Application of composition to preparation of medicine for improving IVF-ET postoperative embryonic development

Abstract

The invention belongs to the technical field of pharmaceutical preparations, and particularly relates to application of a composition to preparation of a medicine for improving IVF-ET postoperative embryonic development. The composition for regulating menstruation and promoting pregnancy is prepared from dehaired pilose antler, roasted herba epimedii, rhizoma curculiginis, radix dipsaci, herba taxilli, semen cuscutae, fructus lycii, fructus rubi, rhizoma dioscoreae, lotus seeds (without cores), poria cocos, radix astragali, radix paeoniae alba, fried spina date seeds, uncaria rhynchophylla, radix salviae miltiorrhizae, radix paeoniae rubra and caulis spatholobi. Based on an in-vitro fertilization-embryo transplantation technology, the ovulation function and the ovum quality after the menstruation regulating and pregnancy promoting pill intervenes a female mouse, and the embryonic development condition, the farrowing rate and the fetal development state after the female mouse is transplanted into the body are evaluated; it is proved that the menstruation-regulating and pregnancy-promoting composition, especially the menstruation-regulating and pregnancy-promoting pill, can improve the in-vivo development degree, in-vivo development quality and pregnancy outcome of an embryo after an in vitro fertilization-embryo transplantation (IVF-ET) operation.

Description

Technical Field

[0001] The present invention belongs to the technical field of pharmaceutical preparations, and specifically relates to the use of a composition for preparing a drug for improving embryo development after IVF-ET surgery, and in particular to the use of a menstruation regulating and pregnancy promoting composition for preparing a drug for improving embryo development quality in vivo and pregnancy outcome after in vitro fertilization-embryo transfer (IVF-ET). Background Art

[0002] At present, the main methods for treating infertility include drug therapy, surgical treatment and assisted reproductive treatment. Among them, assisted reproduction has become the mainstream technology for treating infertility. At least 20% of couples with infertility must rely on assisted reproductive technology to achieve fertility.

[0003] Human assisted reproductive technology (ART) refers to an invasive operation that uses gametes, embryos or genetic materials to perform a series of in vitro and in vivo system operations to obtain new life. It mainly includes artificial insemination and in vitro fertilization-embryo transfer (IVF-ET) and its related derivative technologies. Although assisted reproductive technology represented by IVF-ET has matured after development and improvement, the success rate of assisted reproductive technology in clinical practice is not high, and the clinical pregnancy rate can only reach 29%-35%, indicating that further improvement is still needed. It is reported that the main reasons affecting the success rate of IVF-ET include the patient's own anxiety, depression, poor ovarian reserve function, low quality of the obtained eggs, poor endometrial receptivity in the uterine cavity, low embryo quality and the occurrence of IVF-ET complications related to poor development in the body. Therefore, how to improve the success rate of assisted reproductive technology has attracted widespread attention from scientific researchers, and exploring ways to improve embryo quality and promote embryo development in the body has become one of the entry points.

[0004] In TCM theory, infertility is called "total infertility", "childlessness", "intermittent infertility", etc., and is classified according to the patient's medical history and the clinical manifestations and signs of the four diagnoses. It is mainly divided into syndromes such as kidney qi deficiency syndrome, kidney yang deficiency syndrome, kidney yin deficiency syndrome, kidney deficiency and blood stasis syndrome, liver depression syndrome, phlegm dampness syndrome, and blood stasis syndrome. TCM theory advocates strong male sperm and normal female menstruation. Strong male sperm refers to normal male semen and sexual function, while normal female menstruation refers to normal period, quantity, color, quality, and periodic ovulation. According to the book "Lingshu Jing·Ben Shen", "the coming of life is called Jing, and the collision of two Jing is called Shen", which is the mechanism of female conception; "Nu Ke Zheng Zong" says: "Men's Jing is strong and women's menstruation is regulated, which is the way to have children"; "Fu Ke Bu Jie - Tiao Men" emphasizes: "Most women's diseases are caused by irregular menstruation. Only after menstruation is regulated can they conceive and eliminate diseases, so regulating menstruation is the first priority"; "Zheng Zhi Zhun Sheng·Nu Ke·Prenatal" points out: "Every time a woman's menstruation comes, there must be a day of mist... This is the right time... If you follow it, you will get pregnant." It can be seen that the mechanism of conception lies in the fullness of male and female kidney qi, the maturity of Tian Gui, the fullness of Ren Meridian, the strong Jing and menstruation, the timely harmony, and then the pregnancy will be formed. "The two Shens collide and form" is the beginning of pregnancy, and "Ten months of pregnancy and one day of delivery" is the end of pregnancy. According to "Tai Sheng Xin Fa", "When a woman is pregnant, her blood stays and her Qi gathers, and the uterus is full." The uterine vessels are connected to the kidneys, and both the Chong and Ren meridians originate in the uterus. The fetus lives in the mother's body, relying entirely on the mother's kidneys to connect it, Qi to carry it, blood to nourish it, and Chong and Ren meridians to consolidate it. If the mother's kidney Qi is strong, Qi and blood are abundant, and Chong and Ren meridians are unobstructed, the fetus will be solid and the mother will be safe. Therefore, kidney deficiency, Qi and blood deficiency, Qi stagnation and blood stasis after pregnancy are the causes of the disease, and Chong and Ren meridians are damaged and the fetal essence is not solid. The treatment method is based on the principle of "pre-cultivation of damage, prevention and treatment combined". In addition, it is said in Suwen Four Qi Tiaoshen Da Lun: "So the sage does not treat an existing disease, but treats one that has not yet been sick; does not treat an existing disorder, but treats one that has not yet been disordered. This is what it means. If you treat an illness after it has developed and then take medicine, or treat a disorder after it has developed, it is like digging a well when you are thirsty, or casting an awl when you are fighting. Isn't it too late? Therefore, the best doctor treats an illness before it has occurred, and must prevent it before it happens. Therefore, we should attach importance to conditioning before pregnancy, so that "positive energy is preserved inside, and evil cannot interfere", so as to fill the state of conception, protect the fetus after pregnancy, and achieve "harmony of yin and yang, and the body and spirit together", so as to help the fetus grow steadily and healthily, and achieve both pregnancy and delivery.

[0005] The menstruation regulating and pregnancy-promoting pills are composed of velvet antler (dehaired), epimedium (roasted), curculigo, radix scutellariae, radix scutellariae, radix mulberrye, cuscuta, lycium barbarum, raspberry, yam, lotus seed (core removed), tuckahoe, astragalus, white peony root, spiny jujube seed (fried), Uncaria rhynchophylla, salvia miltiorrhizae, red peony root, and millettia reticulatae. The combination of these medicines can regulate and replenish the Chong and Ren meridians, strengthen the spleen and kidney, nourish blood, regulate menstruation and promote pregnancy. However, the application of the menstruation regulating and pregnancy-promoting pills in assisted reproduction has not been seen so far. Summary of the invention

[0006] To this end, the technical problem to be solved by the present invention is to provide a menstruation regulating and pregnancy promoting composition for use in the preparation of a drug for improving embryo development after IVF-ET, wherein the composition can improve embryo development after IVF-ET, including improving the degree of embryo development in vivo, the quality of embryo development in vivo and pregnancy outcome after in vitro fertilization-embryo transfer (IVF-ET);

[0007] The second technical problem to be solved by the present invention is to provide a use of a menstruation regulating and pregnancy promoting pill for preparing a drug for improving embryo development after IVF-ET surgery;

[0008] The third technical problem to be solved by the present invention is to provide a pharmaceutical preparation for improving embryonic development after IVF-ET surgery and a preparation method thereof.

[0009] In order to solve the above technical problems, the present invention discloses a composition for preparing a drug for improving embryonic development after IVF-ET surgery. The raw materials of the composition are as follows: 5-15 parts by weight of pilose antler, 10-30 parts by weight of epimedium, 10-40 parts by weight of curculigo, 10-30 parts by weight of dipsaccharum, 10-30 parts by weight of parasitic plant, 15-50 parts by weight of dodder seed, 10-30 parts by weight of wolfberry, 10-30 parts by weight of raspberry, 30-60 parts by weight of yam, 10-30 parts by weight of lotus seed, 15-50 parts by weight of poria, 10-30 parts by weight of astragalus, 10-50 parts by weight of white peony root, 10-30 parts by weight of spiny jujube seed, 10-30 parts by weight of Uncaria rhynchophylla, 15-50 parts by weight of salvia miltiorrhiza, 15-50 parts by weight of red peony root, and 30-60 parts by weight of Millettia reticulata.

[0010] Preferably, the raw materials of the composition are composed of: 5 parts by weight of pilose antler, 10 parts by weight of epimedium, 10 parts by weight of curculigo, 10 parts by weight of dipsaccharum, 10 parts by weight of villous herb, 15 parts by weight of dodder seed, 10 parts by weight of wolfberry, 10 parts by weight of raspberry, 30 parts by weight of yam, 10 parts by weight of lotus seed, 15 parts by weight of poria, 10 parts by weight of astragalus, 15 parts by weight of white peony root, 10 parts by weight of spiny jujube seed, 10 parts by weight of Uncaria rhynchophylla, 15 parts by weight of salvia miltiorrhiza, 15 parts by weight of red peony root, and 30 parts by weight of Millettia reticulata.

[0011] Specifically, the improving of embryo development after IVF-ET includes improving the degree of embryo development in vivo, the quality of embryo development in vivo and pregnancy outcome after in vitro fertilization-embryo transfer (IVF-ET).

[0012] The invention also discloses a use of a menstruation regulating and pregnancy promoting pill for preparing a drug for improving embryonic development after IVF-ET surgery. The raw material medicine composition of the menstruation regulating and pregnancy promoting pill is: 5 parts by weight of pilose antler, 10 parts by weight of epimedium, 10 parts by weight of curculigo, 10 parts by weight of dipsaccharum, 10 parts by weight of viburnum, 15 parts by weight of dodder seeds, 10 parts by weight of wolfberry, 10 parts by weight of raspberry, 30 parts by weight of yam, 10 parts by weight of lotus seeds, 15 parts by weight of poria, 10 parts by weight of astragalus, 15 parts by weight of white peony root, 10 parts by weight of spiny jujube seed, 10 parts by weight of Uncaria rhynchophylla, 15 parts by weight of salvia miltiorrhiza, 15 parts by weight of red peony root, and 30 parts by weight of Millettia reticulata.

[0013] The functions and indications of the Tiaojing Cuyun Pill are: warming the kidney and strengthening the spleen, promoting blood circulation and regulating menstruation. It is used for irregular menstruation, amenorrhea, dysmenorrhea, and infertility caused by spleen and kidney yang deficiency and blood stasis, with symptoms of delayed menstruation, scanty menstrual flow, blood clots, cold pain in the lower abdomen during menstruation, prolonged menstruation, long-term infertility, and cold pain in the waist and knees.

[0014] Specifically, the improving of embryo development after IVF-ET includes improving the degree of embryo development in vivo, the quality of embryo development in vivo and pregnancy outcome after in vitro fertilization-embryo transfer (IVF-ET).

[0015] The present invention also discloses a pharmaceutical preparation for improving embryonic development after IVF-ET surgery, wherein the pharmaceutical preparation comprises the composition involved in the use or the menstruation regulating and pregnancy promoting pill.

[0016] Specifically, the pharmaceutical preparation for improving embryonic development after IVF-ET includes pharmaceutically acceptable preparations;

[0017] Optionally, the dosage form includes at least one of tablets, pills, capsules, powders, drops, lyophilized products, granules, suspensions, syrups, decoctions or oral solutions.

[0018] Specifically, the pharmaceutical preparation for improving embryonic development after IVF-ET surgery also includes a pharmaceutically acceptable excipient;

[0019] Optionally, the auxiliary material includes one or a combination of solvents, fillers, solubilizers, cosolvents, emulsifiers, colorants, binders, disintegrants, stabilizers, flavoring agents, preservatives, suspending agents, sweeteners, lubricants, coating materials, fragrances or thickeners.

[0020] Specifically, the pharmaceutical preparation for improving embryo development after IVF-ET includes improving the degree of embryo development in vivo, the quality of embryo development in vivo and the pregnancy outcome after in vitro fertilization-embryo transfer (IVF-ET).

[0021] The present invention also discloses a method for preparing the pharmaceutical preparation for improving embryonic development after IVF-ET surgery, comprising the steps of selecting a quantitative amount of raw material drug components for compounding or extracting active ingredients of the raw material drug components for compounding.

[0022] The invention discloses a new use of a menstruation regulating and pregnancy promoting composition, especially a menstruation regulating and pregnancy promoting pill, for preparing a drug for improving embryonic development after IVF-ET surgery. The menstruation regulating and pregnancy promoting composition and the menstruation regulating and pregnancy promoting pill are composed of velvet antler (dehaired), epimedium (roasted), curculigo, radix dipsaci, parasitic plant, dodder seed, wolfberry fruit, raspberry, yam, lotus seed (de-cored), tuckahoe, astragalus, white peony root, spiny jujube seed (fried), Uncaria rhynchophylla, salvia miltiorrhiza, red peony root, and millettia reticulata. In the prescription, velvet antler and Dipsacus asper strengthen yang and warm the kidney, mistletoe nourishes liver and kidney, epimedium and dodder regulate Chong and Ren, nourish yin and tonify kidney, lotus seed has the effect of tonifying spleen, astragalus has the effect of tonifying qi and raising yang, replenishing qi and strengthening the exterior, spina jujuba seed nourishes yin and blood, white peony root and salvia miltiorrhiza nourish blood and regulate menstruation, Chinese yam nourishes kidney and warms essence, Poria strengthens spleen and eliminates dampness, Millettia reticulata promotes blood circulation and removes blood stasis, and all the medicines are used together to play the effect of regulating and tonifying Chong and Ren, strengthening spleen and tonifying kidney, nourishing blood, regulating menstruation and promoting pregnancy. The present invention evaluates the effect of regulating menstruation and promoting pregnancy pills on ovulation function and egg quality after intervention in female mice and the embryonic development status after transplantation in vivo, litter rate and fetal development status based on in vitro fertilization-embryo transfer technology, and confirms that the regulating menstruation and promoting pregnancy composition, especially the regulating menstruation and promoting pregnancy pills, can improve the degree of embryonic development in vivo, embryonic development quality and pregnancy outcome after in vitro fertilization-embryo transfer (IVF-ET) surgery.

[0023] The present invention discloses a new use of a menstruation regulating and pregnancy promoting composition, especially a menstruation regulating and pregnancy promoting pill, for preparing a drug for improving embryonic development after IVF-ET. The experimental results show that after the embryo donor mouse is administered with the menstruation regulating and pregnancy promoting pill, the egg quality produced by it is higher. After in vitro fertilization to form a fertilized egg and transplanted into the body, compared with the control group, the implantation rate of the fertilized egg, the degree of embryo development, the clinical pregnancy rate, the litter rate, and the fetal mouse skeleton and various organs of the administration group are better, indicating that the menstruation regulating and pregnancy promoting pill can improve the implantation of the embryo and promote embryonic development. Therefore, the effect of the menstruation regulating and pregnancy promoting pill on in vitro fertilization is studied, and the effect characteristics of the drug on the quality of mouse eggs, embryo implantation, and embryonic development in vivo are clarified, and the new use of the drug is expanded, and new effective drugs and means are provided for assisted reproductive technology, thereby providing technical support for the rational application of drugs.

[0024] This method innovatively uses in vitro fertilization and embryo transfer technology to evaluate the effect of Tiaojing Cuyun Pill on the quality of female mouse eggs and the in vivo development of the formed embryos. The pharmacodynamic evaluation system of Tiaojing Cuyun Pill is established by using in vitro fertilization-embryo transfer technology. The eggs discharged from C57BL / 6J mice after Tiaojing Cuyun Pill intervention are collected for in vitro fertilization, and the fertilized eggs are transplanted into the body by embryo transfer technology to detect the effect of Tiaojing Cuyun Pill on the quality of eggs and the level of embryo development in vivo. The results showed that after the embryo donor mice were given the test substance, the eggs they produced were of higher quality. After in vitro fertilization to form fertilized eggs and transplanted into the body, they could implant and develop better in the later stage, achieving the production of more and better-conditioned offspring, indicating that Tiaojing Cuyun Pill significantly improved the implantation and development of embryos in assisted reproduction.

[0025] The present invention is based on in vitro fertilization technology and embryo transplantation. By setting quantifiable indicators such as implantation point and birth size, combined with intuitive indicators of fetal mouse skeleton and organ development morphology, the egg quality and embryo development degree are objectively and clearly evaluated; and by detecting the number of embryo development, development degree, morphology of fetal development at different stages, and birth size of different groups of fetuses, the pharmacodynamic effect of the menstruation regulating and pregnancy promoting pills on the development degree and development quality of fertilized embryos is clarified, the difficulty of how to evaluate the quality of eggs and the quality of the embryos formed is solved, and a method for evaluating the effects of drugs on the quality of eggs and the level of embryo development in vivo is established. The present invention expands the scope of drug application, evaluates the pharmacodynamic effect of the menstruation regulating and pregnancy promoting pills in assisted reproductive technology, and provides new therapeutic drugs and ideas for improving the success rate of assisted reproductive technology. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] In order to make the content of the present invention more clearly understood, the present invention is further described in detail below according to specific embodiments of the present invention in conjunction with the accompanying drawings, wherein:

[0027] Figure 1 The implantation status of embryos in the uterus of mice in each group was observed at the implantation point on day 3.5; A: uterus of the drug-treated group after embryo transplantation; B: uterus of the non-drug-treated group after embryo transplantation;

[0028] Figure 2 The uterine morphology results after embryo transplantation at the 13.5 day implantation point observation; A: uterus after embryo transplantation in the drug-treated group; B: uterus after embryo transplantation in the non-drug-treated group;

[0029] Figure 3 The embryos dissected from mice in each group under implantation observation on day 13.5; A: fetal mice dissected after embryo transfer in the drug-treated group; B: fetal mice dissected after embryo transfer in the non-drug-treated group;

[0030] Figure 4These are the results of microscopic examination of fetal mice under implantation point observation on day 13.5; the left side is the fetal mouse dissected after embryo transplantation in the drug-treated group; the right side is the fetal mouse dissected after embryo transplantation in the non-drug-treated group. DETAILED DESCRIPTION

[0031] Example 1

[0032] This embodiment uses Tongren Tiaojing Cuyun Pills, which are produced by Tongren Tang Pharmaceutical Factory of Beijing Tongrentang Co., Ltd., and the product batch number is 22035305. The Tiaojing Cuyun Pills have the following functions and indications: warming the kidney and strengthening the spleen, promoting blood circulation and regulating menstruation. It is used for irregular menstruation, amenorrhea, dysmenorrhea, and infertility caused by spleen and kidney yang deficiency and blood stasis. Symptoms include delayed menstruation, scanty menstrual flow, blood clots, cold pain in the lower abdomen during menstruation, prolonged menstrual flow, long-term infertility, and cold pain in the waist and knees.

[0033] In this embodiment, the raw material composition of the menstruation regulating and pregnancy promoting pill is: 5 parts by weight of pilose antler, 10 parts by weight of epimedium, 10 parts by weight of curculigo, 10 parts by weight of dipsaccharum, 10 parts by weight of mistletoe, 15 parts by weight of dodder seed, 10 parts by weight of wolfberry, 10 parts by weight of raspberry, 30 parts by weight of yam, 10 parts by weight of lotus seed, 15 parts by weight of poria, 10 parts by weight of astragalus, 15 parts by weight of white peony root, 10 parts by weight of spiny jujube seed, 10 parts by weight of Uncaria rhynchophylla, 15 parts by weight of salvia miltiorrhiza, 15 parts by weight of red peony root, and 30 parts by weight of Millettia reticulata.

[0034] In this embodiment, the dosage of the menstruation regulating and pregnancy promoting pill is: oral, 1 bag at a time, 2 times a day. Take it for 20 consecutive days starting from the fifth day of the menstrual cycle; take it for 20 consecutive days every month for those without a cycle, for three consecutive months or follow the doctor's advice.

[0035] Example 2

[0036] The pharmaceutical preparation of the drug for improving embryonic development after IVF-ET surgery described in this embodiment is a pill, and its raw material drug composition is: 5 parts by weight of pilose antler, 30 parts by weight of epimedium, 10 parts by weight of curculigo, 30 parts by weight of dipsaccharum, 10 parts by weight of villous plant, 50 parts by weight of dodder seed, 10 parts by weight of wolfberry, 30 parts by weight of raspberry, 30 parts by weight of yam, 30 parts by weight of lotus seed, 15 parts by weight of poria, 30 parts by weight of astragalus, 10 parts by weight of white peony root, 30 parts by weight of spiny jujube seed, 10 parts by weight of Uncaria rhynchophylla, 50 parts by weight of salvia miltiorrhiza, 15 parts by weight of red peony root, and 60 parts by weight of Millettia reticulata.

[0037] The method for preparing the pharmaceutical preparation of the drug for improving embryonic development after IVF-ET surgery described in this embodiment includes the steps of compounding the above-mentioned selected amount of raw materials, adding conventional doses of auxiliary materials, and processing into pills.

[0038] Example 3

[0039] The pharmaceutical preparation of the drug for improving embryonic development after IVF-ET surgery described in this embodiment is a tablet, and its raw material composition is: 15 parts by weight of pilose antler, 10 parts by weight of epimedium, 40 parts by weight of curculigo, 10 parts by weight of dipsaccharum, 30 parts by weight of villous herb, 15 parts by weight of dodder seed, 30 parts by weight of wolfberry, 10 parts by weight of raspberry, 60 parts by weight of yam, 10 parts by weight of lotus seed, 50 parts by weight of poria, 10 parts by weight of astragalus, 50 parts by weight of white peony root, 10 parts by weight of spiny jujube seed, 30 parts by weight of Uncaria rhynchophylla, 15 parts by weight of salvia miltiorrhiza, 50 parts by weight of red peony root, and 30 parts by weight of Millettia reticulata.

[0040] The method for preparing the pharmaceutical preparation of the drug for improving embryonic development after IVF-ET surgery described in this embodiment includes the steps of compounding the selected amount of the raw materials mentioned above, adding conventional doses of auxiliary materials, and processing into tablets.

[0041] Example 4

[0042] The pharmaceutical preparation of the drug for improving embryonic development after IVF-ET surgery described in this embodiment is a granule, and its raw material drug composition is: 10 parts by weight of pilose antler, 20 parts by weight of epimedium, 25 parts by weight of curculigo, 20 parts by weight of dipsaccharum, 20 parts by weight of virus, 30 parts by weight of dodder seed, 20 parts by weight of wolfberry, 20 parts by weight of raspberry, 45 parts by weight of yam, 20 parts by weight of lotus seed, 35 parts by weight of poria, 20 parts by weight of astragalus, 40 parts by weight of white peony root, 20 parts by weight of spiny jujube seed, 20 parts by weight of Uncaria rhynchophylla, 35 parts by weight of salvia miltiorrhiza, 40 parts by weight of red peony root, and 45 parts by weight of Millettia reticulata.

[0043] Experimental example

[0044] In the following experimental example of the present invention, based on in vitro fertilization, the effects of the menstruation regulating and pregnancy promoting composition on ovulation function, egg quality, implantation rate after transplantation, embryo development status in vivo, delivery and offspring development were explored to evaluate the pharmacodynamics of the menstruation regulating and pregnancy promoting pills.

[0045] In the following experimental example of the present invention, in order to evaluate the effects of the menstruation regulating and pregnancy promoting pills on ovulation function, egg quality and late embryonic development in vivo, this experiment intends to use in vitro fertilization combined with embryo transplantation technology to establish a pharmacodynamic evaluation system, and use C57BL / 6J mice as research subjects. After multiple drug administration interventions, the mice in the drug administration group are screened for estrus to collect eggs, and co-cultured with donor sperm for a certain period of time, and finally fertilized eggs are collected for embryo transplantation. During the experiment, the number of ovulations, the number of fertilizations, the implantation site, the in vivo development of 3.5-day and 13.5-day embryos, the number of embryos and developmental morphology, and the final litter size and pup morphology are detected.

[0046] Animal Information

[0047] Embryo donor mice C57BL / 6J mice were from the Institute of Laboratory Animal Resources, China Food and Drug Inspection Institute, 3 weeks old, number: 30, female. Laboratory Animal Production License Number: SCXK (Beijing) 2022-0002, Laboratory Animal Use License Number: SYXK (Beijing) 2022-0014. The age of pseudo-pregnant transplant mice ICR was 4-6 weeks, and the number was 15. The age of sperm donor mice C57BL / 6J was 10-12 weeks, and the number was 2. The mice in each group were grouped and adapted to the experimental environment for 5-7 days. They were fed SPF-grade mouse pellet feed and had free access to sterilized tap water. The room temperature was 20-26°C, the relative humidity was 40-70%, and the lighting was 12 hours light / 12 hours dark. This experiment was conducted under the supervision of the Laboratory Animal Welfare Ethics Review Committee of the China Food and Drug Inspection Institute, and the welfare ethics review number was: Zhongjiandong (Fu) No. 2023 (B) 007.

[0048] Dosage regimen

[0049] This experimental example uses the menstruation regulating pregnancy promoting pills described in Example 1 (Tong Ren Tang Pharmaceutical Factory, Beijing Tong Ren Tang Co., Ltd., 22035305) as the experimental drug.

[0050] In each of the following experimental examples of the present invention, two groups are set up, and the quarantine-qualified animals are divided into a group for regulating menstruation and promoting pregnancy and a control group. Test substance treatment method: The regulating menstruation and promoting pregnancy pills are mixed with pure water to form a suspension, and the dosage is 10.275g / kg; the control group is given pure water. Three weeks before the female mice are oocyte-collected, the drug is administered by gavage every day, 5 days a week, and the administration cycle is 3 weeks. Specific experimental grouping information is shown in Table 1 below.

[0051] Table 1 Experimental groups

[0052] Group Number of animals / sex Animal No. Animal cage number Dosage Menstrual regulation and pregnancy promotion dose group 15 / ♀ Y001-Y0030 / ♀ No. 01-No. 03 10.275g / kg Control group 15 / ♀ Y061-Y0090 / ♀ No. 04-06 /

[0053] In vitro fertilization and embryo transfer

[0054] After the above experimental culture, three weeks after the embryo donor mice were gavaged and administered, 15 female mice were killed by cervical dislocation, and the oviducts were taken. The eggs were stripped from the ampulla in M2 culture medium, washed and transferred to KSOM culture medium for culture. Male sperm donor mice were killed by cervical dislocation, and the epididymis was taken. After washing in M2 culture medium, sperm was stripped in KSOM culture medium and capacitated for use. The eggs and capacitated sperm were co-incubated in KSOM culture medium microdrops for 4-6 hours, and the fertilized eggs were washed and transferred to fresh KSOM culture medium for culture.

[0055] Preparation of pseudopregnant transplant mice: One day before egg retrieval, the recipient female mice were placed in the same cage with the vasectomized male mice. On the second day of in vitro fertilization, the vaginal plugs of the transplanted mice were checked, and female mice with plugs were randomly selected as surrogate mice to receive unilateral fallopian tube embryo transfer on the same day. Each surrogate female mouse was transplanted with 20 2-cell embryos.

[0056] Detection of different stages of embryonic development

[0057] Implantation is an important link in the reproductive physiology of viviparous mammals. It is a synchronized development process of interaction between the mother and the blastocyst, which enables the blastocyst free in the fallopian tube or uterine cavity to invade the endometrium and then form the placenta to obtain sufficient nutrition from the mother.

[0058] After transplantation, three mice were dissected at different time points to observe the development of the embryos in vivo. Three female mice were injected with trypan blue dye from the tail vein at 8:00-9:00 am 3.5 days and 13.5 days after transplantation. The mice were killed by cervical dislocation 10 minutes later, and the uterus was removed and naturally flattened. The stained nodules in the uterus were observed, counted and measured, and the counts were observed with the naked eye. The delivery of the surrogate female mice was observed 3 weeks later, and the morphology of the offspring was compared.

[0059] In this experimental case, the embryo implantation in the uterus of each group of mice under the observation of implantation point on day 3.5 is shown in the attached Figure 1 It can be seen that this experiment used the dye trypan blue to perform a staining experiment on the blastocyst implantation site. Three female mice were taken from each experimental group, and the uterus was dissected to analyze the implantation site. The experiment observed that there were 33 and 32 embryo nodules in the drug-treated group and the non-drug-treated group, respectively, with no obvious difference.

[0060] In this experiment, since the mouse embryonic period of 9.5-13.5 days is the stage of organ formation, the intestinal tube gradually begins to have organ precursors with morphological characteristics. Therefore, on the 13.5th day, three female mice from each experimental group were selected, and the uterus was dissected to analyze the morphology and quantity of the embryos. The results are shown in the attached Figure 2 . Figure 2 From the experimental results shown, it can be observed that the three mice in the drug-treated group had a total of 36 embryos, and the three mice in the non-drug-treated group had a total of 20 embryos, of which one mouse was not pregnant.

[0061] The uterus of each group was further opened, and the embryos were removed and observed for morphology. The embryos removed from each group of mice are shown in the attached figure. Figure 3 It can be seen that the morphological development of the bones and organs of the fetal mice in the drug-treated group was more mature than that in the non-drug-treated group.

[0062] One fetus was randomly selected from each of the two groups for microscopic examination. The results of microscopic examination of fetuses in the drug-treated and non-drug-treated groups are shown in the attached Figure 4 It can be seen that the fetuses of the two experimental groups developed normally, but the fetuses of the drug-treated group were significantly larger than those of the non-drug-treated group.

[0063] Statistics of the number of litters born in late pregnancy

[0064] After 21 days of late pregnancy, 5 pregnant mice in the drug group gave birth to 34 offspring. 5 pregnant mice in the non-drug group gave birth to 25 offspring. The experimental results showed that the embryo parameters of the eggs produced by female mice after administration of Tiaojing Cuyu Wan were better than those in the non-drug group, the clinical pregnancy rate and litter rate were higher than those in the non-drug group, and the morphological development of the bones and organs of the fetuses in the drug group was better than that in the non-drug group, and the embryo individuals were better than those in the non-drug group.

[0065] In summary, the experimental results of the present invention show that after the embryo donor mice were administered with the menstruation regulating and pregnancy promoting pills, the quality of the eggs they produced was higher. After in vitro fertilization to form fertilized eggs and transplanted into the body, compared with the control group, the fertilized egg implantation rate, embryo development degree, clinical pregnancy rate, litter birth rate, and fetal mouse bones and organs in the administration group were better, indicating that the menstruation regulating and pregnancy promoting pills can improve embryo implantation and promote embryo development, providing new effective drugs and means for assisted reproductive technology.

[0066] Obviously, the above embodiments are merely examples for the purpose of clear explanation, and are not intended to limit the implementation methods. For those skilled in the art, other different forms of changes or modifications can be made based on the above description. It is not necessary and impossible to list all the implementation methods here. The obvious changes or modifications derived therefrom are still within the scope of protection of the invention.

Claims

1. A composition for preparing a drug for improving embryonic development after IVF-ET surgery, characterized in that: The raw materials of the composition are composed of: 5-15 parts by weight of pilose antler, 10-30 parts by weight of epimedium, 10-40 parts by weight of curculigo, 10-30 parts by weight of dipsaccharum, 10-30 parts by weight of mistletoe, 15-50 parts by weight of dodder seeds, 10-30 parts by weight of wolfberry, 10-30 parts by weight of raspberry, 30-60 parts by weight of yam, 10-30 parts by weight of lotus seeds, 15-50 parts by weight of poria, 10-30 parts by weight of astragalus, 10-50 parts by weight of white peony root, 10-30 parts by weight of spiny jujube seed, 10-30 parts by weight of Uncaria rhynchophylla, 15-50 parts by weight of salvia miltiorrhiza, 15-50 parts by weight of red peony root, and 30-60 parts by weight of Millettia reticulata.

2. The use according to claim 1, characterized in that The raw materials of the composition are composed of: 5 parts by weight of pilose antler, 10 parts by weight of epimedium, 10 parts by weight of curculigo, 10 parts by weight of dipsaci, 10 parts by weight of villous herb, 15 parts by weight of dodder seed, 10 parts by weight of wolfberry, 10 parts by weight of raspberry, 30 parts by weight of yam, 10 parts by weight of lotus seed, 15 parts by weight of poria, 10 parts by weight of astragalus, 15 parts by weight of white peony root, 10 parts by weight of spiny jujube seed, 10 parts by weight of Uncaria rhynchophylla, 15 parts by weight of salvia miltiorrhiza, 15 parts by weight of red peony root, and 30 parts by weight of Millettia reticulata.

3. The use according to claim 1 or 2, characterized in that The improving of embryonic development after IVF-ET includes improving the degree of embryonic development in vivo, the quality of embryonic development in vivo and pregnancy outcome after in vitro fertilization-embryo transfer (IVF-ET).

4. Use of a pill for regulating menstruation and promoting pregnancy for preparing a drug for improving embryo development after IVF-ET surgery, characterized in that: The raw material composition of the menstruation regulating and pregnancy promoting pill is 5 parts by weight of pilose antler, 10 parts by weight of epimedium, 10 parts by weight of curculigo, 10 parts by weight of dipsaccharum, 10 parts by weight of virus, 15 parts by weight of dodder seed, 10 parts by weight of wolfberry, 10 parts by weight of raspberry, 30 parts by weight of yam, 10 parts by weight of lotus seed, 15 parts by weight of poria, 10 parts by weight of astragalus, 15 parts by weight of white peony root, 10 parts by weight of spiny jujube seed, 10 parts by weight of Uncaria rhynchophylla, 15 parts by weight of salvia miltiorrhiza, 15 parts by weight of red peony root, and 30 parts by weight of Millettia reticulata.

5. The use according to claim 4, characterized in that The improving of embryonic development after IVF-ET includes improving the degree of embryonic development in vivo, the quality of embryonic development in vivo and pregnancy outcome after in vitro fertilization-embryo transfer (IVF-ET).

6. A pharmaceutical preparation for improving embryonic development after IVF-ET surgery, characterized in that: The pharmaceutical preparation comprises the composition involved in the use described in any one of claims 1 to 3 or the menstruation regulating and pregnancy promoting pill.

7. The pharmaceutical preparation for improving embryonic development after IVF-ET according to claim 6, characterized in that: The dosage form of the pharmaceutical preparation includes pharmaceutically acceptable preparations; Optionally, the dosage form includes at least one of tablets, pills, capsules, powders, drops, lyophilized products, granules, suspensions, syrups, decoctions or oral solutions.

8. The pharmaceutical preparation for improving embryonic development after IVF-ET according to claim 6 or 7, characterized in that: The pharmaceutical preparation also includes pharmaceutically acceptable excipients; Optionally, the auxiliary material includes one or a combination of solvents, fillers, solubilizers, cosolvents, emulsifiers, colorants, binders, disintegrants, stabilizers, flavoring agents, preservatives, suspending agents, sweeteners, lubricants, coating materials, fragrances or thickeners.

9. The pharmaceutical preparation for improving embryonic development after IVF-ET according to any one of claims 6 to 8, characterized in that: The improving of embryonic development after IVF-ET includes improving the degree of embryonic development in vivo, the quality of embryonic development in vivo and pregnancy outcome after in vitro fertilization-embryo transfer (IVF-ET).

10. A method for preparing the pharmaceutical preparation for improving embryonic development after IVF-ET according to any one of claims 6 to 9, characterized in that: The method comprises the steps of selecting a quantitative amount of raw material drug components for compounding or extracting active ingredients of the raw material drug components for compounding.

Images