Intelligent response type cervical auxiliary fertility gel

Abstract

The invention relates to the technical field of medicines, in particular to an intelligent response type cervical auxiliary fertility gel which comprises the following components in percentage by mass: 0.1-2% of a gel matrix which is selected from one or more of hydroxypropyl methyl cellulose, hydroxyethyl cellulose, sodium alginate and gellan gum; 0.05%-0.5% of a gel adjusting auxiliary agent which is selected from zinc chloride or calcium lactate; the osmotic pressure regulator is selected from one or more of glycerol, glucose, sodium chloride, mannitol, sorbitol, polyethylene glycol and propylene glycol; 0.1%-2% of an acid-base regulator which is selected from one or more of disodium hydrogen phosphate, sodium dihydrogen phosphate, dipotassium phosphate and potassium dihydrogen phosphate; according to the present invention, the toxicity grade of the gel on cervical cells is less than or equal to grade 2 (ISO 10993-5 standard), the cell survival rate is more than or equal to 70%, and the safety is high.

Description

Technical Field

[0001] The present invention relates to the field of medical technology, and in particular to an intelligent responsive cervical assisted reproductive gel. Background Art

[0002] The cervix is ​​the primary barrier for sperm to enter the female reproductive system, and the physical and chemical properties of its mucus directly affect the sperm's ability to penetrate. Under pathological conditions, abnormal cervical mucus will hinder the passage of sperm, leading to infertility in approximately 10%-15% of women. Existing solutions such as intrauterine insemination (IUI), cervical mucus regulating drugs (such as estrogen), and in vitro fertilization (IVF) have problems such as high cost, limited effect, or large side effects. In addition, there are limited treatment options for patients with weak sperm and oligospermia. Traditional methods (such as hormone therapy and surgery) are only effective for some patients, and assisted reproductive technologies (such as IUI and IVF) are costly and invasive.

[0003] Limitations of existing technologies include:

[0004] Intrauterine insemination (IUI): This procedure bypasses the cervix and injects sperm directly into the uterus. However, it requires a medical procedure, is costly, and may cause infection or uterine cramping.

[0005] Cervical mucus regulating drugs (such as estrogen): limited effect, ineffective for immune infertility, and there is a risk of hormonal side effects.

[0006] In vitro fertilization (IVF): completely avoids the cervix, but is expensive and ethically controversial.

[0007] Saline irrigation, enzyme treatment, etc.: temporarily change the viscosity of mucus, but lack targeting and may disrupt the balance of vaginal microecology.

[0008] Therefore, a safe and non-invasive solution that can simulate the characteristics of ovulatory cervical mucus in situ is urgently needed. The ideal product should have the following characteristics:

[0009] Sperm survival rate>80%;

[0010] Biocompatibility: Does not affect the original flora of the vagina / cervix;

[0011] Intelligent response: Dynamically adjust mucus osmotic pressure and pH according to the cervical environment;

[0012] Long-acting: It is placed before sexual intercourse and lasts for several hours to several days. Summary of the Invention

[0013] The purpose of the present invention is to provide an intelligent responsive cervical fertility-assisting gel, which is injected deep into the vagina through a vaginal applicator. Through the intelligent regulation of the in situ gel, the characteristics of cervical mucus during ovulation are simulated to promote sperm penetration, thereby assisting fertility.

[0014] To achieve the above objectives, the present invention is implemented through the following technical solutions:

[0015] The present invention provides an intelligent responsive cervical assisted reproductive gel, comprising the following components and their mass percentages:

[0016] Gel matrix: 0.1% to 2%, one or more selected from hydroxypropyl methylcellulose, hydroxyethyl cellulose, sodium alginate, and gellan gum;

[0017] Gel regulating adjuvant: 0.05% to 0.5%, selected from zinc chloride or calcium lactate;

[0018] Osmotic pressure regulator: 0.1% to 5%, selected from one or more of glycerol, glucose, sodium chloride, mannitol, sorbitol, polyethylene glycol, and propylene glycol;

[0019] Acid-base regulator: 0.1% to 2%, selected from one or more of disodium hydrogen phosphate, sodium dihydrogen phosphate, dipotassium hydrogen phosphate, and potassium dihydrogen phosphate;

[0020] Preservatives: 0.01% to 0.05%, one or more selected from polyhexamethylene biguanide, methylparaben, propylparaben, phenoxyethanol, and octanol;

[0021] The balance is water;

[0022] The dynamic viscosity of the gel is 50-200 Pa·s, the pH value is 7.0-8.0, the osmotic pressure is 270-290 mOsm / kg, the sperm survival rate is ≥80%, and the toxicity level to cervical cells is ≤level 2.

[0023] Furthermore, the gel matrix is ​​hydroxypropyl methylcellulose with a concentration of 0.5% to 1%, and the gel regulating adjuvant is zinc chloride with a concentration of 0.1% to 0.5%.

[0024] Furthermore, the gel matrix is ​​sodium alginate with a concentration of 0.3% to 1.0%, and the gel regulating adjuvant is calcium lactate with a concentration of 0.05% to 0.1%.

[0025] Furthermore, the gel matrix is ​​a mixture of hydroxypropyl methylcellulose and hydroxyethyl cellulose, wherein the concentration of hydroxypropyl methylcellulose is 0.5% to 1%, and the concentration of hydroxyethyl cellulose is 0.5% to 1.2%.

[0026] Furthermore, the osmotic pressure regulator is a mixture of glycerol and glucose, with the glycerol concentration being 0.5% to 2% and the glucose concentration being 0.5% to 2%.

[0027] Furthermore, the acid-base regulator is a mixture of disodium hydrogen phosphate and sodium dihydrogen phosphate, the concentration of disodium hydrogen phosphate is 0.5% to 1.5%, and the concentration of sodium dihydrogen phosphate is 0.05% to 0.2%.

[0028] Furthermore, the preservative is polyhexamethylene biguanide with a concentration of 0.01% to 0.03%.

[0029] Furthermore, the preparation method of the gel comprises the following steps:

[0030] a. Dissolve the gel matrix and gel conditioning agent in a portion of water and allow to swell for 12 hours;

[0031] b. Dissolve the osmotic pressure regulator, acid-base regulator, and preservative in the remaining water and stir evenly;

[0032] c. Mix the solutions from step 1 and step 2, heat to 40°C to 60°C, homogenize and emulsify, and then fill.

[0033] Furthermore, the gel can dynamically adjust the osmotic pressure and pH in the cervical environment, simulate the characteristics of cervical mucus during ovulation, and promote sperm penetration.

[0034] Furthermore, the gel has no negative impact on the native microbial environment of the vagina and cervix, and the growth rate of lactobacilli is less than 5% different from that of the control group.

[0035] Compared with the prior art, the technical solution provided by the present invention has the following beneficial effects:

[0036] Excellent biocompatibility and safety

[0037] The gel's toxicity level to cervical cells is ≤ level 2 (ISO 10993-5 standard), the cell survival rate is ≥70%, and the survival rate of some experimental groups (such as E1, E2, E5, E7, and E9) exceeds 80%, indicating high safety.

[0038] Animal experiments have shown that the gel is non-irritating to the cervical mucosa (Draize score ≤ 0.7 points) and non-sensitizing (sensitization rate ≤ 10%), complies with ISO 10993-10 standards, and is suitable for long-term use.

[0039] Highly simulates the characteristics of ovulation cervical mucus

[0040] The dynamic viscosity (50-200 Pa·s), pH value (7.0-8.0) and osmotic pressure (270-290 mOsm / kg) are highly consistent with natural ovulation cervical mucus, effectively promoting sperm penetration (penetration speed ≥3.8 mm / h, penetration rate ≥76%).

[0041] By intelligently adjusting the osmotic pressure and pH, it adapts to different cervical environments and solves the problem of abnormal physical and chemical properties of mucus in the non-ovulation period.

[0042] Significantly improve sperm survival rate and motility

[0043] The sperm survival rate for 6 hours is ≥80%, and the proportion of forward-moving sperm (PR%) is 62% to 72%, which is better than traditional assisted reproductive methods (such as hormone therapy).

[0044] The experimental groups E4 and E5 performed particularly well, with survival rates reaching 85% and 86% and PR% reaching 70% and 72%, respectively, which are close to the optimal state under physiological conditions.

[0045] Protect the balance of vaginal microecology

[0046] It has no inhibitory effect on lactobacilli (such as Lactobacillus crispatus and Lactobacillus jensenii) (the diameter of the inhibition zone is 0 mm), and the growth rate difference with the control group is <5%, ensuring the stability of the native vaginal microbial environment and reducing the risk of infection.

[0047] The preparation process is simple and the cost is controllable

[0048] It adopts conventional homogenization and emulsification technology, does not require complex equipment, and is suitable for large-scale production.

[0049] The components are all commonly used raw materials in the pharmaceutical field (such as hydroxypropyl methylcellulose, zinc chloride, etc.), and the cost is significantly lower than artificial insemination (IUI) or in vitro fertilization (IVF) technology.

[0050] Non-invasive and widely applicable

[0051] It can be used through a vaginal applicator, avoiding the side effects of surgery or hormonal intervention. It is especially suitable for patients with asthenospermia, oligospermia and infertile women with abnormal cervical mucus.

[0052] It can dynamically adjust the properties of mucus, prolong the effective action time (from hours to days), and increase the chance of conception. DETAILED DESCRIPTION

[0053] To make the objectives, technical solutions, and advantages of the embodiments of the present invention more clear, the technical solutions in the embodiments of the present invention are described clearly and completely below. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making any creative efforts are within the scope of protection of the present invention.

[0054] The present invention will be further described below with reference to the embodiments.

[0055] Example 1

[0056] Preparation of intelligent responsive cervical gel containing hydroxypropyl methylcellulose

[0057] Prescription: Amount: 1000g

[0058]

[0059] Preparation process:

[0060] 1. Weigh the prescribed amount of hydroxypropyl methylcellulose, use 60% of the prescribed amount of water to swell for 12 hours, add the prescribed amount of zinc chloride, stir thoroughly to dissolve and set aside.

[0061] 2. Weigh the prescribed amount of glucose, sodium chloride, disodium hydrogen phosphate, sodium dihydrogen phosphate, and polyhexamethylene biguanide, add the remaining amount of water, and stir evenly. Heat to 50°C to 60°C, then add the reserved solution from step 1. Homogenize in a homogenizer at 2500 rpm for 30 minutes, and fill into a vaginal applicator to obtain the intelligent responsive cervical gel containing hydroxypropyl methylcellulose, which can be used to assist fertility.

[0062] Example 2

[0063] Preparation of intelligent responsive cervical gel containing hydroxyethyl cellulose

[0064] Prescription: Amount: 1000g

[0065]

[0066] Preparation process:

[0067] 1. Weigh the prescribed amount of hydroxyethyl cellulose, use 60% of the prescribed amount of water to swell for 12 hours, add the prescribed amount of zinc chloride, stir thoroughly to dissolve and set aside.

[0068] 2. Weigh the prescribed amount of glucose, sodium chloride, disodium hydrogen phosphate, sodium dihydrogen phosphate, methylparaben, and ethylparaben, add the balance of water, and stir evenly. Heat to 50°C to 60°C, then add the reserved solution from step 1. Homogenize in a homogenizing emulsifier at 2500 rpm for 30 minutes, and fill into a vaginal applicator to obtain the hydroxyethyl cellulose-containing intelligent responsive cervical gel, which can be used to assist fertility.

[0069] Example 3

[0070] Preparation of intelligent responsive cervical gel containing sodium alginate

[0071] Prescription: Amount: 1000g

[0072]

[0073] Preparation process:

[0074] 1. Weigh the prescribed amount of sodium alginate and use 60% of the prescribed amount of water to swell for 12 hours. Add the prescribed amount of calcium lactate and stir thoroughly to dissolve.

[0075] 2. Weigh the prescribed amount of mannitol, glucose, dipotassium hydrogen phosphate, potassium dihydrogen phosphate, and phenoxyethanol, add the balance of water, and stir evenly. Heat to 40°C to 50°C, then add the reserved solution from step 1. Homogenize in a homogenizer at 2500 rpm for 30 minutes, and pour into a vaginal applicator to obtain a sodium alginate-containing intelligent responsive cervical gel, which can be used to assist fertility.

[0076] Example 4

[0077] Preparation of smart responsive cervical gel containing gellan gum

[0078] Prescription: Amount: 1000g

[0079]

[0080]

[0081] Preparation process:

[0082] 1. Weigh the prescribed amount of gellan gum and use 50% of the prescribed amount of water to swell for 12 hours.

[0083] 2. Weigh the prescribed amount of sorbitol, propylene glycol, dipotassium hydrogen phosphate, potassium dihydrogen phosphate, and ethanol, add the balance of water, and stir evenly. Heat to 40°C-50°C, then add the reserved solution from step 1. Homogenize in a homogenizer at 2500 rpm for 30 minutes, and pour into a vaginal applicator to obtain the gellan gum-containing intelligent responsive cervical gel, which can be used to assist fertility.

[0084] Example 5

[0085] Preparation of intelligent responsive cervical gel containing hydroxypropyl methylcellulose and hydroxyethyl cellulose: Preparation amount: 1000g

[0086]

[0087]

[0088] Preparation process:

[0089] 1. Weigh the prescribed amount of hydroxypropyl methylcellulose and hydroxyethyl cellulose, use 60% of the prescribed amount of water to swell for 12 hours, add the prescribed amount of zinc chloride, stir thoroughly to dissolve and set aside.

[0090] 2. Weigh the prescribed amount of sodium chloride, glucose, disodium hydrogen phosphate, sodium dihydrogen phosphate, and polyhexamethylene biguanide, add the remaining amount of water, and stir evenly. Heat to 40°C to 50°C, then add the reserved solution from step 1. Homogenize in a homogenizer at 2500 rpm for 30 minutes, and fill into a vaginal applicator to obtain a smart responsive cervical gel containing hydroxypropyl methylcellulose and hydroxyethyl cellulose, which can be used to assist fertility.

[0091] Example 6

[0092] Preparation of intelligent responsive cervical gel containing hydroxypropyl methylcellulose and hydroxyethyl cellulose: Preparation amount: 1000g

[0093]

[0094]

[0095] Preparation process:

[0096] 1. Weigh the prescribed amount of hydroxypropyl methylcellulose and hydroxyethyl cellulose, use 60% of the prescribed amount of water to swell for 12 hours, add the prescribed amount of zinc chloride, stir thoroughly to dissolve and set aside.

[0097] 2. Weigh the prescribed amount of glycerin, sorbitol, dipotassium hydrogen phosphate, potassium dihydrogen phosphate, methylparaben, and ethylparaben, add the balance of water, and stir evenly. Heat to 40°C to 50°C, then add the reserved solution from step 1. Homogenize in a homogenizing emulsifier at 2500 rpm for 30 minutes, and fill into a vaginal applicator to obtain a smart responsive cervical gel containing hydroxypropyl methylcellulose and hydroxyethylcellulose, which can be used to assist fertility.

[0098] Example 7

[0099] Preparation of intelligent responsive cervical gel containing hydroxypropyl methylcellulose and gellan gum

[0100] Prescription: Amount: 1000g

[0101]

[0102]

[0103] Preparation process:

[0104] 1. Weigh the prescribed amount of hydroxypropyl methylcellulose and gellan gum, use 60% of the prescribed amount of water to swell for 12 hours, add the prescribed amount of zinc chloride, stir thoroughly to dissolve and set aside.

[0105] 2. Weigh the prescribed amount of glycerin, polyethylene glycol, dipotassium hydrogen phosphate, potassium dihydrogen phosphate, methylparaben, and ethylparaben, add the balance of water, and stir evenly. Heat to 40°C to 50°C, then add the reserved solution from step 1. Homogenize in a homogenizer at 2500 rpm for 30 minutes, and fill into a vaginal applicator to obtain a smart responsive cervical gel containing hydroxypropyl methylcellulose and gellan gum, which can be used to assist fertility.

[0106] Example 8

[0107] Preparation of intelligent responsive cervical gel containing hydroxyethyl cellulose and gellan gum

[0108] Prescription: Amount: 1000g

[0109]

[0110] Preparation process:

[0111] 1. Weigh the prescribed amount of hydroxypropyl methylcellulose and gellan gum, use 60% of the prescribed amount of water to swell for 12 hours, add the prescribed amount of zinc chloride, stir thoroughly to dissolve and set aside.

[0112] 2. Weigh the prescribed amount of sodium chloride, mannitol, dipotassium hydrogen phosphate, potassium dihydrogen phosphate, and polyhexamethylene biguanide, add the balance of water, and stir evenly. Heat to 40°C to 50°C, then add the reserved solution from step 1. Homogenize in a homogenizer at 2500 rpm for 30 minutes, and pour into a vaginal applicator to obtain the intelligent responsive cervical gel containing hydroxyethyl cellulose and gellan gum, which can be used to assist fertility.

[0113] Example 9

[0114] Preparation of intelligent responsive cervical gel containing hydroxypropyl methylcellulose, hydroxyethyl cellulose and gellan gum

[0115] Prescription: Amount: 1000g

[0116]

[0117] Preparation process:

[0118] 1. Weigh the prescribed amount of hydroxypropyl methylcellulose, hydroxyethyl cellulose and gellan gum, use 60% of the prescribed amount of water to swell for 12 hours, add the prescribed amount of zinc chloride, stir thoroughly to dissolve and set aside.

[0119] 2. Weigh the prescribed amount of sodium chloride, dipotassium hydrogen phosphate, potassium dihydrogen phosphate, and polyhexamethylene biguanide, add the balance of water, and stir evenly. Heat to 40°C to 50°C, then add the reserved solution from step 1. Homogenize in a homogenizer at 2500 rpm for 30 minutes, and fill into a vaginal applicator to obtain a smart responsive cervical gel containing hydroxyethyl cellulose, hydroxypropyl methylcellulose, and gellan gum, which can be used to assist fertility.

[0120] Example 10

[0121] Preparation of intelligent responsive cervical gel containing sodium alginate and gellan gum

[0122] Prescription: Amount: 1000g

[0123]

[0124] Preparation process:

[0125] 1. Weigh the prescribed amount of sodium alginate and gellan gum and swell them with 60% of the prescribed amount of water for 12 hours. Add the prescribed amount of calcium lactate and stir thoroughly to dissolve.

[0126] 2. Weigh the prescribed amount of sodium chloride, glucose, dipotassium hydrogen phosphate, potassium dihydrogen phosphate, and phenoxyethanol, add the balance of water, and stir evenly. Heat to 50°C to 60°C, then add the reserved solution from step 1. Homogenize in a homogenizer at 2500 rpm for 30 minutes, and pour into a vaginal applicator to obtain a smart responsive cervical gel containing sodium alginate and gellan gum, which can be used to assist fertility.

[0127] Study on the physical properties of intelligent responsive cervical assisted reproductive gel

[0128] During the experiment, fresh cervical mucus, whether human or bovine, is difficult to obtain in large quantities, and once collected from the source, it degrades rapidly, which may lose the accuracy of comparative studies. Therefore, the need for synthetic substitutes is very important when developing and testing vaginal preparations. Reference (Katz DF, Contraception, 55(5):315-319, 1997) developed a polymer-based cervical mucus substitute, so the study of intelligent responsive cervical gel can be used as a control.

[0129] Experimental methods

[0130] Parameter settings

[0131]

[0132] Control and experimental groups

[0133] Preparation of reference substance (synthetic cervical mucus preparation C1)

[0134] composition Concentration (W / W) guar gum 1％ Porcine gastric mucosa (type III) 0.5％ Midalamide 0.3％ Methylparaben 0.15％ Propylparaben 0.02％ Dipotassium hydrogen phosphate 0.26％ Potassium dihydrogen phosphate 1.57％ water 96.2％

[0135] Experimental group preparation: from Examples 1 to 10, the corresponding numbers are experimental groups E1 to E10.

[0136] Experimental method for dynamic viscosity:

[0137] 1.1 Experimental equipment and materials: rotational rheometer (TA Instruments AR-G2), cone and plate, water bath.

[0138] 1.2 Experimental methods:

[0139] Samples C1, E1 to E10 were pretreated by centrifugation (3000 rpm, 5 min) to remove air bubbles and allowed to stand for 10 min before loading.

[0140] Loading: Pipette 500 μL of sample onto the center of the lower plate, slowly lower the plate to a 1 mm gap, scrape off any excess material from the edges, and ensure there are no gaps. Run: Automatically scan the shear rate according to the preset program, repeating 3 times per set.

[0141] 1.3 Experimental Results

[0142] The dynamic viscosity (η) of cervical mucus samples is shown in Table 1

[0143] Test conditions: TA AR-G2, parallel plates 20 mm, gap 1 mm, 37°C, shear rate 0.1-100 s -1 .

[0144] Table 1 Dynamic viscosity (η) of cervical mucus samples

[0145]

[0146] Conclusion: From the data in Table 1, we can see that all samples are within 0.1s. -1 The viscosity coefficient of variation (CV) was 12.3% (<15% target) under the conditions of 0.1 to 100 s, indicating rheological consistency. -1 The viscosity decreased by 98.5-99.3%, showing shear sensitivity, and the viscosity range was (50-200) Pa·s. Based on the WHO Guidelines for Semen Analysis (2010), the dynamic viscosity was <200 Pa·s (0.1s -1 ) does not hinder sperm migration. The viscosity of the samples used in this study (100-150 Pa·s) preserves the mucus barrier function while ensuring reproductive compatibility. Therefore, it closely matches the rheological properties of natural cervical mucus and meets the application requirements of assisted reproductive technology.

[0147] Permeability test method:

[0148] Refer to the WHO (2010) sperm penetration test standard to simulate the migration ability of sperm in cervical mucus.

[0149] 1.1 Sample preparation:

[0150] Cervical mucus samples (C1 / E1-E10) were injected into capillary tubes (10 mm long, 1 mm inner diameter, and sealed at one end).

[0151] Control group: HTF medium (Human Tubal Fluid).

[0152] 1.2 Sperm loading:

[0153] Use semen from healthy donors (concentration ≥ 15 × 10 6 / mL, activity ≥40%), and placed at the open end of the capillary tube after liquefaction.

[0154] 1.3 Migration Observation:

[0155] Place vertically at 37°C and 5% CO2 for 1 hour.

[0156] Measure the sperm leading edge migration distance (mm) and penetration density (sperm number / mm 2 ).

[0157] 1.4 Data Analysis

[0158] Penetration speed (mm / h) = migration distance / time

[0159] Penetration rate (%) = (penetration distance of experimental group / penetration distance of control group) × 100

[0160] 2.1 Fluorescence-labeled macromolecular diffusion assay (FRAP)

[0161] 2.2 Sample processing: The sample was mixed with fluorescently labeled IgG (150 kDa) or nanoparticles (200 nm) (final concentration 0.1 mg / mL).

[0162] 2.3 Photobleaching and recovery: Laser bleaching was performed on a selected area (50 μm in diameter) using a confocal microscope.

[0163] The fluorescence intensity recovery curve was recorded (30 s, 1 s interval).

[0164] 2.4 Diffusion coefficient calculation:

[0165] Fit the recovery curve and calculate the apparent diffusion coefficient (D) according to the formula:

[0166]

[0167] (r: bleaching area radius, t 1 / 2: half recovery time)

[0168] 3 Experimental results

[0169] The sperm penetration test results are shown in Table 2 , and the FRAP diffusion coefficient (D) is shown in Table 3

[0170] Table 2: Sperm penetration test results

[0171] Test conditions: Time: 1 hour, Temperature: 37°C. Control group (HTF medium) Penetration distance: 5.0 mm

[0172] Sperm concentration: ≥15×10 6 / mL, activity ≥40%

[0173]

[0174]

[0175] Table 3: FRAP Diffusion Coefficients (D)

[0176] Test conditions: Fluorescent marker: IgG (150kDa), 200nm fluorescent particles, bleaching area diameter: 50μm, recovery time: 30s

[0177] Group <![CDATA[IgG(×10 -3 μm 2 / s)]]> <![CDATA[200nm particles (×10 -3 μm 2 / s)]]> C1 1.2±0.1 0.5±0.05 E1 1.5±0.2 0.7±0.06 E2 1.0±0.1 0.4±0.04 E3 1.4±0.1 0.6±0.05 E4 1.1±0.1 0.5±0.04 E5 1.6±0.2 0.8±0.07 E6 1.3±0.1 0.5±0.05 E7 0.9±0.1 0.3±0.03 E8 1.4±0.1 0.6±0.05 E9 1.2±0.1 0.5±0.04 E10 1.3±0.1 0.6±0.05

[0178] Conclusion: The sperm penetration velocity (3.8–5.0 mm / h) of all samples (E1-E10) reached the clinical standard (≥3 mm / h), with E1, E5, and E8 performing the best and being consistent with natural cervical mucus (4–6 mm / h). The IgG diffusion coefficient (0.9–1.6×10-3 μm 2 / s) simulates the nutrient transport function of cervical mucus, while the 200nm particles are diffusion-limited (0.3–0.8×10-3μm 2 The cervical mucus membrane ( / s) replicates its physical barrier effect against pathogens. Therefore, it can achieve the permeability of natural cervical mucus and meet the application requirements of assisted reproduction.

[0179] Study on osmotic buffering

[0180] With reference to Katz, DF (1997) - Osmotic Pressure Regulation Mechanism of Cervical Mucus and WHO (2010) - Standards for Human Reproduction Technology, the test samples were tested to see whether they had an osmotic pressure buffering effect on cervical fluid to reach the range of 270-290 mOsm / kg during ovulation.

[0181] 1.1 Sample preparation: Mix C1 with E1-E10 at a ratio of 1:9 (v / v) (simulating the clinical addition ratio)

[0182] 1.2 Osmotic pressure determination

[0183] Instrument: Wescor 5520 Vapor Pressure Osmometer.

[0184] step:

[0185] Calibrate the instrument (300mOsm / kg standard solution).

[0186] 10 μL of sample was loaded and read after 5 min of equilibration (repeated 3 times).

[0187] Calculate mean ± SD

[0188] 1.3 Data Analysis: One-way ANOVA was used to compare the differences between the mixed group and the standard range of ovulation (270-290 mOsm / kg).

[0189] 1.4 Experimental Results

[0190] The osmotic pressure values ​​(mOsm / kg) of the mixed samples are shown in Table 4

[0191] Table 4 Osmotic pressure measurement values ​​(mOsm / kg)

[0192]

[0193] Conclusion: Since the osmotic pressure of non-ovulatory cervical mucus is typically lower than that of ovulatory cervical mucus, ranging from approximately 240-260 mOsm / kg, the osmotic pressure regulator in the sample stabilizes the osmotic pressure of the mixed sample within the standard range for ovulatory cervical mucus (270-290 mOsm / kg). Therefore, the osmotic pressure characteristics of the mixed sample are highly consistent with those of natural ovulatory cervical mucus, meeting the application requirements of assisted reproductive technology.

[0194] Study on pH buffering

[0195] Refer to Gipson et al. (2001) - Biol Reprod (Cervical mucus buffer mechanism) and Chretien et al. (1973)

[0196] -Fertil Steril (pH and reproductive function) tests whether the sample has a buffering effect on the cervical fluid to bring it to the pH range of 7.0 to 8.0 during ovulation.

[0197] 1.1 Sample preparation: Mix C1 with E1-E10 at a ratio of 1:4 (v / v) (simulating the clinical addition ratio)

[0198] 1.2 pH determination

[0199] Instrument: Metrohm pH meter (microelectrode, accuracy ±0.01).

[0200] Procedure: Calibrate the instrument (pH 4.0 / 7.0 / 10.0 standard buffer).

[0201] Take 10 mL of sample, equilibrate at 37°C for 10 min, and then measure (repeat 3 times).

[0202] Mean ± SD were recorded.

[0203] Data Analysis

[0204] The pH changes before and after mixing were compared by paired t-test.

[0205] 1.3 Experimental Results

[0206] The pH of the mixed sample is shown in Table 5

[0207] Table 5 pH measurement values

[0208]

[0209] Conclusion: Because the pH of non-ovulatory cervical mucus is typically lower than that of ovulatory cervical mucus, ranging from approximately 6.5 to 7.0, pH adjustment of the sample stabilizes the pH of the mixed sample within the standard range for ovulatory cervical mucus (7.0 to 8.0). Therefore, this pH closely matches the pH characteristics of natural ovulatory cervical mucus and meets the application requirements of assisted reproductive technology.

[0210] Study on the biological properties of intelligent responsive cervical assisted reproductive gel

[0211] Study on the effect of intelligent responsive cervical gel on sperm survival rate

[0212] Sample preparation:

[0213] 1.1 The reference and experimental groups were derived from the aforementioned physical properties study: reference C1, and experimental groups E1 to E10.

[0214] 1.2 Sperm Sample:

[0215] Source: Healthy donors (abstinence for 3-5 days), semen parameters meet WHO standards (concentration ≥15×10 6 / mL, motility ≥40%, progressive motility ≥32%).

[0216] Treatment: After liquefaction, high-motility sperm were extracted by density gradient centrifugation (80% / 40% Percoll) and resuspended in HTF medium (Human Tubal Fluid).

[0217] 1.3 Incubation: 50ul sperm suspension (10×10 6 / mL) was mixed with 150ul sample in a transparent capillary (length 10mm, inner diameter 1mm) and incubated at 37°C, 5% CO2 for 6 hours.

[0218] 1.4 Survival rate detection

[0219] Time points: 0h, 2h, 4h, 6h.

[0220] Methods: Eosin staining: Take 5 μL of the mixture, stain dead sperm red, and reject live sperm (count 200 sperm). CASA (Computer-Assisted Semen Analysis): Assess the proportion of progressively motile sperm (PR%).

[0221] Survival rate (%) = (number of live sperm / total sperm) × 100, motility parameters (VCL, VAP, ALH).

[0222] Experimental results

[0223] The 6-hour sperm survival rate and motility parameters are shown in Table 6

[0224] Table 6 Sperm survival rate and motility parameters in 6 hours

[0225] Group Initial survival rate (0h) 2h survival rate 4h survival rate 6-hour survival rate 6h forward motion (PR%) C1 95±2 90±3 85±3 82±2 65±5 E1 96±1 91±2 87±2 83±2 67±4 E2 94±2 89±3 84±3 81±3 63±5 E3 95±2 90±2 86±2 82±2 66±4 E4 97±1 92±2 88±2 85±2 70±4 E5 98±1 93±2 89±2 86±2 72±3 E6 95±2 90±3 84±3 80±3 65±5 E7 96±2 91±2 87±2 81±2 64±4 E8 94±2 89±2 85±2 83±2 68±4 E9 95±2 90±2 86±2 80±2 62±5 E10 96±2 91±2 87±2 81±2 63±4

[0226] Conclusion: The 6-hour survival rate of all groups (E1-E10) was ≥80%, which proves that the intelligent responsive cervical gel of the present invention is completely consistent with the adaptability of normal ovulation cervical fluid to sperm, achieving the effect of assisting reproduction.

[0227] Study on the toxicity of intelligent responsive cervical gel to cervical cells

[0228] 1. Materials and reagents:

[0229] 1.1 The reference and experimental groups were derived from the aforementioned physical properties study: reference C1, and experimental groups E1 to E10.

[0230] 1.2 Cell line: human cervical epithelial cells (HCEpiC)

[0231] 1.3 Culture medium: DMEM + 10% FBS

[0232] 1.4 Detection reagent: CCK-8 cell proliferation detection kit

[0233] Experimental procedures

[0234] 2.1. Cell culture: HCEpiC cells were seeded in 96-well plates (1×10 4 cells / well) and cultured at 37°C, 5% CO2 for 24 h.

[0235] 2.2 Sample Treatment: Gels from the experimental groups (E1-E10) and control group (C1) were extracted with culture medium (0.1 g / mL, 37°C for 24 h) and filter-sterilized. 100 μL of the extract was added to each well. Six replicate wells were set up, with a negative control (culture medium) and a positive control (containing 10% DMSO).

[0236] 2.3 Cytotoxicity Assay (CCK-8 Assay): After 24 hours of treatment, add 10 μL of CCK-8 reagent to each well and incubate for 2 hours. Measure the absorbance at 450 nm (OD value) using a microplate reader to calculate cell viability.

[0237] 2.4. Determination of toxicity level (ISO 10993-5 standard):

[0238] Level 0 (non-toxic): survival rate ≥ 100%

[0239] Level 1 (very light): 80% to 99%

[0240] Grade 2 (mild): 50% to 79%

[0241] Grade 3 (moderate): 30% to 49%

[0242] Grade 4 (severe): <30%

[0243] Experimental results

[0244] The cytotoxicity of the experimental samples is shown in Table 7

[0245] Table 7: Cytotoxicity levels of each group

[0246]

[0247]

[0248] Conclusion: Cell viability in all experimental groups (E1-E10) was ≥70%, with toxicity levels of 1-2, meeting the ≤2 requirement. In particular, the control substance C1 (95%) and some experimental groups (E1, E2, E5, E7, and E9) achieved a toxicity level of 1, demonstrating superior safety. This demonstrates that the intelligent responsive cervical gel described herein has excellent biocompatibility with the cervix and meets the requirements for a gel for assisted reproductive technology.

[0249] Study on the stimulation of cervical mucosa by intelligent responsive cervical gel

[0250] 1. Materials and reagents:

[0251] 1.1 The reference and experimental groups were derived from the aforementioned physical properties study: reference C1, and experimental groups E1 to E10.

[0252] 1.2 Animal Model: New Zealand White Rabbits (female, 6 months old, n=6 / group)—The cervical mucosa is similar to that of humans and is widely used in irritation testing.

[0253] 1.3 Negative control: normal saline

[0254] 1.4 Positive control: 1% sodium dodecyl sulfate (SDS, a known irritant)

[0255] Experimental procedures

[0256] 2.1 Animal grouping and treatment: Each group consisted of 6 rabbits, and 0.1 mL of gel (C1 / E1-E10) or control was administered locally to the cervix once.

[0257] 2.2 Negative control group: normal saline; positive control group: 1% SDS.

[0258] 2.3 Observation period: After 24 h, 48 h, and 72 h, the animals were anesthetized and cervical tissues were obtained for pathological examination.

[0259] 2.4 Irritation scoring criteria (modified Draize scoring method):

[0260] 0 points (no irritation): no redness, swelling, congestion, or ulcer.

[0261] 1 point (very mild): slight congestion, no tissue damage.

[0262] 2 points (mild): obvious congestion or edema.

[0263] 3 points (moderate): congestion + edema + slight erosion.

[0264] 4 points (severe): ulcer or necrosis.

[0265] 2.5 Histopathological analysis

[0266] Cervical tissue was fixed in 4% paraformaldehyde and stained with HE for observation:

[0267] Epithelial integrity

[0268] Inflammatory cell infiltration

[0269] Vascular congestion / bleeding

[0270] Experimental results

[0271] The irritation of cervical mucosa of the experimental samples is shown in Table 8

[0272] Table 8: Macroscopic observation scores of cervical mucosal irritation in each group (mean ± SD)

[0273] Group 24h 48h 72h in conclusion Negative control 0±0 0±0 0±0 No stimulation C1 0.2±0.1 0±0 0±0 No stimulation E1 0.3±0.2 0.1±0.1 0±0 No stimulation E2 0.5±0.2 0.2±0.1 0±0 No stimulation E3 0.7±0.3 0.3±0.2 0.1±0.1 Very mild (acceptable) E4 0.4±0.2 0.2±0.1 0±0 No stimulation E5 0.6±0.3 0.3±0.2 0.1±0.1 Very mild (acceptable) E6 0.2±0.1 0±0 0±0 No stimulation E7 0.3±0.2 0.1±0.1 0±0 No stimulation E8 0.5±0.2 0.2±0.1 0±0 No stimulation E9 0.4±0.2 0.1±0.1 0±0 No stimulation E10 0.3±0.2 0.1±0.1 0±0 No stimulation Positive control 3.8±0.4 4.0±0.0 3.5±0.5 Severe stimulation

[0274] 4. Conclusion: Histopathological results: Negative control / C1 / E1~E10: The epithelial layer is intact, without erosion or ulcer.

[0275] Occasionally, a very small amount of neutrophil infiltration was observed (≤5 / high-power field). Positive control (SDS): epithelial exfoliation, a large number of inflammatory cell infiltration, vascular dilation and bleeding. The irritation score of all experimental groups (E1 to E10) and the control substance C1 was ≤ 0.7 points (all dropped to 0 points after 72 hours), meeting the non-irritation standard (ISO 10993-10). Therefore, it can be proved that the intelligent responsive cervical gel of the present invention has good biocompatibility with the cervix and meets the requirements for making assisted reproductive gel.

[0276] Study on the sensitization of intelligent responsive cervical gel to cervical mucosa

[0277] 1. Materials and reagents:

[0278] 1.1 The reference and experimental groups were derived from the aforementioned physical properties study: reference C1, and experimental groups E1 to E10.

[0279] 1.2 Animal model: Guinea pigs (female, Hartley strain, n=10 / group)—an internationally recognized allergy testing model.

[0280] 1.3 Adjuvant: Freund's incomplete adjuvant (FIA, enhances immune response).

[0281] Experimental method (guinea pig maximization test, GPMT)

[0282] 2.1. Sensitization stage (Days 0 to 7):

[0283] Day 0: The back of the neck was shaved and 0.1 mL of the mixture (50% sample + 50% FIA) was injected intradermally.

[0284] Day 7: Apply 0.2 mL of gel sample to the same area and cover with occlusive dressing for 48 h.

[0285] 2.2, stimulation stage (days 21 to 28):

[0286] Day 21: The abdomen was shaved and 0.1 mL of gel sample (not mixed with adjuvant) was applied and blocked for 24 h.

[0287] Day 28: Repeat the stimulation and observe the skin reaction within 72 hours.

[0288] 2.3. Scoring criteria (Magnusson & Kligman classification):

[0289] Grade 0: No erythema / edema

[0290] Grade 1: Mild erythema

[0291] Grade 2: Moderate erythema / edema

[0292] Grade 3: severe erythema / edema with ulceration

[0293] 2.4. Calculation of sensitization rate:

[0294] Sensitization rate (%) = (number of animals with grade 1 reaction or higher / total number of animals) × 100%

[0295] Judgment criteria:

[0296] No sensitization: sensitization rate <10%

[0297] Mild sensitization: 10% to 30%

[0298] Moderate sensitization: 30% to 60%

[0299] Strong sensitization: >60%

[0300] Experimental results

[0301] The sensitization of the experimental samples is shown in Table 9

[0302] Table 9: Sensitization score of each group (72h after challenge)

[0303] Group Number of animals (n=10) Level 0 Level 1 Level 2 Level 3 Sensitization rate (%) Negative control 10 10 0 0 0 0 C1 10 10 0 0 0 0 E1 10 10 0 0 0 0 E2 10 9 1 0 0 10 E3 10 10 0 0 0 0 E4 10 10 0 0 0 0 E5 10 9 1 0 0 10 E6 10 10 0 0 0 0 E7 10 10 0 0 0 0 E8 10 10 0 0 0 0 E9 10 10 0 0 0 0 E10 10 10 0 0 0 0 Positive control 10 0 2 5 3 100

[0304] 4. Conclusion: Histopathology (HE staining) of negative controls (C1 / E1-E10) revealed no epidermal thickening and no lymphocytic infiltration in the dermis. The positive control (DNCB) showed epidermal spongiosis and abundant eosinophil infiltration. The sensitization rate for all experimental groups (E1-E10) and the control (C1) was ≤10%, meeting the non-sensitization standard (ISO 10993-10). This demonstrates that the intelligent responsive cervical gel of the present invention has good biocompatibility with the cervix and meets the requirements for an assisted reproductive gel.

[0305] Study on the impact of intelligent responsive cervical gel on the native microbial environment in the cervix

[0306] 1. Materials and reagents:

[0307] 1.1 The reference and experimental groups were derived from the aforementioned physical properties study: reference C1, and experimental groups E1 to E10.

[0308] 1.2. Bacterial species: Lactobacillus crispatus (ATCC 33820)

[0309] Lactobacillus jensenii (ATCC 25258)

[0310] 1.3. Culture medium: MRS liquid / agar medium (pH 6.2, anaerobic culture at 37°C)

[0311] 2. Experimental methods

[0312] 2.1. Inhibition zone test (agar diffusion method):

[0313] Apply Lactobacillus culture solution (1×10 6 CFU / mL) on MRS agar plates and placed in Oxford cups.

[0314] Add 100 μL of gel extract, culture anaerobically for 48 h, and measure the diameter of the inhibition zone (accurate to 0.1 mm).

[0315] 2.2 Growth curve analysis (dynamic monitoring):

[0316] Lactobacillus was inoculated into MRS liquid medium containing 10% gel extract (initial OD 600 =0.1).

[0317] OD was measured every 2 h 600 , continued for 24 h, and calculated the relative growth rate (vs PBS control group).

[0318] 3. Experimental results

[0319] The antibacterial properties of the experimental samples are shown in Tables 10 and 11

[0320] Table 10: Diameter of inhibition zone in each group (mm, mean ± SD, n = 3)

[0321]

[0322] Table 11 Relative growth rate, 24h

[0323]

[0324] 4. Conclusion: Antibacterial experiments: All gel samples (E1-E10) showed no inhibition zones (diameter = 0 mm) against either Lactobacillus species, indicating no direct inhibitory effect. Growth curves: The difference in growth rate between the experimental and control groups was <5%, indicating no significant effect on Lactobacillus proliferation (p>0.05). Therefore, the intelligent responsive cervical gel has no negative impact on beneficial cervical bacteria (Lactobacillus). It meets the requirements for a fertility-enhancing gel.

[0325] The above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit the same. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or make equivalent replacements for some of the technical features therein. However, these modifications or replacements will not cause the essence of the corresponding technical solutions to deviate from the protection scope of the technical solutions of the various embodiments of the present invention.

Claims

1. An intelligent responsive cervical assisted reproductive gel, characterized in that: Includes the following components and their mass percentages: Gel matrix: 0.1% to 2%, selected from one or more of hydroxypropyl methylcellulose, hydroxyethyl cellulose, sodium alginate, and gellan gum; Gel regulating adjuvant: 0.05% to 0.5%, selected from zinc chloride or calcium lactate; Osmotic pressure regulator: 0.1% to 5%, selected from one or more of glycerol, glucose, sodium chloride, mannitol, sorbitol, polyethylene glycol, and propylene glycol; Acid-base regulator: 0.1% to 2%, selected from one or more of disodium hydrogen phosphate, sodium dihydrogen phosphate, dipotassium hydrogen phosphate, and potassium dihydrogen phosphate; Preservatives: 0.01% to 0.05%, one or more selected from polyhexamethylene biguanide, methylparaben, propylparaben, phenoxyethanol, and octanol; The balance is water; The dynamic viscosity of the gel is 50-200 Pa·s, the pH value is 7.0-8.0, the osmotic pressure is 270-290 mOsm / kg, the sperm survival rate is ≥80%, and the toxicity level to cervical cells is ≤level 2.

2. The intelligent responsive cervical assisted reproductive gel according to claim 1, characterized in that: The gel matrix is ​​hydroxypropyl methylcellulose with a concentration of 0.5% to 1%, and the gel regulating adjuvant is zinc chloride with a concentration of 0.1% to 0.5%.

3. The intelligent responsive cervical assisted reproductive gel according to claim 1, characterized in that: The gel matrix is ​​sodium alginate with a concentration of 0.3% to 1.0%, and the gel regulating adjuvant is calcium lactate with a concentration of 0.05% to 0.1%.

4. The intelligent responsive cervical assisted reproductive gel according to claim 1, characterized in that: The gel matrix is ​​a mixture of hydroxypropyl methylcellulose and hydroxyethyl cellulose, wherein the concentration of hydroxypropyl methylcellulose is 0.5% to 1%, and the concentration of hydroxyethyl cellulose is 0.5% to 1.2%.

5. The intelligent responsive cervical assisted reproductive gel according to claim 1, characterized in that: The osmotic pressure regulator is a mixture of glycerol and glucose, with a glycerol concentration of 0.5% to 2% and a glucose concentration of 0.5% to 2%.

6. The intelligent responsive cervical assisted reproductive gel according to claim 1, characterized in that: The acid-base regulator is a mixture of disodium hydrogen phosphate and sodium dihydrogen phosphate, wherein the concentration of disodium hydrogen phosphate is 0.5% to 1.5%, and the concentration of sodium dihydrogen phosphate is 0.05% to 0.2%.

7. The intelligent responsive cervical assisted reproductive gel according to claim 1, characterized in that: The preservative is polyhexamethylene biguanide with a concentration of 0.01% to 0.03%.

8. The intelligent responsive cervical assisted reproductive gel according to claim 1, characterized in that: The preparation method of the gel comprises the following steps: a. Dissolve the gel matrix and gel conditioning agent in a portion of water and allow to swell for 12 hours; b. Dissolve the osmotic pressure regulator, acid-base regulator, and preservative in the remaining water and stir evenly; c. Mix the solutions of step 1 and step 2, heat to 40℃~60℃, homogenize and emulsify, and then fill.

9. The intelligent responsive cervical assisted reproductive gel according to claim 1, characterized in that: The gel can dynamically adjust the osmotic pressure and pH in the cervical environment, simulate the characteristics of cervical mucus during ovulation, and promote sperm penetration.

10. The intelligent responsive cervical assisted reproductive gel according to claim 1, characterized in that: The gel has no negative impact on the native microbial environment of the vagina and cervix, and the growth rate of lactobacilli is less than 5% different from that of the control group.

Images