KR102654800B1 - Composition of in vitro fertilization and in vitro culture medium for aged oocyte containing lipoic acid and resveratrol - Google Patents

Abstract

The present invention relates to a medium composition for in vitro fertilization and in vitro culture of aged eggs containing lipoic acid and resveratrol, and specifically to a medium containing lipoic acid and resveratrol, or additionally phosphate. When used for in vitro fertilization and in vitro culture of aged eggs, it was confirmed that the rate of embryo division and blastocyst formation increased, and the quality of the embryo was improved, increasing the pregnancy rate and implantation rate. Therefore, the above medium was used for in vitro fertilization and in vitro culture of aged eggs. It is suitable as a culture medium composition and can ultimately improve the quality of in vitro cultured embryos, contributing to pregnancy and implantation rates.

Description

{Composition of in vitro fertilization and in vitro culture medium for aged oocyte containing lipoic acid and resveratrol}

The present invention relates to a medium composition for in vitro fertilization and in vitro culture of aged eggs containing lipoic acid and resveratrol, and more specifically to aged eggs and sperm containing lipoic acid and resveratrol as active ingredients. It relates to a medium composition for in vitro fertilization and in vitro culture.

Various assisted reproductive technologies, such as in vitro production of embryos, including in vitro fertilization, are being implemented to promote the animal industry and to treat infertility or subfertility in humans. In particular, in a situation where the number of infertility patients is increasing every year as the age at conception increases due to the recent increase in age at first marriage and aging of childbirth, etc., eggs collected from outside the body are fertilized with sperm, and only the fertilized embryos are cultured outside the body to make the woman pregnant. In vitro fertilization (IVF) implantation within the uterus is the most common treatment method to overcome infertility. However, as the eggs of elderly infertility patients age, their mitochondrial function deteriorates and the production of oxygen radicals increases, resulting in poor egg quality and poor pregnancy outcomes.

In vitro production of embryos is a technology that secures embryos that have developed beyond a certain level by carrying out the series of processes of egg maturation, fertilization, and embryo development that occur inside the body outside the body. In in vitro culture, differences in production efficiency appear depending on the culture environment, and the improvement in the production efficiency of embryos and blastocysts through in vitro culture leads to an increase in implantation and pregnancy rates. When applying in vitro production technology, the quality of embryos is improved to produce embryos. It is necessary to develop a suitable culture medium that can increase efficiency.

Meanwhile, resveratrol is a type of stilbene and is found in many plants, including berries such as mulberries, peanuts, grapes, raspberries, and cranberries. Resveratrol is known to have anticancer and powerful antioxidant properties.

Lipoic acid, also called alpha-lipoic acid, is one of the compounds used in the energy production process in mitochondria, and has recently been used as an ingredient in health foods and multivitamins.

Accordingly, the present inventors made efforts to develop a medium composition suitable for in vitro fertilization and in vitro culture of aged oocytes, and as a result, a medium containing lipoic acid and resveratrol, or additional phosphate, was used for in vitro fertilization of oocytes from aged mouse models and elderly infertility patients. It was confirmed that when used for fertilization and in vitro culture, the rate of embryo division and blastocyst formation increased, and the quality of the embryo improved, increasing the pregnancy rate and implantation rate. Accordingly, the present inventors have arrived at the present application by revealing that lipoic acid and resveratrol, or lipoic acid, resveratrol, and phosphate, are suitable as active ingredients in a medium composition for in vitro fertilization and in vitro culture of aged eggs.

Republic of Korea Patent No. 10-2054710 Republic of Korea Patent No. 10-2469369

The purpose of the present invention is to provide a medium composition for in vitro fertilization and in vitro culture that can improve the quality and embryo development rate of aged eggs.

In order to achieve the object of the present invention, the present invention provides a medium composition for in vitro fertilization and in vitro culture of mammalian eggs and sperm containing lipoic acid and resveratrol.

In addition, the present invention provides a medium composition for in vitro fertilization and in vitro culture of mammalian eggs and sperm, further comprising phosphate in the medium composition.

When the medium containing lipoic acid and resveratrol, or additionally phosphate, of the present invention is used for in vitro fertilization and in vitro culture of aged eggs, the rate of embryo division and blastocyst formation increases, and the embryo's Since it was confirmed that the pregnancy rate and implantation rate increased due to improved quality, the medium is suitable as a medium composition for in vitro fertilization and in vitro culture of aged eggs, and ultimately contributes to the pregnancy rate and implantation rate by improving the quality of in vitro cultured embryos. You can.

Hereinafter, the present invention will be described in detail.

The present invention provides a medium composition for in vitro fertilization and in vitro culture of mammalian eggs and sperm, comprising lipoic acid and resveratrol as active ingredients.

In the present invention, “in vitro fertilization (IVF)” is a method that is distinguished from the state in which eggs and sperm are fertilized inside the body, and is fertilized and cultured in a laboratory incubator under conditions similar to the environment of the uterus in the body. It refers to a series of laboratory processes, and the medium composition of the present invention is a medium composition optimized for such in vitro fertilization, specifically, in vitro fertilization of aged eggs and sperm.

In addition, "in vitro culture (IVC)" refers to culturing the fertilized egg produced after the above in vitro fertilization in a medium outside the body, and refers to the process of forming embryos and blastocysts through in vitro culture of the fertilized egg, and the present invention The medium composition of is a medium composition optimized for such in vitro culture, specifically for the in vitro culture of fertilized eggs produced after in vitro fertilization of aged eggs and sperm.

In the present invention, the medium composition may contain 0.1 to 2 μM lipoic acid, specifically 0.3 to 1.5 μM lipoic acid, and more specifically 0.8 to 1.2 μM lipoic acid. Not limited.

In the present invention, the medium composition may contain 0.1 to 1 μM of resveratrol, specifically 0.2 to 0.8 μM of resveratrol, and more specifically 0.4 to 0.6 μM of resveratrol. Not limited.

In the present invention, the medium composition may additionally include phosphate. At this time, phosphate may be included at 0.1 to 1 mM, specifically 0.1 to 0.7 mM, and more specifically 0.1 to 0.5 mM, but is not limited thereto.

In the present invention, the medium composition can be added to the in vitro culture medium. The in vitro culture medium refers to a substance that enables the growth and proliferation of cells in vitro, including essential elements such as carbon sources, amino acids, various nutrients, serum, growth factors, and inorganic salts, and specifically, conventional The basic medium used for in vitro fertilization of mammals or for culturing in vitro fertilized eggs can be used without limitation. As for the medium, a person skilled in the art can appropriately select and use a known medium depending on the type of in vitro fertilization or in vitro fertilized egg.

Examples of the carbon source include, but are not limited to, glucose, sodium pyruvate, calcium lactate, or sodium lactate.

Examples of the inorganic salts include, but are not limited to, sodium chloride (NaCl), potassium chloride (KCl), or sodium hydrogen carbonate (NaHCO ₃ ).

In one embodiment of the present invention, MRC (Maria Research Center) medium having the composition shown in [Table 1] was used as an in vitro culture medium.

In the present invention, the mammal may be, for example, a human, a livestock animal such as a pig, a cow, a goat, a sheep, a horse, a pet, a laboratory animal such as a rat, a guinea pig, etc., but is not limited thereto.

In a specific example of the present invention, the present inventors prepared a medium for in vitro fertilization and in vitro culture containing lipoic acid and resveratrol, and a medium for in vitro fertilization and in vitro culture further containing phosphate.

In addition, the present inventors used a medium for in vitro fertilization and in vitro culture containing lipoic acid and resveratrol to induce in vitro fertilization of aged eggs and sperm collected from an elderly mouse model, and when culturing the in vitro fertilized eggs in vitro, the embryo It was confirmed that the rate of division and blastocyst formation increased.

In addition, the present inventors induced in vitro fertilization of eggs and sperm collected from elderly infertile patients using a medium for in vitro fertilization and in vitro culture containing lipoic acid, resveratrol, and phosphate, and when culturing the in vitro fertilized eggs in vitro, the embryo It was confirmed that the quality improved and the pregnancy rate and implantation rate increased.

Therefore, the medium composition for in vitro fertilization and in vitro culture containing lipoic acid and resveratrol, or additionally phosphate, of the present invention is suitable for in vitro fertilization and in vitro culture of aged eggs, and ultimately improves the quality of in vitro cultured embryos, thereby increasing the pregnancy rate and May contribute to implantation rate.

In addition, the present invention

1) Preparing mature mammalian eggs;

2) in vitro fertilization by adding the mature eggs and sperm of step 1) to the medium composition for in vitro fertilization of mammalian eggs and sperm and in vitro culture of fertilized eggs according to the present invention; and

3) It provides a method of in vitro culturing of mammalian in vitro fertilized eggs, comprising the step of culturing the in vitro fertilized eggs fertilized in step 2) in the medium composition.

In the present invention, the mammalian oocyte matured in step 1) may be prepared by a method comprising the following steps, but is not limited thereto:

a) collecting mammalian eggs; and

b) In vitro maturation of the egg.

In the present invention, “in vitro maturation (IVM)” refers to a method of inducing oocytes collected before performing in vitro fertilization to perform meiosis and reach the second meiotic metaphase II. do. The oocyte refers to a cell that differentiates from an oval cell and ceases cellular activity, and the term “oocyte” in the present invention can be considered to mean a cell that is substantially the same as the “oocyte.”

The in vitro fertilization and in vitro culture method of the present invention improves the quality of embryos, It can improve embryo division and blastocyst formation rates.

Hereinafter, the present invention will be described in detail through examples and experimental examples.

However, the following examples and experimental examples only illustrate the present invention, and the content of the present invention is not limited to the following examples and experimental examples.

<Example 1> Preparation of in vitro fertilization and in vitro culture medium containing lipoic acid and resveratrol

The medium for in vitro fertilization and in vitro culture supplemented with lipoic acid and resveratrol is 1 each of MRC (Maria Research Center) #D01, MRC #D13, and MRC #D46 medium with the composition shown in Table 1 below. It was prepared by adding µM lipoic acid (Sigma-Aldrich, USA) and 0.5 µM resveratrol (Sigma-Aldrich, USA).

badge composition MRC#D01 102.7mM sodium chloride 0.58mM glucose 0.50 mM alanyl-glutamine 0.50 mM glycyl-glutamine 10.50 mM sodium lactate 5.0 mg/mL human serum albumin 1.0 mg/mL alpha and beta globulins MRC#D13 102.7mM sodium chloride 0.58mM glucose 0.50 mM alanyl-glutamine 0.50 mM glycyl-glutamine 10.50 mM sodium lactate 0.10 mg/mL hyaluronic acid 5.0 mg/mL human serum albumin 1.0 mg/mL alpha and beta globulins MRC#D46 106.0mM sodium chloride 3.15mM glucose 0.50 mM alanyl-glutamine 0.50 mM glycyl-glutamine 5.87 mM sodium lactate 0.10 mg/mL hyaluronic acid 5.0 mg/mL human serum albumin 1.0 mg/mL alpha and beta globulins

<Example 2> Preparation of medium for in vitro fertilization and in vitro culture with lipoic acid, resveratrol and phosphate added

Media for in vitro fertilization and in vitro culture supplemented with lipoic acid, resveratrol, and phosphate are MRC#D01, MRC#D13, and MRC#D46 media, each containing 1 μM lipoic acid, 0.5 μM resveratrol, and 0.3 mM phosphate (Sigma -Aldrich, USA) was added to prepare it.

<Comparative Example 1> Preparation of in vitro fertilization and in vitro culture medium containing lipoic acid

A medium for in vitro fertilization and in vitro culture to which lipoic acid was added was prepared in the same manner as <Example 1>, except that only 1 μM lipoic acid was added.

<Comparative Example 2> Preparation of in vitro fertilization and in vitro culture medium containing resveratrol

A medium for in vitro fertilization and in vitro culture to which resveratrol was added was prepared in the same manner as in <Example 1> except that only 0.5 μM of resveratrol was added.

<Comparative Example 3> Preparation of medium for in vitro fertilization and in vitro culture with added phosphate

A medium for in vitro fertilization and in vitro culture to which resveratrol was added was prepared in the same manner as in <Example 1> except that only 0.3 mM phosphate was added.

<Experimental Example 1> Confirmation of the effect of lipoic acid and resveratrol treatment on embryonic development of eggs in an elderly mouse model during in vitro fertilization and in vitro culture

The aging of eggs due to aging causes problems such as decreased mitochondrial function and increased production of oxygen radicals, leading to poor egg quality and poor pregnancy. Therefore, in order to determine whether the in vitro fertilization and in vitro culture medium of the present invention is suitable for in vitro fertilization and in vitro culture of aged eggs, eggs from an aged mouse model were used using the in vitro fertilization and in vitro culture medium prepared in <Example 1> above. After in vitro fertilization and in vitro culture, the developmental capacity of the embryo was confirmed.

<1-1> Preparation of aged oocytes from aged mouse model

All animal experiments were performed in compliance with the NIH Guide for the Care and Use of Research Animals. The animal study was approved by the Animal Experiment Ethics Committee of the Maria Medical Foundation Fertility Research Institute. As an elderly mouse model, 62-week-old B6D2 F1 (C57BL/6×DBA) female mice were used. 5 IU of gonadotropin was administered to female mice, and 48 hours after intraperitoneal injection of pregnant horse serum gonadotropin (PMSG; Folligon, Intervet, UK), human chorionic hormone was administered. (human chorionic gonadotropin, hCG; Chorulon, Intervet, UK) was injected. 14 hours after hCG injection, the animals were euthanized by cervical dislocation, and metaphase II eggs were collected from in vitro fertilization medium.

<1-2> In vitro fertilization and in vitro culture

Sperm were collected from the cauda epididymis of B6D2 F1 males. Next, each of the eggs collected in Example <1-1> was divided into MRC, MRC+Lipoic acid, MRC+Resveratrol, MRC+phosphate, and MRC+Lipoic acid+Resveratrol groups, and fertilization was performed.

Specifically, the MRC group received MRC#D01, MRC#D13, and MRC#D46 media, and the MRC+Lipoic acid group (comparative group 1) received MRC#D01 and MRC# to which the lipoic acid prepared in <Comparative Example 1> was added. D13 and MRC#D46 medium, the MRC+Resveratrol group (comparative group 2) used MRC#D01, MRC#D13 and MRC#D46 medium supplemented with resveratrol prepared in <Comparative Example 2>, and the MRC+phosphate group used Fertilization and culture were performed using MRC#D01, MRC#D13, and MRC#D46 media supplemented with phosphate prepared in <Comparative Example 3>. As a basic medium, MRC#D01 medium was used when cultivating early embryos 5 hours after fertilization, MRC#D13 medium was used, and MRC#D46 medium was used when cultivating late embryos. Additionally, embryos in both stages were cultured in groups of 10 embryos per 50 μl drop of medium under mineral oil.

Meanwhile, during culture after fertilization, the quality of the embryo is evaluated and recorded by observing the blastomere regularity, blastomere fragmentation, and cytoplasmic granularity of embryonic blastomere cells under an optical microscope, thereby ensuring the division and The ability to develop into a blastocyst was confirmed.

Group No. of
Zygotes No. of
2-cells No. of
4-cells < No.(%) of embryos on D4 TBR HdB HgB EdB Deg- MRC 91 83
(91.2) 80
(96.4) 64
(77.1) One
(1.2) 43
(51.8) 20
(24.1) 17
(20.5) MRC+Lipoic acid 89 83
(93.3) 79
(95.2) 65
(78.3) 2
(2.4) 49
(59.0) 14
(16.9) 17
(20.5) MRC+Resveratrol 66 62
(93.9) 62
(100.0) 51
(82.3) 0
(0.0) 38
(61.3) 13
(21.0) 11
(17.7) MRC+Lipoic acid+Resveratrol 87 85
(97.7) 84
(98.8) 76
(89.4) One
(1.2) 62
(72.9) 13
(15.3) 9
(10.6) *TBR: Total Blastocyst rate / HdB: Hatched blastocyst / HgB: Hatching blastocyst / EdB: Expanded blastocyst / Deg-: Degeneration )

Group No. of
MII No. of
2-cells No. of
4-cells < No.(%) of embryos on D4 TBR >HgB EdB Deg- MRC 121 115
(95.0) 115
(100.0) 107
(93.0) 64
(55.7) 43
(37.4) 8
(7.0) MRC+Phosphate 217 211
(97.2) 210
(99.5) 200
(94.8) 160
(75.8) 40
(19.0) 11
(5.2)

As a result, as shown in Tables 2 and 3, the hatching rate of embryos on the 4th day of culture after fertilization was lower than that in the MRC+Lipoic acid+Resveratrol group (74.1%, sum of HdB and HgB in Table 2) and the MRC group (53.0%). %) increased significantly. In addition, the blastocyst rate on the 4th day of culture after fertilization was significantly increased in the MRC+Lipoic acid+Resveratrol group (89.4%) compared to the MRC group (77.1%). Meanwhile, the effect of increasing the blastocyst ratio (78.3%) in the MRC+Lipoic acid group compared to the MRC group (77.1%) was found to be minimal. The hatching rate and blastocyst rate of the MRC+Phosphate group were 75.8% and 94.8%, respectively. Although the effect of increasing the hatching rate was superior to that of the MRC group, the effect of increasing the blastocyst rate was somewhat insignificant.

<Experimental Example 2> Confirmation of the effects of lipoic acid, resveratrol, and phosphate treatment on embryos of elderly infertility patients during in vitro fertilization and in vitro culture

In order to determine whether the in vitro fertilization and in vitro culture medium of the present invention is suitable for in vitro fertilization and in vitro culture of aged eggs in clinical practice, eggs from elderly infertility patients were used using the in vitro fertilization and in vitro culture medium prepared in <Example 2> above. After in vitro fertilization and in vitro culture, the embryos were confirmed.

<2-1> Egg preparation

Eggs were collected from elderly infertile patients.

Specifically, from January 2019 to December 2021, the Maria Medical Foundation, a specialized center for in vitro fertilization, administered follicle stimulating hormone (FSH) to 966 infertile patients aged 40 or older from the 2nd to 3rd day of their menstrual cycle. and human menopausal gonadotropin (HMG) is administered together with an agonist or antagonist of gonadotropin releasing hormone (GnRH) to induce superovulation, and human chorionic hormone After administering human chorionic gonadotropin (HCG), eggs were collected using transvaginal ultrasound.

<2-2> Preparation of sperm

Immediately after collecting eggs from the wife, if mature eggs were present, the husband's semen was collected. The collected semen was placed on a sterile workbench and liquefaction of solid substances contained in the semen was induced for 30 to 60 minutes. The liquefied semen was treated with 3ml of culture medium for semen processing (MRC#SW) and diluted. The volume of the diluted semen was examined, and sperm concentration, survival rate, and motility were tested. Next, centrifugation was performed at 300 × g for 5 minutes to remove the supernatant. MRC#SW was added to the remaining sperm mass and centrifugation was performed at 300 × g for 10 minutes. After removing the supernatant, 0.7 ml of culture medium for sperm culture (MRC-SC) was carefully added to the remaining sperm mass, and floating of sperm was induced in an incubator controlled at a temperature of 36.8°C and 5% CO ₂ . After 2 hours, the upper part of the sperm solution was recovered and placed in a test tube at room temperature without light until fertilization was induced.

<2-3> Inducing correction

Each of the collected eggs was divided into MRC, MRC+Lipoic acid+Resveratrol+phosphate groups, and fertilization was performed 5 hours later.

Specifically, the MRC+Lipoic acid+Resveratrol+phosphate group added the eggs collected in Experimental Example <2-1> to 1 ml of MRC#D01 medium containing lipoic acid, resveratrol, and phosphate prepared in <Example 2>. did. After 5 hours, 1.0 × 10 ⁵ sperm prepared in Experimental Example <2-2> were injected into the medium containing the egg, and the partial pressure was adjusted to 6% CO ₂ , 5% O ₂ and 89% N ₂ for 19 hours. and placed in an incubator with a temperature controlled at 36.8°C to induce fertilization.

In addition, the MRC group (control group) used MRC#D01 medium to induce fertilization in the same manner as above.

<2-4> Confirmation of fertilization and culture of early embryos

The morning after fertilization in Experimental Example <2-3>, normal fertilization was determined by observing the second polar body and the hermaphroditic nucleus in the egg.

Embryos of the MRC+Lipoic acid+Resveratrol+phosphate group, in which normal fertilization was confirmed, were placed in a new culture dish with 10-50 ㎕ microdroplets of MRC#D13 medium supplemented with lipoic acid, resveratrol, and phosphate prepared in <Example 2>. was made and placed therein, placed in an incubator where the partial pressure was adjusted to 6% CO ₂ , 5% O ₂ , and 89% N ₂ and the temperature was adjusted to 36.8°C, and cultured for 48 hours.

Additionally, the MRC group was cultured in the same manner as above using MRC#D13 medium.

On the 3rd day of culture after fertilization, embryo quality was evaluated by observing blastomere cell uniformity, fragmentation, and cytoplasmic granulation under a light microscope. Next, the best embryos among the embryos on the third day of culture after fertilization were transferred.

<2-5> Culture and transplantation of late embryos

The remaining embryos of Experimental Example <2-4>, in the case of the MRC+Lipoic acid+Resveratrol+phosphate group, were cultured in a new culture dish with MRC#D46 medium supplemented with lipoic acid, resveratrol, and phosphate prepared in <Example 2> for 10 days. ~50㎕ of microdroplets were made and placed in an incubator where the partial pressure was adjusted to 6% CO ₂ , 5% O ₂ and 89% N ₂ and the temperature was adjusted to 36.8°C and cultured for 48 hours. The MRC group was cultured in the same manner as above using MRC#D46 medium.

When embryos were transferred on the fifth day of culture after fertilization, the quality of the embryos was evaluated as above, and the best embryos were transferred. Implantation and pregnancy rates were evaluated after transplantation.

Group p-value MRC+Lipoic acid+Resveratrol+phosphate MRC No. of patients 502 464 Age(yr)±SD 42.5±2.2 42.3±2.0 0.358 No. of previous ART cycles±SD 3.6±2.3 3.7±2.5 0.680 Thickness of endometrium±SD 9.2±1.5 9.4±1.4 0.312 Maturation rate per oocytes(%) 74.6 75.8 0.290 Fertilization rate(%) 81.8 80 0.165 No. of transferred embryos±SD 2.4±0.6 2.4±0.6 0.818 Good quality embryo rate(%) 44.0 36.4 0.05 Pregnancy rate(%) 26.8 22.7 0.167 Implantation rate(%) 11.9 9.9 0.131

As a result, as shown in Table 4, it was confirmed that the quality of embryos was improved and the pregnancy rate and implantation rate were increased in the MRC+Lipoic acid+Resveratrol+phosphate group compared to the MRC group.

Based on the above results, when the medium containing lipoic acid and resveratrol and the medium containing phosphate are used for in vitro fertilization and in vitro culture of aged eggs, the rate of embryo division and blastocyst formation increases, and the quality of the embryo improves, leading to pregnancy. Since it was confirmed that the rate and implantation rate increased, it can be seen that the medium is suitable for in vitro fertilization and in vitro culture of aged eggs.

Claims (7)

A medium composition for in vitro fertilization and in vitro culture of mammalian eggs and sperm containing lipoic acid, resveratrol and phosphate as active ingredients,
A medium composition in which the egg is an aged egg.
The medium composition according to claim 1, wherein the medium composition contains 0.1 to 2 μM lipoic acid.
The medium composition according to claim 1, wherein the medium composition contains 0.1 to 1 μM of resveratrol.
delete The medium composition according to claim 1, wherein the medium composition further contains 0.1 to 1 mM phosphate.
delete The medium composition according to claim 1, wherein the medium composition is added to an in vitro culture medium.