In vitro maturation (IVM) is an assisted reproductive technology in which immature oocytes retrieved from small antral follicles undergo maturation in vitro prior to fertilization.1,2 Compared with conventional in vitro fertilization (IVF), IVM offers notable clinical advantages, including minimal or no gonadotropin stimulation, lower overall treatment costs, and complete elimination of the risk of ovarian hyperstimulation syndrome (OHSS).3,4,5,6 These characteristics render IVM particularly advantageous for women with polycystic ovary syndrome (PCOS)—recently recognized under international consensus as polyendocrine metabolic ovarian syndrome (PMOS)7—as well as individuals with high ovarian reserve and patients requiring urgent fertility preservation. Nevertheless, the widespread clinical adoption of IVM remains constrained by lower blastocyst yields and inferior cumulative live birth rates compared with conventional IVF.8,9

A principal factor compromising IVM oocyte developmental competence is the desynchronization between nuclear and cytoplasmic maturation during in vitro culture. In vivo, oocyte meiotic arrest is sustained within the ovarian follicle while the ooplasm progressively accumulates maternal transcripts, proteins, and metabolic organelles essential for fertilization and subsequent embryogenesis. Rapid spontaneous meiotic resumption upon retrieval disrupts this balance. To address this limitation, a biphasic culture strategy termed capacitation-IVM (CAPA-IVM) was developed.10 By incorporating a pre-IVM phase that maintains elevated cyclic adenosine monophosphate (cAMP) levels and sustains gap-junction communication between the oocyte and surrounding cumulus cells, CAPA-IVM promotes coordinated cytoplasmic and nuclear maturation.11 Although CAPA-IVM significantly improves oocyte developmental competence compared with standard IVM, usable blastocyst numbers and cumulative pregnancy rates remain lower than those achieved with conventional IVF, underscoring the necessity for further optimization of the in vitro maturation milieu.8,9

Oocyte developmental competence is governed by dynamic bidirectional paracrine signaling between the oocyte and cumulus cells, mediated predominantly by oocyte-secreted factors (OSFs). Among these, growth differentiation factor 9 (GDF9) and bone morphogenetic protein 15 (BMP15) are pivotal regulators of follicular development and granulosa cell function, acting through downstream SMAD signaling cascades.12,13,14 Exogenous supplementation of IVM media with OSFs has been shown to improve oocyte maturation, fertilization, and embryonic developmental capacity in both human and animal models.15,16,17 Notably, GDF9 and BMP15 form a heterodimer, termed cumulin, that exhibits dramatically higher bioactivity in activating granulosa cell signaling than either homodimer alone.8,14 However, the translational utility of recombinant cumulin is hindered by substantial biochemical challenges related to production yield and purification stability. To circumvent these constraints, a bioengineered variant of human GDF9, designated Super-GDF9 (sGDF9), was developed. This engineered factor exhibits over 1,000-fold greater potency than wild-type GDF9 in activating SMAD2/3 signaling in granulosa cells.14,18 Although sGDF9 operates primarily as a homodimeric GDF9 mimic and activates the SMAD2/3 pathway without concomitant BMP15-mediated SMAD1/5/8 activation, its enhanced bioactivity was hypothesized to strengthen oocyte–cumulus communication and establish a more physiologically supportive follicular environment during IVM.14,18 While supplementation with cumulin or sGDF9 enhances oocyte competence and blastocyst formation in conventional IVM systems,8 its specific impact within biphasic systems such as CAPA-IVM remains poorly defined.

Recent advances in time-lapse imaging technology enable continuous, non-invasive observation of preimplantation embryo development under undisturbed, highly controlled culture conditions. This platform permits precise characterization of morphokinetic parameters—such as the exact timing of early cleavage divisions, cell cycle durations, synchronization intervals, and abnormal division behaviors—that are closely correlated with developmental competence, chromosomal constitution, and implantation potential.19 Consequently, morphokinetic assessment serves as a sensitive tool to evaluate how specific culture media modifications during oocyte maturation influence downstream embryo development.

Given the critical role of OSFs in orchestrating cumulus–oocyte communication and the enhanced potency of sGDF9, rigorous evaluation is needed to determine whether supplementing CAPA-IVM culture with sGDF9 confers developmental benefits during subsequent embryogenesis. Therefore, this study aimed to investigate the impact of sGDF9 supplementation during the pre-IVM and IVM phases of the CAPA-IVM system on subsequent human embryo morphokinetics and cleavage dynamics using time-lapse imaging technology.

This study was a secondary analysis of a prospective, sibling-oocyte clinical trial evaluating CAPA-IVM in women with PMOS, previously described by Vuong et al. (2026).20 The original study protocol was reviewed and approved by the Institutional Ethics Committee of My Duc Hospital (approval reference: 12/24/DD-BVMD; November 25, 2024) and prospectively registered at ClinicalTrials.gov (identifier: NCT06766604). The study was conducted in accordance with the Declaration of Helsinki and the International Council for Harmonisation–Good Clinical Practice (ICH-GCP) guidelines. All participants received comprehensive counseling and provided written informed consent prior to enrollment.

In this secondary analysis of a prospective sibling-oocyte cohort, supplementation of the biphasic CAPA-IVM culture system with 50 ng/mL sGDF9 did not significantly alter early embryo cleavage kinetics or the incidence of cleavage abnormalities after adjusting for patient-level clustering using mixed-effects models. Nevertheless, notable kinetic trends were observed, specifically a non-significant delay in cleavage to the 8-cell stage and a statistically significant prolongation in the time required to achieve full blastocyst expansion (tB).

The substantial overlap in early cleavage timing distributions (t2PN through t8) between the control and sGDF9 groups indicates that sGDF9 supplementation during oocyte maturation does not substantially perturb early preimplantation mitotic divisions. This observation aligns with the established physiological role of GDF9, a member of the transforming growth factor-beta (TGF-β) superfamily, which functions primarily during pre-fertilization folliculogenesis and oocyte–cumulus cell communication rather than directly orchestrating post-fertilization cleavage events.24 Prior investigations have demonstrated the essential contribution of GDF9 to oocyte meiotic resumption and developmental competence, although evidence regarding its direct impact on post-cleavage embryogenesis has remained conflicting.25 A compelling mechanistic explanation for the lack of benefit observed here lies in signaling pathway selectivity: sGDF9 exclusively activates the SMAD2/3 signaling cascade, whereas physiological oocyte paracrine signaling relies on the synergistic actions of GDF9 and BMP15 (natively represented as cumulin), which concurrently stimulate both the SMAD2/3 and SMAD1/5/8 pathways.14 Balanced, dual-pathway activation is critical for orchestrating normal cumulus cell metabolism, extracellular matrix expansion, and steroidogenesis. The isolated activation of SMAD2/3 by sGDF9 in the absence of complementary BMP15 signaling may generate an incomplete or unbalanced signaling state, thereby limiting its capacity to enhance downstream embryonic competence.

Although not reaching statistical significance after multi-testing correction, embryos in the sGDF9 group exhibited an adjusted 5.25-hour delay in reaching the 8-cell stage (t8; p = 0.070), alongside a significant 18.98-hour delay in achieving full blastocyst expansion (tB; p = 0.002). In mammalian embryogenesis, the transition to the 8-cell stage represents the critical window of major embryonic genome activation (EGA), during which developmental control shifts from maternal messenger RNA and protein reserves to de novo embryonic transcription.26,27 Disruptions or delays during EGA are known to impair developmental progression, compromise blastocyst cell allocation, and attenuate implantation competence.27 Morphokinetic investigations have demonstrated that even minor kinetic deviations at key developmental junctions can reflect underlying metabolic or transcriptional stress.28 The delayed blastulation observed in the sGDF9 cohort may indicate that supplementation did not adequately facilitate the accumulation of essential maternal transcripts and metabolic substrates during oocyte maturation required to sustain robust EGA. However, this interpretation must be framed as an exploratory hypothesis: the blastocyst cohort was modest (n = 16 blastocysts), and the adjusted difference at t8 did not reach statistical significance. Blastulation kinetics are inherently multifactorial, governed by embryo intrinsic genetics, microenvironmental culture conditions, and epigenetic remodeling.24,29 Adequately powered studies are required to establish whether this kinetic lag has lasting functional consequences.

Regarding mitotic fidelity, unadjusted analysis revealed a higher frequency of direct cleavage 1 (dc1) in the sGDF9 group (62.0% vs. 45.9%), although this difference was not statistically significant in multivariable mixed-effects modeling (conditional OR: 1.81; 95% CI: 0.84–3.88; p = 0.129). Direct unequal cleavage, particularly during the first mitotic division from 1 to 3 blastomeres, is strongly linked to multipolar spindle formation, chromosome mis-segregation, embryonic aneuploidy, and diminished implantation rates.30 While the elevated incidence of dc1 in the sGDF9 group raises questions regarding potential mitotic destabilization, the lack of statistical significance after accounting for patient clustering indicates that patient-specific variation and baseline cohort characteristics likely account for this observation.

An intriguing insight from our findings is the contrast between single-phase IVM and biphasic CAPA-IVM systems regarding responsiveness to OSF supplementation. In preclinical mouse models, supplementing standard one-step IVM media with sGDF9 or cumulin consistently enhanced cumulus cell mucification, stimulated glycolytic gene expression, and increased blastocyst formation rates.8,24 Conversely, adding sGDF9 during biphasic CAPA-IVM failed to improve meiotic maturation, cleavage, or blastocyst yield in murine experiments, despite inducing transcriptional upregulation of cumulus expansion markers such as Ptgs2, Ptx3, and Adamts1.9,21 This discrepancy likely stems from the specialized biochemical architecture of CAPA-IVM. In CAPA-IVM, the pre-IVM phase employs cAMP modulators to maintain meiotic arrest and preserve transzonal projections, already optimizing cumulus–oocyte metabolic exchange and mitochondrial function.11 Under these optimized conditions, exogenous sGDF9 supplementation may provide redundant stimulation or perturb the fine-tuned metabolic balance achieved during the capacitation phase. Consequently, current evidence suggests that while sGDF9 holds clear promise for optimizing conventional, uncoordinated IVM protocols, its integration into biphasic CAPA-IVM systems requires careful reassessment of dosing, timing, and companion factors.

A principal methodological strength of this study is the application of linear and generalized linear mixed-effects models with patient-level random intercepts. Because human sibling oocytes and embryos derived from the same patient share intrinsic genetic and clinical backgrounds, failing to account for intra-cluster correlation can artificially inflate type I error rates.31 By modeling patient-level clustering, we ensured robust estimation of effect sizes. Furthermore, continuous time-lapse incubation enabled objective, fine-grained temporal tracking under undisturbed culture conditions.

Nevertheless, several limitations warrant consideration. First, as a secondary analysis of a clinical trial originally powered to evaluate oocyte maturation rates rather than embryo morphokinetics, the study had limited statistical power to detect small effect sizes in kinetic intervals. Second, inevitable attrition across extended preimplantation culture markedly reduced sample size at advanced developmental stages, with only 16 embryos (12 control and 4 sGDF9 across seven patients) reaching the fully expanded blastocyst stage. This late-stage attrition compromised the balanced sibling pairing present at earlier stages, meaning that blastocyst-stage kinetic findings must be interpreted strictly as exploratory and conditional upon blastocyst attainment. Finally, preimplantation genetic testing for aneuploidies (PGT-A) was not performed on these research embryos, precluding correlation between observed morphokinetic deviations and chromosomal ploidy.

In conclusion, supplementation of the biphasic CAPA-IVM culture system with 50 ng/mL sGDF9 was not associated with significant alterations in early human embryo cleavage kinetics or the incidence of abnormal cleavage patterns after adjusting for patient-level clustering. Although time to full blastocyst expansion was significantly prolonged in the sGDF9 group, this finding was based on a small subset of blastocysts and should be considered hypothesis-generating. Taken together, these data suggest that the pro-developmental effects of sGDF9 demonstrated in conventional single-step IVM do not readily translate into improved embryogenesis within biphasic CAPA-IVM protocols. Larger, prospectively powered trials evaluating balanced OSF combinations (such as cumulin) across titrated concentrations and incorporating genetic outcomes will be essential to establish the therapeutic utility of growth factor modulation in assisted reproductive technology.