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A recent porcine study asks how much culture can recover from a small follicle's starting disadvantage.
An oocyte enters an in vitro maturation dish at a convenient experimental beginning. The clock starts, the medium is defined, and treatment groups become visible. Yet the cell has already spent most of its life elsewhere. Its follicle supplied signals, nutrients, and contact with somatic cells before collection. A culture system inherits that history even when the protocol begins at aspiration.
A recent paper in *Scientific Reports* makes this hidden starting point part of the design. Rosenbaum Bartkova and colleagues compared porcine cumulus-oocyte complexes from small and large antral follicles, then asked whether adding fibroblast growth factor 2, leukemia inhibitory factor, and insulin-like growth factor 1 to the maturation medium could improve their developmental competence. The combination is abbreviated FLI. The paper was published online on September 28, 2026, as a peer-reviewed, open-access Article in Press (journal page).
The two-factor design turns a favorable culture result into a harder question: when an intervention improves a cell, how much of the original biological difference has actually been recovered?
The researchers collected ovaries from prepubertal gilts and separated follicles by diameter. Small follicles measured 1 to 2 millimeters; large follicles measured 3 to 6 millimeters. Only cumulus-oocyte complexes with a compact, multilayered cumulus investment were selected. Each follicle group was then split between a TCM199-based control medium and the same medium supplemented with FGF2, LIF, and IGF1.
That produced four groups: small-follicle control, small-follicle FLI, large-follicle control, and large-follicle FLI. The team matured the complexes for 44 hours and assessed nuclear maturation. Mature oocytes were parthenogenetically activated, which allowed cleavage and blastocyst formation to be followed without variation from sperm. The developmental experiment included 350 complexes per group across three independent experiments. Separate fluorescence assays examined mitochondrial distribution, intracellular reactive oxygen species, glutathione, and mitochondrial superoxide, while RT-qPCR measured a selected set of transcripts related to mitochondrial function, oxidative defense, and oocyte competence (full paper).
The structure matters because follicle size and medium are tested together. A treatment-only comparison could show that FLI helps, while a source-only comparison could show that large follicles perform better. The four groups reveal how those two influences combine.
Under the control condition, small-follicle oocytes had the lowest maturation rate: 44.60 percent reached metaphase II, compared with 80.00 percent of large-follicle oocytes. FLI raised the small-follicle rate to 79.40 percent. Statistically, that group did not differ from the large-follicle control. The same broad pattern appeared after activation. Cleavage rose from 51.28 to 79.13 percent in the small-follicle group, and blastocyst formation rose from 21.25 to 35.45 percent.
Those gains still have a boundary. Large-follicle oocytes given FLI produced the highest maturation, cleavage, and blastocyst rates: 89.42, 87.54, and 42.34 percent, respectively. The supplement moved small-follicle oocytes toward the untreated large-follicle reference. It did not make follicular origin irrelevant.
This distinction changes how the result should be described. FLI partly compensated for a starting disadvantage under these laboratory conditions. The experiment does not show that culture can erase developmental history. The best-performing group suggests that origin and environment both continued to shape the outcome.
The cellular measurements give that interpretation more texture. During maturation, mitochondria normally redistribute as energy demands and cytoplasmic organization change. FLI shifted oocytes from both follicle groups away from predominantly homogeneous mitochondrial patterns and toward heterogeneous or clustered patterns. In small-follicle oocytes at metaphase II, for example, clustered mitochondria increased to 19.33 percent with FLI. The large-follicle FLI group reached 33.33 percent.
Redox measurements moved in the same general direction, though not every assay gave the same strength of evidence. In small-follicle oocytes, FLI reduced normalized intracellular reactive oxygen species from 1.11 to 0.71 arbitrary units and increased glutathione from 0.56 to 0.97. Both differences were statistically significant. Mitochondrial superoxide fell from 0.81 to 0.69, but that comparison was not significant. A broad claim that FLI simply suppressed oxidative stress would lose this assay-level difference.
The transcript results were mixed as well. Several genes related to mitochondrial activity or maturation changed with FLI at particular meiotic stages, including *MFN1*, *MTCO1*, *TFB1M*, *GDF9*, and *SOD2*. Other comparisons were unchanged; *BMP15*, for instance, showed no statistically significant difference across the experimental conditions. These measurements support an association between supplementation and cytoplasmic remodeling. They do not establish which growth factor caused each change or which transcript was required for the developmental improvement.
The paper tests a three-factor mixture, not FGF2, LIF, and IGF1 separately. Its strongest evidence concerns the performance of the combined medium across oocytes with different follicular origins.
Parthenogenetic activation is useful here because it isolates maternal developmental capacity from fertilization. It also limits the claim. Cleavage and blastocyst formation after artificial activation cannot establish normal fertilization, embryo transfer success, fetal development, or live birth. The work used selected complexes from pooled abattoir-derived ovaries of prepubertal gilts, so it also cannot show that the same response will hold across individual donors, reproductive ages, health states, or clinical human IVM.
The paper links FLI to redox state, mitochondrial organization, gene expression, and development, but it does not yet order them into a causal chain. Fluorescent intensity and transcript abundance are informative snapshots. They do not show whether redox improvement drives mitochondrial redistribution, whether the redistribution drives competence, or whether both reflect another response to the supplemented medium.
These limits point toward a useful next design. Individual donors could remain identifiable rather than being pooled, and follicle size could stay in the analysis instead of being treated as collection noise. Fertilization and embryo-transfer endpoints would test whether the apparent recovery survives later developmental demands. A component or subtraction experiment could separate the effects of FGF2, LIF, and IGF1. Mechanistic perturbations could then ask which redox or mitochondrial changes are necessary rather than merely present.
For porcine oocyte research, the broader lesson concerns experimental baselines. A treatment effect depends partly on the material that receives the treatment. If small- and large-follicle oocytes respond differently, mixing them before randomization can blur both the starting deficit and the recovery. The source of a cumulus-oocyte complex belongs in the causal model.
Culture can change what an oocyte is able to do next. This paper shows that clearly. It also preserves a second fact that is easier to miss: the cell's earlier environment remains legible after collection. The most informative protocol records both the intervention and the history it is trying to modify.
- Rosenbaum Bartkova A, Sevcikova A, Fecko P, Morovic M, Strejcek F, Nemcova L, Benc M, Laurincik J. "FGF2, LIF, and IGF1 enhance mitochondrial function and developmental competence in porcine oocytes from small antral follicles." *Scientific Reports*. Published online September 28, 2026. Peer-reviewed Article in Press. DOI: 10.1038/s41598-026-73920-x. Open full-text PDF.