC\nalso possesses direct antioxidant properties, preserves mitochondrial metabolism,\nand suppresses ROS-producing enzyme activities ( 14 ). Beneficial effects of LC on embryonic development in culture have\nbeen observed in many mammalian species ( 15 ).\nIn mice, supplementation of the IVM medium with LC promotes spindle microtubule\nassembly and chromosome alignment in MII oocytes and improves subsequent embryonic\ndevelopment by preventing apoptosis ( 15 ).\nOocyte metabolism is linked to oocyte quality, and it was recently discovered that\nbeta-oxidation of lipids is required for oocyte developmental competence ( 16 ).\nMesenchymal stem cells (MSCs) are adult and multipotent stem cells with self-renewal\ncapacity that can develop into cells of numerous unique mesodermal lineages,\nincluding bone, cartilage, and adipose tissues ( 17 ). According to various studies, MSCs secrete various types of\ncytokines, growth factors, bioactive factors, and tissue regenerative components\ninto mesenchymal stem cell-conditioned medium (MSC-CM) ( 17 , 18 ). Moreover, MSCs\nrelease anti-apoptotic molecules, including Bcl-xL and Bcl-2, as well as antioxidant\nproteins like peroxiredoxin-5 (PRDX5) ( 17 ).\nSince cytokines and growth factors are known to enhance meiotic progression and the\nprocesses involved with IVM ( 17 , 18 ), we examined whether IVM,  in\nvitro  fertilization (IVF), and subsequent embryonic processes with\noocytes derived from EMS-induced mice could be improved by MSC-CM.\nHence, the present study was aimed to investigate the comparative effect of RP, LC,\nand bone marrow MSC-CM (BMSC-CM) supplementation during IVM on the developmental\ncompetence of oocytes derived from normal and EMS-induced mice in terms of IVM, IVF,\nand subsequent developmental rate, as well as on the TAC and NO levels in the IVM\nmedium.\n\nAll chemicals were purchased from Sigma Chemical Corporation (USA) and Gibco\n(USA), except repaglinide that was purchased from Farabi Corporation (Iran).\nAdult female NMRI mice (6-8 weeks old) were purchased from Pasteur Institute\n(Iran). The animals were first habituated for one week and then divided into\ncontrol and experimental groups. The animals were held under standard conditions\n(12-h light-dark cycles, 23±1°C, and 50-60% humidity) and had  ad\nlibitum  access to water and food (standard diet) throughout the\nstudy. All experimental procedures pursued international guidelines for the care\nand use of laboratory animals and were approved by the Animal Welfare and Ethics\nCommittee of Basic Sciences, Razi University, Kermanshah, Iran. The studied\ngroups included two normal mice and mice under EMS induction. Oocytes obtained\nfrom normal mice were cultured in the IVM medium supplemented with RG, LC, and\nBMSC-CM. Likewise, oocytes derived from EMS-induced mice were cultured in the\nIVM medium containing RG, LC, and BMSC-CM.\nTwo groups of mice (6-8 weeks old) were used to induce EMS. To establish the EMS\nmodel, the mice in the donor group were intraperitoneally injected with\nestradiol-17β depot diluted in sesame oil (100 μg/kg) for one week. Then, they\nwere sacrificed on day 14, and their uterine horns were removed. In the next\nstep, tissue fragments from both uterine horns were harvested in a petri dish\ncontaining warm sterile saline. The provided suspension was injected\nintraperitoneally to the mice of the recipient group (approximately 40-50\nfragments per mouse) according to Somigliana et al. ( 19 ) method with some modification.\nBone marrow mesenchymal stem cells (BMSCs) were isolated from 6-8-week old NMRI\nmice. Briefly, bone marrow was harvested by flushing femurs and tibias that were\ncultured in Dulbecco's Modified Eagles Medium (DMEM; Gibco, USA) consisting of\n10% fetal bovine serum (FBS), L-glutamine 2 mM, 1% non-essential amino acids,\nand 1% penicillin/streptomycin (incubation at 37°C and 5% CO 2 ). After\n3 days of culture, non-adherent cells were removed by washing twice with PBS,\nand culture of adherent cells continued for 5-7 days until 80% confluence; the\nmedium was changed every 2-3 days. At the third passage, the cells were\ntrypsinized and seeded at a density of 1×10 4  cells/cm 2  in\na culture flask. After reaching 80% confluence to prepare a conditioned medium\nof BMSCs, the cells were washed three times with PBS and incubated for 48 h at\n37°C and 5% CO 2  in a serum‐free DMEM culture medium. After 48 h of\nincubation, the supernatant (conditioned medium) was collected and filtered\nthrough a 0.2-μm filter for immediate use. The BMSCs cells were derived from\nfemale and male NMRI mice. For detection, we used flow cytometry for CD14, CD45,\nCD34, CD73, CD90, CD105, and CD 29 to detect the phenotype of the 5th passage\ncells. The results showed that about 98% of BMSCs were CD90-positive and lacked\nexpression of CD14, CD45, and CD34. These results showed that mouse bone marrow\ncells had the characteristics of mesenchymal stem cells (Supplementary Figure\nS1). The results have already been published in our previous article ( 20 ).\nIn order to ensure the induction of EMS and its impacts on the ovaries, some mice\nin both normal and EMS groups were randomly selected, and after sacrifice, their\novaries were fixed in Bouin's solution, embedded in paraffin wax, and serially\nsectioned at 5 μm. Then, the serial sections of ovaries were stained with\nhematoxylin and eosin (HE). Afterward, the diagnosis of EMS was determined under\na light microscope according to the morphological criteria such as different\nstages of follicular growth (folliculogenesis) and follicular quality, dead or\natretic follicles, changes of oocyte quality, presence of residual cyst, and\nbleeding in the ovarian tissue ( Figure\n1 ).\nFemale NMRI mice from normal and EMS groups were sacrificed by an inhaled\noverdose of carbon dioxide (CO 2 , 10-30%), followed by cervical\ndislocation (all efforts were made to minimize suffering). Then, their ovaries\nwere removed and immediately transferred to the dissection medium of Alpha\nMinimal Essential Medium (α-MEM) containing 5% FBS and 1%\npenicillin/streptomycin. The immature oocytes (GV stage) were mechanically\nisolated from ovaries under a stereomicroscope (Motic: SMZ-143, China at 10×\nmagnification) in 50-μL micro drops of dissection medium by using a 27-gauge\nneedle. After washing three times with droplets of dissection medium by mouth\npipette, GV oocytes were transferred into 30-μL drops of IVM medium consisting\nof α-MEM, supplemented with 4 mg/mL bovine serum albumin (BSA), 10 ng/mL\nrecombinant epidermal growth factor (rEGF), 7.5 IU/mL human chorionic\ngonadotropin (HCG), and 100 IU/mL penicillin and 100 μg/mL streptomycin (in\nmineral oil at 37°C and 5% CO 2 ). In both normal and EMS groups,\nexperimental groups included control (IVM medium alone) and treatments (IVM\nmedium supplemented by 1 µM RG, 0.3 and 0.6 mg/ml LC, and 25, 50% BMSC-CM).\nAfter 24-h incubation, IVM rate was assessed under an inverted microscope\n(Olympus, Japan) according to the observation of different stages of maturation\nsuch as germinal vesicle (GV), germinal vesicle breakdown (GVBD), metaphase II\n(MII), and degenerated (Deg) oocytes ( Figure\n2 ).\nTwenty-four hours after incubation of oocytes, the IVM condition media from all\nexperimental groups were collected and antioxidant capacity (TAC, NO levels) was\nassessed ( 21 ).\nSpectrophotometer analysis with a colorimetric assay kit (Naxifer™, Navand\nSalamat Co., Iran) was used to estimate the concentrations of testicular levels\nof ferric reducing antioxidant power (FRAP). This procedure is based on the\nability of testis lysis to reduce iron III (Fe 3+ ) to iron II\n(Fe 2+ ) in the presence of 2,4,6-tripyridyl-S-triazine (TPTZ). A\ncomplex with blue color and maximum absorbance appeared in 593 nm with a\nreaction of Fe 2+  and TPTZ. Finally, the values are shown as nanomoles\nof Fe 2+  equivalents per wet tissue weight (nmol/mg protein) ( 22 ).\nThe total NO content of the homogenized testis was measured according to the\nGriess reaction using the Natrix™ assay kit (Navand Salamat Co.). In the Griess\nreaction, NO rapidly converts into nitrite, which is an acidic environment, and\nthen converts into HNO 2 . After adding sulfanilamide, HNO 2 \nforms a diazonium salt that reacts with N-(1-Naphthyl) ethylenediamine\ndihydrochloride to form an azo dye, which can be measured at 570 nm. The NO\ncontent of the examined organs was reported in nmol/mg protein in samples ( 22 ).\nIn vitro  matured oocytes (MII) were transferred to the 50-μL\ndrops of global IVF medium supplemented with 16 mg/mL of BSA. The cauda\nepididymis was isolated from 8-12-week-old male NMRI mice, and motile sperm\nfraction was obtained by the swim-up technique after a 45-min incubation at 37°C\nand 5% CO 2  in the Ham's F10 medium containing 16 mg/mL of BSA, 10 μL\nof motile sperm (final concentration of 1×10 6 ) added to each drop of\nIVF medium. After 4-6 h of sperm-oocyte incubation, the resulting zygotes were\nremoved and washed three times in 50-μL drops of global medium with 4 mg/mL of\nBSA and subsequently transferred to the 30-μL drops of culture medium consisting\nof global media with 4 mg/mL of BSA in groups of 10 zygotes/drops that were\ncovered with mineral oil (incubation at 37°C and 5% CO 2 ). The embryos\ncleavage rates were assessed under an inverted microscope (Olympus, IX71) for 1\nto 5 days, post-IVF (method described by Giritharan et al. ( 23 ) with some modification) ( Figure 3 ).\nDifferential staining was used to count the number of blastocysts and\ntrophectoderms (TE) and assess the inner cell mass (ICM) of cells. At 96 h of\nembryo culture, blastocysts were washed several times in phosphate buffered\nsaline (PBS, pH 7.2) and incubated at 37°C and 5% CO 2  for 30 s in 500\nµL of 100 µg/mL of propidium iodide (PI, Sigma) and 1% Triton X-100, then washed\nwith PBS and transferred in 500 µL of absolute ethanol containing 25 µg/mL\nbisbenzamide (Hoechst 33258; Sigma) and incubated for 30 min at 37°C. Fixed and\nstained blastocysts were mounted in glycerol and observed under an inverted\nfluorescence microscope (Olympus IX71), and observed using UV light. The nuclei\nof TE cells labeled by Hoechst 33258 have a blue color and the nuclei of ICM\ncells labeled by propidium iodide have a red color. Finally, the quality of\nblastocysts was evaluated based on the ICM and TE cells ( 24 ) ( Figure 4 ).\nData analysis was done using the SPSS statistical software (version 19: SPSS\nInc., USA). Normality and homogeneity of data were determined by\nKolmogorov-Smirnov test. IVM, IVF, and embryo cleavage rates were analyzed by\nthe chi-squared test. The TAC and NO levels and blastocyst cell numbers were\nevaluated by one-way ANOVA and Tukey's  post hoc  test.\nQuantitative data are reported as means±SE and 95% confidence interval (CI).\nDifferences were considered statistically significant when the P-value was\n≤0.05.\n\nMicroscopic studies of random ovarian tissue samples showed that\nfolliculogenesis, quality of follicles, and number and quality of intact oocytes\n(atretic follicles or oocytes) were reduced in the EMS-induced group compared\nwith the normal group. Also, the number of ovarian cysts, bleeding in the ovary,\nand adhesion of the ovary to the surrounding tissues and pelvic organs were\nincreased. Consequently, all these factors led to a noticeable reduction in the\novulation rate.\nTable 1  shows the characteristics of the\ndifferent stages of oocyte maturation in the normal group. The percentages\nindicate that there was a significant difference between control and all\ntreatment groups. Indeed, the highest IVM rate in both the normal and EMS mice\nwas observed in the 50% BMSC-CM group. In the normal group, only 0.6 mg/mL LC\nand 25 and 50% BMSC-CM were able to considerably reduce the GV rate compared to\nthe control group.\nData are reported as means±SE. Co: Control; RG: Repaglinide at 1 µM;\nLC-0.3 and 0.6 mg/mL: LC: L-Carnitine at 0.3 and 0.6 mg/mL; CM-25%,\n-50%: Conditioned medium of bone marrow mesenchymal stem cells at 25\nand 50%; GV: germinal vesicle; GVBD: germinal vesicle break down;\nMII: metaphase II; DEG: degenerated oocytes.  a P<0.05\ncompared with Co;  b P<0.05 compared with RG;\n c P<0.05 compared with LC-0.3;\n d P<0.05 compared with LC-0.6;  e P<0.05\ncompared with CM-25%;  f P<0.05 compared with CM-50%\n(chi-squared test).\nTable 1  also provides IVM data from the\nEMS group. The 0.3 and 0.6 mg/mL LC, and 25 and 50% BMSC-CM enhanced the\npercentage of MII oocytes significantly. However, no considerable difference was\nobserved between the control and RG groups. The RG, LC, and BMSC-CM\nsignificantly reduced the percentage of GV oocytes compared to control\ngroup.\nTable 2  shows the levels of TAC and NO\nin the normal and EMS groups. Our results revealed that 0.3 and 0.6 mg/mL LC and\n25 and 50% BMSC-CM significantly decreased NO levels and significantly increased\nTAC levels compared to the control group. Yet, this significant alteration was\nnot observed between the RG treatment and the control group. More notably, in\nboth the normal and EMS groups, the highest TAC level was observed in the 0.6\nmg/mL LC treatment.\nData are reported as means±SE. Co: Control; RG: Repaglinide at 1µM;\nLC-0.3, 0.6 mg/mL: L-Carnitine at 0.3 and 0.6 mg/mL; CM-25%, -50%:\nConditioned medium of bone marrow mesenchymal stem cells at 25 and\n50%; NO: nitric oxide levels; TAC: total antioxidant capacity.\n a P<0.05 compared with Co;  b P<0.05\ncompared with RG;  c P<0.05 compared with LC-0.3;\n d P<0.05 compared with LC-0.6;\n e P<0.05 compared with CM-25%;  f P<0.05\ncompared with CM-50% (ANOVA).\nTable 3  shows the different stages of\nembryonic development in the normal groups. There was a substantial improvement\nin IVF, cleavage, and blastocyst rates in all treatment groups compared to the\ncontrol group. in addition, the highest blastocyst formation rate was obtained\nafter supplementation of IVM medium with a 50% BMSC-CM (73.19±0.82) (P<0.05).\n Table 3  also shows the results of\n in vitro  embryo development in the EMS groups. A\nsignificant enhancement in IVF rate was observed in the 0.6 mg/mL LC and 25 and\n50% BMSC-CM treatment groups, and there were significant differences in cleavage\nrate in all treatment groups compared to the control. Moreover, 0.3 and 0.6\nmg/mL LC and 25 and 50% BMSC-CM exhibited a rise in blastocyst rate compared to\nthe control group, and the highest blastocyst percentage was associated with 50%\nBMSC-CM (53.28±0.24).\nData are reported as means±SE. Co: Control; RG: Repaglinide at 1 µM;\nLC-0.3, 0.6 mg/mL: L-Carnitine at 0.3 and 0.6 mg/mL; CM-25%, -50%:\nConditioned medium of bone marrow mesenchymal stem cells at 25 and\n50%; MII: metaphase II; IVF:  in vitro  fertilization\nrate.  a P<0.05 compared with Co;  b P<0.05\ncompared with RG;  c P<0.05 compared with LC-0.3;\n d P<0.05 compared with LC-0.6;\n e P<0.05 compared with CM-25%;  f P<0.05\ncompared with CM-50% (chi-squared test).\nThere was a dramatic increase in the mean total cell number and TE cells in the 1\nµM RG, 0.3 and 0.6 mg/mL LC, and 25 and 50% BMSC-CM. More importantly, among all\ntreated normal groups, only 50% BMSC-CM had a significant effect on ICM compared\nto the control group (P<0.05) ( Table\n4 ). Results of EMS groups are also reported in  Table 4 . Accordingly, except for the 1M RG group, all\ntreatment groups exhibited a significant difference in the mean total cell\npopulation and TE cells compared to the control group. Nevertheless, there was\nno significant difference in blastocysts ICM among treated groups compared with\nthe control group.\nData are reported as means±SE. Co: Control; RG: Repaglinide at 1 µM;\nLC-0.3, 0.6 mg/mL: L-Carnitine at 0.3 and 0.6 mg/mL; CM-25%, -50%:\nConditioned medium of bone marrow mesenchymal stem cells at 25 and\n50%; TE: trophectoderm; ICM: inner cell mass.  a P<0.05\ncompared with Co;  b P<0.05 compared with RG;\n c P<0.05 compared with LC-0.3;\n d P<0.05 compared with LC-0.6;  e P<0.05\ncompared with CM-25%;  f P<0.05 compared with CM-50%\n(ANOVA).\n\nMany infertile women with EMS undergo IVF to increase their chances of achieving a\npregnancy ( 25 ). However, in general, EMS is\nlinked to low oocyte yield, implantation rates, and pregnancy rates following IVF\n( 3 ). Among the factors associated with\ninfertility in EMS women, oocyte quality is the most critical since it represents\nthe intrinsic developmental potential and is responsible for proper\nfertilization/embryonic development during IVF ( 1 , 3 ). Surprisingly, limited\nstudies have been conducted to examine the impacts of EMS on oocyte quality.\nROS has detrimental effects on oocytes and oxidative stress plays an important role\nin the pathogenesis of abnormal oocyte development ( 26 ). In accordance with this, our current findings indicated that EMS\ninduction led to a considerable drop in TAC levels of the IVM medium. Since TAC is\nthe result of the interactions among its numerous components, it reflects the\npotential to protect against free radical damage more effectively than individual\nplasma antioxidant measurements ( 11 ).\nSimilarly, EMS also causes severe impairment in the generation and metabolism of NO\n( 27 ). NO is a ubiquitous free radical in\nthe oocyte microenvironment involved in the physiology and biology of the ovary and\nevery stage of oocyte development, including meiotic maturation, fertilization,\nembryonic cleavage, and implantation ( 27 ). As\na result of diminished bioavailability of NO under certain pathologic conditions,\noocyte viability and developmental capacity may be compromised ( 27 ). In this regard, NO oxidation by\nO 2 ·- produces peroxynitrite (ONOO-), a highly reactive molecule that\ndepletes lipid-soluble antioxidants, contributing to oxidative stress and lipid\nperoxidation in the oocyte microenvironment, which mediates an adverse impact on\noocyte quality ( 27 ). Our data support the\nresults of previous animal experiments and human trials showing that the level of NO\nin the IVM medium was significantly raised in EMS model groups, which could reflect\nnitrosative stress. Therefore, to preserve follicles from oxidative damage, the\nfollicular fluid is naturally provided with an effective antioxidant system\ncomprised of enzymatic antioxidants and vitamins ( 28 ). It is important to note that  in vitro \nenvironmental conditions such as increased exposure to oxygen, light, and culture\nmedium composition trigger metabolic alterations in oocytes and embryos, resulting\nin an imbalance between the ROS formation and antioxidant capacity ( 29 ). Thus, adding anti-oxidative components to\nthe IVM medium of EMS subjects is likely to provide more appropriate conditions and\nboost maturation, fertilization, and further embryo development ( 29 ).\nWe recently discovered that supplementing IVM medium with RG promotes oocyte\nmaturation and embryo cleavage rate by elevating the intracellular calcium\nconcentration ( 9 ). In line with this, the\npresent findings revealed that RG significantly improved nuclear oocyte maturation\nin normal mice. More importantly, the rates of fertilization, cleavage, and\nblastulation were positively changed in the RG-supplemented normal mice. Since\nalterations of the oocyte cytoskeleton have been documented to be one of the reasons\nfor poor oocyte quality in EMS subjects, we assumed that RG may reverse this impact\nby raising intracellular calcium concentration ( 1 , 3 ). In addition, dysregulation\nof intracellular Ca 2+  concentration with resulting poor oocyte quality\nhas been recently attributed to oxidative stress in oocytes and their\nmicroenvironment ( 30 ). Accordingly, exposure\nto ROS might be a primary cause of abnormal patterns of Ca 2+  release at\nfertilization ( 4 ). Thus, RG could combat this\nphenomenon by increasing intracellular calcium concentration. However, when\ndetermining the percentages of GV, GVBD, and MII oocytes in the EMS-induced groups,\nour results demonstrated that the addition of RG did not affect the rates of nuclear\noocyte maturation compared to the control group. Similarly, adding RG to the IVM\nmedium did not improve fertilization rates and embryo development. Even though some\nrecent studies claim that RG possesses anti-oxidative properties and significantly\naffects lipid peroxidation levels in an  in vivo  study, our results\nindicated that RG did not improve antioxidant status in IVM medium ( 31 ). In other words, the levels of TAC and NO\nin the IVM medium were not significantly affected by RG in both normal and EMS\ngroups.\nPrevious research reported an EMS-dependent decline in oocyte quality attributed to\nthe improper energy metabolism of fatty acids and/or the mitochondrial dysfunction\ndetected in the oocytes and cumulus oophorus cells of EMS women ( 32 ). On the contrary, LC can facilitate fatty\nacid and energy application by transporting long-chain fatty acids through the inner\nmitochondrial membrane for β-oxidation, subsequently increasing the concentration of\nadenosine triphosphate (ATP) ( 32 ).\nInterestingly, the β-oxidation process is essential in the nuclear and cytoplasmic\nmaturation of oocytes, leading to oocyte developmental competence ( 33 ). The dual role of LC as an antioxidant and\nas an important element of lipid metabolism makes it an option as a novel\nnon-invasive agent for optimizing oocyte competence efficiency and subsequent\nembryonic development ( 34 ). In this regard,\nthe obtained results indicated that the treatment of normal and EMS-induced immature\noocytes with LC during IVM increased the proportion of oocytes that reached the MII\nstage and reduced oocyte degeneration rate. These results are consistent with prior\ncanine and porcine studies demonstrating that adding LC to the IVM medium improved\nnuclear maturation and subsequent embryo development following IVF ( 35 , 36 ).\nIn addition, here, LC improved cleavage and blastocyst rates as well as total\nblastocyst cell numbers when added to the maturation medium of EMS-induced and\nEMS-free mice. In line with this, LC supplementation (1.5-3 mM) to embryo culture\nenhanced lipid metabolism in bovine embryos, most likely by β-oxidation and ATP\nproduction, leading to improved blastocyst development and blastocyst cell numbers\n( 37 ). In this context, Jiang et al.\n( 38 ) reported that supplementation of the\nIVC medium with LC enhanced the development of zygotes from bovine aged oocytes to\nthe blastocyst stage, as well as the quality of the blastocysts. Our results also\nshowed that LC elevated TAC levels in maturation medium, which is in accordance with\na recent study that indicated that supplementing IVM medium with 0.5 mg/mL LC\nsignificantly increased intracellular GSH levels of porcine matured oocytes and\nimproved development competence of parthenogenetic embryos ( 36 ). This effect was attributed to the effect of LC on ROS and\nthus preserving GSH reserves in porcine mature oocytes. Moreover, LC supplementation\nhas been reported to boost the activities of antioxidant enzymes, including\nsuperoxide dismutase, catalase, and glutathione peroxidase, which constitute a\nnatural defense system against oxidant activity ( 38 ). Consistently, our findings revealed that LC was the most effective\nsupplement for enhancing TAC levels in the IVM medium among all treated groups.\nAs shown previously, MSCs secrete various cytokines and growth factors into MSC-CM\nthat can improve  in vitro  meiotic maturation and subsequent\nembryonic developmental potential ( 18 ). In\nthe current study, the co-culture of normal and EMS-induced immature oocytes with\nBMSC-CM improved the quality of the medium and IVM and IVF rates and increased the\nrates of blastocyst production compared to the control group. Our results agree with\nthe study of Ling et al. ( 39 ) in which the\nmaturation rate of mouse oocytes was higher in MSC-CM compared to that in the\ncontrol group. Indeed, recent studies have established that MSC-CM contains a\nvariety of cytokines, growth factors, and anti-apoptotic and antioxidant components\nthat may help in the maintenance of IVM and fertilization rates that are comparable\nto those observed in the control group ( 17 , 18 ). In our findings,\ntreatments containing BMSC-CM also increased TAC levels while diminishing NO levels\nin the IVM medium. The bioactive factors of MSC-CM have the potential to modulate\noxidative stress by decreasing ROS and boosting the expression of antioxidant\nenzymes ( 17 ). Furthermore, a Ca 2+ \nincrease is an early detectable indicator of oocytes activation, stimulating the\nresumption of meiosis and the formation of pronuclear ( 40 ). It is noteworthy that MSC-CM can operate as an effective\nparthenogenetic agent, mimicking the critical events of oocyte activation, including\nCa 2+  elevation, meiosis resumption, pronuclear formation, and\nparthenogenetic development ( 40 ). To sum up,\nwe demonstrated that 25 and 50% BMSC-CM supplementation during IVM improved\nmaturation, fertilization, and the subsequent development of EMS-induced oocytes. We\nalso showed that 50% BMSC-CM was the most beneficial concentration to be used. In\nfact, it resulted in higher maturation and embryo developmental rates than the\ncontrol and other treated groups. However, one of the study limitations was that we\ndid not measure the actual levels of growth factors, pro-inflammatory cytokines,\nanti-apoptotic agents, and antioxidants in the IVM medium. We, therefore, do not\nknow which of the constituents of the BMSC-CM in particular had promoter effects on\nthe oocyte maturation, fertilization, and developmental competence of IVF\nembryos.\nIn conclusion, although the relevance of the results here obtained is limited by the\nuse of an animal model, we demonstrated for the first time that supplementing\nendometriosis-induced oocytes with LC and BMSC-CM during IVM improved their\nmaturation and fertilization rates and subsequent preimplantation embryo development\nfollowing IVF and embryo culture. Among the different supplementations and\nconcentrations examined, 50% BMSC-CM seemed to be the most beneficial one, as it\nresulted in higher rates of morula development on day 5. These novel approaches may\nhave clinical applications in the ARTs setting and may improve fertility outcomes in\nendometriosis-related infertile couples. Nonetheless, more studies are required to\ndetermine the precise molecular and subcellular mechanisms underlying the role of\nRG, LC, and BMSC-CM in oocyte maturation and embryo development of\nendometriosis-derived oocytes.","source_license":"CC-BY-4.0","license_restricted":false}