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Human in-vitro seminal liquid and preparing method thereof

A technology for fertilization of semen and human body, applied in biochemical equipment and methods, embryonic cells, germ cells, etc., can solve problems such as long import procurement cycle, inability of patients to achieve fertilization effect, high price, etc.

Active Publication Date: 2019-04-16
SHENZHEN VITAVITRO BIOTECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, the fertilized fluid used in domestic clinical practice is all imported reagents, which are expensive and bring huge economic burden to patients.
In addition, the import procurement cycle is long, and the uncertainty of long-distance transportation conditions may lead to product performance degradation
[0004] The fertilization fluid in the current market is basically human tubal fluid (HTF), which is only used for men with normal sperm count according to routine fertilization procedures, and the ideal fertilization effect cannot be achieved for patients with low sperm count

Method used

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  • Human in-vitro seminal liquid and preparing method thereof
  • Human in-vitro seminal liquid and preparing method thereof
  • Human in-vitro seminal liquid and preparing method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0018] [Example 1] In vitro sperm formulation and preparation method:

[0019] A. In vitro semen formula:

[0020]

[0021] B. Preparation method:

[0022] a. Weigh all the solid components according to the formula and place them separately;

[0023] b. According to the principle of adding solid first, then liquid, and adding sodium bicarbonate last, add and dissolve all ingredients except gentamicin sulfate, phenol red, sodium bicarbonate and human serum albumin in ultrapure water;

[0024] c. Add gentamicin sulfate and phenol red to the solution in step b, and finally add sodium bicarbonate and dissolve to obtain a base solution;

[0025] d. Put the above base solution into an incubator with 5% carbon dioxide, 95% air and saturation temperature to balance for 7-12 hours;

[0026] e. Measure and adjust the pH value of the above-mentioned base solution at 7.3±0.1, and the osmotic pressure at 260-290mOsm / kg, and make a record;

[0027] f. For use in the isolator. Filter ...

Embodiment 2

[0028] [Example 2] Quality control detection of semen in vitro

[0029] 1. pH value detection

[0030] Take an appropriate amount of the sample to be tested and measure it three times with a pH meter, and take the average value of the three data obtained as the result, and it is considered qualified if it is 7.3±0.1.

[0031] Osmotic pressure test

[0032] After the osmometer is calibrated, take 500μl of the sample to be tested and put it into the test tube to start the test. When the value is stable, read the data, measure 3 data according to the above method, take the average as the result, and it is considered qualified if it is 260-290mOsm / kg .

[0033] Bacterial endotoxin detection

[0034] According to the requirements of the 2015 edition of the Pharmacopoeia of the People's Republic of China, the test is carried out by the limulus reagent gel method, and the result is ≤0.1EU / ml, which is considered qualified.

[0035] Cytotoxicity

[0036] Tested according to the p...

Embodiment 3

[0061] [Example 3] mouse embryology experiment

[0062] Experimental steps of in vitro fertilization in mice

[0063] 1. Experimental materials

[0064] (1) Mice: female C57, 4-5 weeks old, male C57, 10 weeks old. 12 light, temperature 20-25 ℃, free access to food and water;

[0065] (2) Consumables and equipment: 1ml syringe, ophthalmic scissors, ophthalmic forceps;

[0066] (3) Reagents: PMSG, hCG, HTF culture fluid, KSOM-AA embryo culture fluid, fertilization fluid of the experimental group.

[0067] 2. Petri dish preparation (prepared in the afternoon of the day before IVF and equilibrated in the incubator overnight)

[0068] (1) Fresh semen collection dish: 500μl HTF (35mm domestic), 500μl experimental group fertilized fluid (35mm domestic);

[0069] (2) Egg collection dish: 1ml M2 (35mm domestic) preheated to 37°C 1 hour in advance;

[0070] (3) In vitro fertilization dishes and petri dishes for cleaning: 60mm (HTF) dish--the upper part is used for fertilization drop...

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Abstract

The invention discloses a human in-vitro seminal liquid. The human in-vitro seminal liquid comprises 90-113 mM / L of inorganic salt of sodium chloride, 4.20-5.18 mM / L of potassium chloride, 1.80-2.28 mM / L of calcium chloride dihydrate, 0.26-0.48 mM / L of monopotassium phosphate, 0.18-0.87 mM / L of magnesium sulfate heptahydrate, 20-30 mM / L of pH buffering agent sodium bicarbonate, 0.01-0.05 g / L of bacteriostat component gentamicin sulphate, 0.21-0.45 mM / L of energy substance sodium pyruvate, 18.50-24.3mM / L of L-sodium lactate, 2.56-3.00 mM / L of glucose, 0.45-1.05%(v / v) of other components of non-essential amino acid, 0-1.0 mM / L of glutamine dipeptide, 0-0.01 mM / L of ethylene diamine tetraacetic acid, 0.05-0.10 mM / L of taurine, 5-10 g / L of human serum protein and 42-58 IU / L of human recombinant insulin.

Description

technical field [0001] The invention belongs to the field of human assisted reproduction, and in particular relates to a human in vitro semen and a method for preparing the in vitro semen. Background technique [0002] The first human in vitro fertilization (IVF) baby was born in 1978. Since then, assisted reproduction has flourished, and egg single sperm injection (ICSI), preimplantation genetic diagnosis (PGD), egg or embryo cryopreservation and other technologies have been born one after another. The earth facilitates the assisted reproductive industry. [0003] In vitro fertilization fluid is one of the most routine reagents in the field of human assisted reproduction. It provides energy and fertilization environment for in vitro fertilization, and is an indispensable reagent in clinical or experimental research in the field of assisted reproduction. At present, the fertilized fluid used in domestic clinical practice is all imported reagents, which are expensive and bri...

Claims

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Application Information

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IPC IPC(8): C12N5/073C12N5/075C12N5/076
CPCC12N5/0604C12N5/0609C12N5/061C12N2500/05C12N2500/30C12N2500/32C12N2500/33C12N2501/33C12N2501/998
Inventor 林小贞陈文彬
Owner SHENZHEN VITAVITRO BIOTECH CO LTD
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