Sichuan University Licenses Novel Ratiometric pH Probe RCPH for Male Infertility Diagnosis at 25,000 RMB Plus Royalties

Recently, the Research Institute of Scientific and Technological Development of Sichuan University released a public notice on the transformation of scientific and technological achievements. The institute intends to license the implementation of patents for ““A Compound RCPH, Its Synthesis Method, Application as a Ratiometric pH Probe, and Product”Relevant patent assignment, with a transaction price of“RMB 25,000 + commission”。

This technology discloses a compound, RCPH, along with its synthesis method and applications as a ratiometric pH probe in related products, falling within the technical field of intracellular pH detection in sperm. The team synthesized a novel compound, RCPH, and employed it as a ratiometric pH probe in sperm pH detection reagents, enabling precise quantitative measurement of intracellular pH in sperm cells. The detection limit is less than 0.15 pH units, thereby allowing differentiation between healthy individuals and patients with asthenozoospermia, and facilitating drug screening and genetic optimization.

Male infertility has become a highly prevalent reproductive health issue worldwide. According to data from the World Health Organization, approximately 9% of couples globally face fertility challenges, with male factors accounting for 30%–50% of these cases.

Sperm Function AssessmentIt is a core component in the diagnosis of male infertility. Intracellular pH (pHi) of sperm, as a key factor regulating sperm motility activation and fertilizing capacity, requires precise detection, which is crucial for identifying the etiology of infertility and guiding assisted reproductive therapy.

In the normal physiological process of transitioning from the acidic environment of the epididymis (pH 6.5–6.8) to the alkaline environment of the female reproductive tract (pH 7.6–7.8), normal spermatozoa must precisely regulate intracellular pH (pHi) to achieve capacitation activation. Any abnormality in pHi will directly result in reduced sperm motility and fertilization failure.

However, sperm pHi detection technology has long faced"Insufficient precision, low spatiotemporal resolution, and poor clinical adaptability"and other multifaceted challenges, making it difficult to meet the needs of clinical diagnosis.

FromIn terms of detection accuracyFrom this perspective, existing technologies exhibit significant limitations. Clinical studies have shown that the difference in sperm intracellular pH (pHi) between successful and failed in vitro fertilization (IVF) groups is merely 0.11 pH units. However, the limits of detection for conventional assays, such as BCECF probes and AuNP-based probes, exceed 0.15 pH units, rendering them incapable of discerning this subtle discrepancy. Consequently, 15% of infertile men with “abnormal pHi but normal conventional semen parameters” are misdiagnosed or overlooked, thereby missing the window for precise intervention.

Insufficient spatiotemporal resolutionThis further limits the clinical application of the technology. In terms of temporal resolution, existing AuNP-based probes require more than 10 minutes to detect changes in intracellular pH (pHi) following progesterone stimulation. However, during sperm capacitation, pHi fluctuates dynamically (for instance, rising from pH 6.70 to 6.84 within just a few minutes). Such a slow response rate precludes real-time tracking of physiological processes, thereby hindering the elucidation of the mechanistic link between pHi and the activation of sperm function.

In terms of spatial resolution, nearly all existing methods can only provide an average pH reading for the entire sperm cell, failing to precisely localize local pH changes in key regions such as the head and the midpiece (the mitochondria-rich region). However, abnormal mitochondrial pH is the core cause of impaired energy metabolism in sperm from patients with asthenozoospermia. The lack of local information limits etiological diagnosis to addressing symptoms rather than the root cause.

Moreover, existing detection technologies still have“High cytotoxicity, limited applicability”drawbacks. Although traditional BCECF probes can achieve a certain degree of intracellular pH (pHi) quantification, they have a significant negative impact on sperm viability. While AuNP probes exhibit slightly lower toxicity, they still interfere with sperm motility. In assisted reproductive technologies (such as IVF), maintaining sperm viability is a prerequisite; this contradiction has rendered most existing techniques difficult to apply in clinical practice.

Meanwhile, most existing detection methods rely on large-scale instruments (such as high-end confocal microscopes) and involve complex operational procedures (requiring professional personnel for sample pretreatment), making them difficult to popularize in primary hospitals and reproductive centers, thereby limiting the clinical coverage of this technology.

More importantly,The core value of “subtype-guided therapy” is missing in the prior artCurrent diagnostic methods can only determine whether intracellular pH (pHi) is abnormal, but struggle to correlate these findings with treatment strategies. For instance, they cannot distinguish whether pHi abnormalities are caused by “bicarbonate transporter defects” or “ion channel dysfunction,” nor can they predict patient responses to interventions such as “glutamine-mediated pHi regulation.” As a result, clinical treatment remains in a stage of “blind trial and error,” with a therapeutic efficacy rate of less than 40%.

The patented technology for the "compound RCPH ratiometric pH probe" transferred this time has its core advantages inConstruct a complete solution based on “dual-fluorescence ratio design” and “multi-dimensional performance optimization,”Achieving breakthroughs across all dimensions—from detection sensitivity and clinical adaptability to functional expansion—it completely overcomes the limitations of traditional sperm pH testing, which relies on low-precision tools.

This technology was first introduced inField of Molecular DesignAchieved disruptive innovation,Successfully overcame the limitations of traditional fluorescent probes, such as “single-signal susceptibility to interference and high toxicity,” and pioneered the design of a “green-red dual-channel ratiometric structure.”

Traditional BCECF probes rely solely on a single fluorescence signal, making them highly susceptible to variations in probe concentration and light source intensity, while also significantly suppressing sperm viability. Although AuNP probes exhibit slightly lower toxicity, they require more than 10 minutes to respond to pH changes.

The invention developed in this patentCompound RCPHBy precisely coupling 7-(diethylamino)-2-oxo-2H-chromene-3-carboxylic acid with fluorescein derivatives, the pH value can be calculated based on the ratio of two fluorescence signals (Ired/Igreen), effectively avoiding errors associated with single-signal measurements.

More critically,Its molecular structure does not contain cytotoxic groups.. Validated by CASA testing, staining sperm at a concentration of 20 μM for 10 minutes resulted in no significant differences in core parameters such as total motility and progressive velocity compared to the blank control group (P > 0.05). This approach thoroughly resolves the clinical challenge of balancing detection with viability preservation, making it particularly suitable for scenarios requiring the maintenance of sperm activity, such as in vitro fertilization (IVF).

InCore Performance Optimizationabove, the probe is used to"Three Highs and One Narrow"Features precisely match clinical needs, achieving“Synergistic Integration of High-Sensitivity Quantification and High Spatiotemporal Resolution”

First, it overcomes the bottleneck of detecting subtle pH differences through high sensitivity:The detection limit of this probe is less than 0.15 pH units, significantly superior to the traditional BCECF probe (which has a detection limit exceeding 0.15 pH units). The standard curve established via flow cytometry (y = 190.3x - 1217, R² = 0.9908) demonstrates that even an intracellular pH fluctuation of only 0.11 units in sperm—a key difference between successful and failed IVF groups—can be accurately captured. Testing results from 68 normal sperm samples and 30 samples from patients with asthenozoospermia confirmed that the probe can clearly distinguish a pH difference of 0.15 units between the two groups (P < 0.01), providing a diagnostic basis for the 15% of infertile men who present with normal conventional semen parameters but abnormal intracellular pH (pHi).

Second, enabling tracking of dynamic physiological processes through high spatiotemporal resolution:In terms of the time dimension, traditional AuNP probes require 10 minutes to detect progesterone-induced sperm alkalinization, whereas the RCPH probe has a response time of less than 1 minute, enabling real-time capture of the dynamic pH increase from 6.70 to 6.84 during sperm capacitation.

In the spatial dimension, laser confocal imaging (excitation wavelength: 405 nm; emission wavelengths: 465 nm/543 nm) enables precise localization of local pH changes in the sperm head (acrosomal region) and midpiece (mitochondria-rich region). Experiments have shown that following progesterone stimulation, the pH value of mitochondria in the sperm midpiece increases by 0.2 units. This localized information provides critical data for elucidating the etiology of “energy metabolism disorders” in asthenozoospermia, thereby overcoming the limitations of traditional assays that can only obtain average pH values.

Third, the narrow pH-responsive range enhances clinical adaptability:The probe exhibits pH-sensitive responsiveness within the range of 6.0–8.0, which precisely covers the physiological pH ranges of spermatozoa (6.5–6.8 in the epididymis and 7.6–7.8 in the female reproductive tract). This characteristic avoids signal interference from traditional probes in non-target pH ranges, effectively reducing such interference and enhancing detection specificity.

Meanwhile, by optimizing the final concentration of the probe (2 μM for flow cytometry and 20 μM for confocal microscopy), it can be adapted to different clinical scenarios:Primary care hospitals can utilize flow cytometers for high-throughput sample screening, while reproductive centers can employ confocal microscopes to conduct detailed etiological analyses, thereby balancing universality with precision.

Furthermore, this technology is in“Multi-Functional Expansion”and"Industrialization Adaptation"Breakthroughs have also been achieved in this aspect.

At the level of functional expansion, the RCPH probe can be applied not only in clinical diagnosis but also supports drug screening and genetic selection. For example, it evaluates the efficacy of drugs in improving sperm motility by quantitatively detecting glutamine-induced changes in sperm intracellular pH (pHi); and provides a molecular basis for sperm selection in IVF technology by monitoring the impact of pH regulation on the frequency of sperm DNA mutations.

In terms of industrialization,The synthesis process of the probe is simple and controllable. It can be prepared through a four-step chemical reaction (carboxyl activation, hydrolysis, cyclization substitution, and coupling). The raw materials used (such as fluorescein and 3-hydroxyphenylpiperazine) are all commercially available analytical grade reagents, requiring no special equipment. Moreover, the reagents exhibit good stability; after storage at 4°C for six months, their detection performance shows no significant decline, demonstrating potential for large-scale production.

In the field of male infertility diagnosis, centered on the core need for "precise assessment of sperm function," multiple enterprises and research institutions have expanded from traditional semen analysis to molecular functional testing, forming a competitive landscape characterized by "basic parameter testing + innovative functional assessment."

Currently, similar products on the market primarily focus on conventional parameters such as sperm motility and DNA fragmentation. A few companies have begun to explore deeper indicators, including sperm pH and ion channel function. With varying technical approaches and R&D progress among different stakeholders, these efforts are collectively advancing the diagnosis of male infertility from “morphological assessment” to “functional analysis.”

Beijing Weili New Century Technology Development Co., Ltd.Core Products"Computer-Aided Sperm Analysis System (CASA)", and has been widely adopted in reproductive centers at hospitals of all levels. Leveraging image recognition technology, the system detects routine parameters such as sperm concentration, motility, and movement trajectories. This product has obtained registration with the National Medical Products Administration (NMPA) and offers significant advantages, including rapid detection (single-sample analysis time ≤ 5 minutes) and automated data processing.

Currently, the product is in a mature stage of promotion, with high market penetration, and serves as the primary equipment for semen analysis in primary healthcare institutions.

CooperSurgical, Inc. (formerly Origio), USAProvided"Sperm Motility Staining Kit", evaluate sperm membrane integrity using SYBR-14/propidium iodide dual staining as a routine functional screening method in assisted reproduction.

From a technical perspective, the ratiometric pH probe technology based on RCPH employed in this transaction integrates high-sensitivity quantitative detection with low-toxicity clinical compatibility. This approach offers a novel paradigm that combines diagnostics, drug screening, and genetic optimization, thereby addressing the core challenges associated with precision, spatiotemporal resolution, and cellular safety in sperm pH testing.