Combined Calcium Ionophore and Pentoxifylline Improve Embryo Quality in Dual Gamete Deficiency Mouse Model
核心洞察
A University of Indonesia study is the first to examine embryo quality when both immature oocytes and immotile sperm are present, using a controlled mouse model.
Calcium ionophore (搜索) (5 μM) significantly improved oocyte maturation (45.3% vs. 32.0%, P < 0.05), while pentoxifylline (10 μM) significantly increased sperm motility versus washed and unwashed controls.
The combined CaI + PTX group achieved the highest proportion of good-quality embryos (42%), compared with CaI alone (25%), PTX alone (18%), and controls (10%).
Researchers at the Faculty of Medicine, University of Indonesia, have demonstrated that combining calcium ionophore (搜索) (CaI) supplementation for immature oocytes with pentoxifylline (PTX) treatment for immotile sperm significantly improves embryo quality in a mouse model. The experimental study, approved by the institution's Ethics Committee (No. KET-1185/UN2.F1/ETIK/PPM.00.02/2020), is the first to investigate the synergistic potential of these two adjuvants to address simultaneous gamete deficiencies—a scenario that remains unexplored despite the individual study of each agent.
In vitro fertilization (IVF) and intracytoplasmic sperm injection (ICSI) are key assisted reproductive technologies, yet global success rates remain modest at around 24%–29%, with challenges intensifying in difficult cases such as immature oocytes and asthenozoospermia (搜索). Immature oocytes can be matured in vitro with CaI supplementation potentially improving outcomes, while PTX can enhance sperm motility by inhibiting phosphodiesterase.
Study Design and Methods
The study used 48 male and 48 female five-week-old Daiichi Doubutsu Yakuhin (DDY) mice (16–27 g) obtained from Bogor Agricultural University and acclimated for two weeks at the University of Indonesia's Indonesia Medical Education and Research Institute (IMERI). Males were divided into three groups—unwashed low-motility sperm, washed sperm without PTX, and washed sperm with PTX—while females were divided into mature oocytes and immature oocytes with and without CaI.
Female mice (8-week-old, average 29 g) were stimulated with 5 IU follicle-stimulating hormone (FSH) (Gonal F) twice, 48 hours apart, followed by 10 IU luteinizing hormone (LH) analog (Ovidrel) 16 hours before collection. Oocytes were denuded using hyaluronidase. Immature oocytes in the treatment group were incubated with 5 μM CaI for 5 minutes, then cultured for 20–24 hours, with maturation assessed by polar body extrusion.
Sperm were collected from the cauda epididymis of male mice (~35.1 g) through swim-out, washed by centrifugation at 1800 × g for 10 minutes, and resuspended in human tubal fluid (HTF) medium. Motility was assessed in a Makler chamber under ×400 magnification, with 200 sperm per sample categorized as rapid progressive, nonprogressive, or immotile. PTX (10 μM) was added to the treatment group 30 minutes before IVF.
For conventional IVF, oocytes were placed in 10 μl cleavage medium containing human serum albumin (HSA), gentamicin, and nutrients, followed by addition of 1 μl (~0.1 × 10⁶ sperm). After insemination, oocytes were transferred to G1 medium (Vitrolife) and incubated at 37°C in 5% O₂, 6% CO₂, and 89% N₂. At the 6–8 cell stage (day 2), embryos were moved to G2 medium (Vitrolife). Embryo quality was assessed on days 1 and 3 using a modified Gardner system based on cell count, uniformity, and fragmentation, with embryos classified as good, moderate, or poor (moderate and poor combined into a single "poor" category for analysis).
Key Findings
Calcium ionophore (搜索) significantly improved oocyte maturation. After 24 hours, maturation—measured by polar body extrusion—was significantly higher in the CaI group (45.3% vs. 32.0%, P < 0.05). Notably, parthenogenesis occurred in 39.6% of CaI-treated oocytes. Of the collected oocytes, 67.5% were immature, and first polar body (PB1) extrusion was used as a noninvasive maturation marker.
Pentoxifylline significantly enhanced sperm motility. Sperm motility differed significantly across the three groups (P < 0.05, Kruskal–Wallis test). Post hoc analysis showed no difference between unwashed and washed-without-PTX samples (P > 0.05), but PTX-supplemented sperm had significantly higher motility than both other groups (P < 0.05 for each). The study revealed that 84.5% of unwashed sperm were immotile and only 26.5% viable.
For embryo development, mature oocytes yielded more 2-cell (64% vs. 42%) and 8-cell (28% vs. 16%) embryos than immature oocytes. Among poor-quality gametes, all treatments improved embryo quality versus controls by day 3 (P < 0.05). The CaI + PTX combination group had the highest proportion of good-quality embryos (42%), followed by CaI alone (25%), PTX alone (18%), and controls (10%).
Mechanistic Insights
CaI (5 μM) improved oocyte maturation by triggering maturation-promoting factor (搜索) (MPF) activation and meiotic progression. Calcium regulates maturation through ion channels and store-operated Ca²⁺ entry (SOCE), which modulates oscillations and signaling. CaI activates Ca²⁺/Calmodulin-Dependent Protein Kinase II (CaMKII) to mediate chromosome transitions and cell cycle progression. However, CaI alone produces a single abnormal calcium transient, unlike physiological oscillations, which may impair long-term development and induce cytotoxicity.
PTX increased intracellular cyclic AMP (cAMP) levels, enhancing motility, hyperactivation, capacitation, and acrosome reaction, while exerting antioxidant effects by reducing reactive oxygen species and lipid peroxidation. PTX also increased lateral head displacement and curvilinear velocity but reduced linearity and straightness, indicating more erratic movement. These changes are linked to increased ATP and mitochondrial membrane potential, supporting embryonic spindle organization and cleavage. Improved motility also reflects higher phospholipase C zeta (搜索) (PLCζ) levels, which trigger oocyte calcium oscillations through cAMP and phosphoinositide pathways enhanced by PTX.
Clinical Implications and Limitations
Immature oocytes are rarely used in assisted reproductive technology (ART) due to lower pregnancy rates, but may be utilized in difficult cycles. In this study, in vitro maturation (IVM) oocytes had lower fertilization rates than mature oocytes (31% vs. 63.9%), consistent with maturation failure causing aneuploidy and poor development. PTX only transiently stimulates motility without improving genomic integrity, meaning selected sperm may still carry DNA damage that compromises embryo development.
The authors note several limitations. First, reliance on PB1 extrusion and early cleavage stages as markers provided only a partial assessment of cytoplasmic maturation and long-term embryo potential, and could not reliably distinguish embryos from parthenotes. Second, the use of a mouse model was necessary due to the rarity of human cases with 100% immotile sperm and immature oocytes. Finally, individual variability in response to superovulation may have influenced results.
The study concludes that PTX supplementation can increase sperm motility in immotile sperm, while CaI supplementation can increase oocyte maturation in immature oocytes. The combined PTX and CaI intervention strategy significantly improves subsequent embryo quality in a mouse model, offering a promising approach for managing cases of combined sperm and oocyte deficiencies—though the combination did not yield significantly better outcomes than either treatment alone.