Jinan Energy Technology Co., Ltd Introduction to the clinical significance and methods of assisted reproductive technology
(AI) is a method of inserting sperm into the female reproductive tract through non sexual intercourse, allowing them to naturally combine with the egg and achieve conception. The earliest successful AI treatment in humans was John Hunter's diagnosis of severe hypospadias in 1790
Image of assisted reproductive technology (sperm bank)
Image of assisted reproductive technology (sperm bank)
Artificial insemination between spouses by the wife. Although it has been more than 200 years now, it is still a commonly used and effective pregnancy aid technique. Due to different sources of semen, AI is divided into artificial insemination with husband sperm (AIH) and artificial insemination with donor sperm (non spouse) (AID). The two have different indications, and AIH treatment includes: (1) sexual intercourse disorders; (2) Obstruction of sperm movement in the female reproductive tract; (3) Oligozoospermia and asthenozoospermia. AID treatment: (1) azoospermia; (2) The man has a genetic disease; (3) Special blood type or immune incompatibility between spouses. When implementing AID treatment, donors must choose young adults who are physically healthy, have good intellectual development, and have no family history of genetic diseases. Chromosome variation, hepatitis B, hepatitis C, gonorrhea, syphilis, especially AIDS must be excluded. The blood type should be the same as the recipient's husband. Sperm donors should be frozen for 6 months and tested HIV negative before use. Due to the incubation period of about 6 months for HIV infection, diagnosis is difficult to determine, so donor sperm should generally be obtained from a sperm bank.
Whether AIH or AID treatment is implemented, sperm must undergo optimal induction and capacitation treatment before fertilization, which is an important routine technique for intrauterine or in vitro fertilization. Its function is to remove seminal plasma containing inhibitory and affecting fertilization components, and activate and induce sperm capacitation. In natural fertilization, sperm undergo these changes during the process of passing through cervical mucus and staying in the fallopian tube waiting for the egg. In clinical treatment, a combination of ion washing and fertilization culture medium with components similar to fallopian tube fluid is used, including sperm upstream method and Percoll gradient centrifugation method. The former method is relatively simple, but the sperm recovery rate is low. For those with few or weak sperm, the latter method should be used. The insemination time should be selected between 48 hours before ovulation and 12 hours after ovulation based on preoperative monitoring of the female's ovulation. The commonly used method for insemination is to inject sperm into the cervix or into the uterine cavity under strict aseptic measures.
Main methods
artificial insemination
(AI) is a method of inserting sperm into the female reproductive tract through non sexual intercourse, allowing them to naturally combine with the egg and achieve conception. The earliest successful AI treatment in humans was John Hunter's diagnosis of severe hypospadias in 1790的妻子进行的配偶间artificial fertilization。Although it has been more than 200 years now, it is still a commonly used and effective pregnancy aid technique. Due to different sources of semen, AI is divided into artificial insemination with husband sperm (AIH) and artificial insemination with donor sperm (non spouse) (AID). The two have different indications, and AIH treatment includes: (1) sexual intercourse disorders; (2) Obstruction of sperm movement in the female reproductive tract;(3)Oligozoospermia and asthenozoospermia. AID treatment: (1) azoospermia; (2) The man has a genetic disease; (3) Special blood type or immune incompatibility between spouses. When implementing AID treatment, donors must choose young adults who are physically healthy, have good intellectual development, and have no family history of genetic diseases. Chromosome variation, hepatitis B, hepatitis C, gonorrhea, syphilis, especially AIDS must be excluded. The blood type should be the same as the recipient's husband. Sperm donors should be frozen for 6 months and tested HIV negative before use. Due to the incubation period of about 6 months for HIV infection, diagnosis is difficult to determine, so donor sperm should generally be obtained from a sperm bank.
Whether AIH or AID treatment is implemented, sperm must undergo optimal induction and capacitation treatment before fertilization, which is an important routine technique for intrauterine or in vitro fertilization. Its function is to remove seminal plasma containing inhibitory and affecting fertilization components, and activate and induce sperm capacitation. In natural fertilization, sperm undergo these changes during the process of passing through cervical mucus and staying in the fallopian tube waiting for the egg. In clinical treatment, a combination of ion washing and fertilization culture medium with components similar to fallopian tube fluid is used, including sperm upstream method and Percoll gradient centrifugation method. The former method is relatively simple, but the sperm recovery rate is low. For those with few or weak sperm, the latter method should be used. The insemination time should be selected between 48 hours before ovulation and 12 hours after ovulation based on preoperative monitoring of the female's ovulation. The commonly used method for insemination is to inject sperm into the cervix or into the uterine cavity under strict aseptic measures.
Oligozoospermia and asthenozoospermia. AID treatment: (1) azoospermia; (2) The man has a genetic disease; (3) Special blood type or immune incompatibility between spouses. When implementing AID treatment, donors must choose young adults who are physically healthy, have good intellectual development, and have no family history of genetic diseases. Chromosome variation, hepatitis B, hepatitis C, gonorrhea, syphilis, especially AIDS must be excluded. The blood type should be the same as the recipient's husband. Sperm donors should be frozen for 6 months and tested HIV negative before use. Due to the incubation period of about 6 months for HIV infection, diagnosis is difficult to determine, so donor sperm should generally be obtained from a sperm bank.
2.3 Controlled superovulation and follicle monitoring: Eggs are collected according to natural cycles, and only one egg can be obtained in one cycle. In order to improve pregnancy, currently in IVF-ET technology, controlled ovarian hyperstimulation is commonly used, which uses human gonadotropins to enhance and improve ovarian function, allowing multiple follicles to develop in one cycle and retrieving multiple eggs for fertilization. To obtain more embryos for transplantation. But sometimes, the method of promoting superovulation may result in premature maturation and poor quality of follicles. In 1991, the First Affiliated Hospital of Zhongshan Medical University used Buserelin, a nasal spray of hypothalamic gonadotropin agonist (GnRH-a), for downregulation. Combined with the use of gonadotropins, the number of dominant follicles significantly increased [3]. During the implementation of ovarian hyperstimulation, it is necessary to use vaginal ultrasound scans to monitor the number and size of follicular development, as well as changes in urine LH and blood hormones, in order to adjust medication in a timely manner, accurately estimate the time of egg retrieval, and try to match the function of the corpus luteum and endometrium, pregnancy occurrence, and pregnancy maintenance.
2.4 Egg retrieval: Our hospital uses B-ultrasound guided transvaginal puncture for egg retrieval. This method does not require anesthesia or incision, nor does it pass through the bladder, avoiding the harm of urine to sperm, and is superior to the method of using laparoscopy or B-ultrasound to guide transabdominal egg retrieval.
2.5 In vitro fertilization: Inject the obtained follicular fluid into a culture dish, and quickly identify with the naked eye the oocytes and their surrounding zona pellucida, as well as the coronal complex of the radiating corona. After confirming the presence of egg cells under the dissecting microscope, they are placed in a CO 2 incubator for 4-8 hours. Mature egg cells are selected based on the morphological changes of the complex. According to the ratio of 100000 to 200000 sperm per egg, washed and selected induced sperm are added. After 16 to 18 hours of fertilization, the situation is observed, and the fertilized egg is transferred to a culture tube/dish for cultivation.
2.6 Embryo transfer occurs 48 hours after egg retrieval, when the embryo develops into 2-8 cell stages, or when the embryo develops to 8-16 cells 72 hours after egg retrieval, it is implanted into the uterus. The latter is more in line with the time when naturally fertilized embryos enter the uterus, and under traditional in vitro culture conditions, only the healthiest embryos can survive to 3 days, so the success rate of transplantation is high. It has been reported that the application of co culture can enable embryo culture to the blastocyst stage before transplantation, with a pregnancy rate of up to 50% [4]. It is advisable to transfer 2-3 embryos at a time. Due to the increase in the number of embryo transfers, although the pregnancy rate does not increase proportionally, the multiple pregnancy rate will also increase accordingly. After comparing the data of several groups of 1-6 embryo transfers [5], the pregnancy rate of 3 embryos transferred is relatively high, while the multiple pregnancy rate is relatively low.
2.7 Embryo freeze-thaw is not only necessary for AID treated sperm, but also for IVF-ET due to the application of superovulation promotion. Follicles recovered in one cycle and embryos developed after fertilization also need to be frozen and stored after transfer. If the transfer fails, it can be transferred in the next natural cycle or HRT cycle to improve the pregnancy rate of one egg retrieval. In addition, when encountering ovarian stimulation syndrome caused by ovarian hyperstimulation, in order to prevent pregnancy from worsening the condition, embryos can also be frozen and left for future transplantation. The mechanism of embryo cryopreservation is that ultra-low temperature can inhibit cell metabolism, causing life to enter a dormant state and be preserved. The storage temperature is -196 ℃, and the storage device is a liquid nitrogen tank using liquid nitrogen as the cooling source. However, during the freezing and heating recovery process of embryos, if the temperature drops too fast within the temperature range of 0 ℃ to 60 ℃, the water in the intracellular fluid will quickly freeze again, causing cell death due to volume expansion and breaking of the cell membrane; If the temperature drops too slowly, the water in the extracellular fluid will first form small ice crystals, causing the osmotic pressure of the extracellular fluid to increase, causing the water in the intracellular fluid to penetrate outward and the solute concentration to increase relatively, leading to the decomposition and denaturation of cell proteins, making it difficult for cells to escape the fate of death.Therefore, in the freezing and thawing of embryos, it is necessary to choose appropriate cooling and heating rates, and use certain techniques that can reduce the formation of ice crystals within cells while also delaying the concentration of solutes in extracellular fluids
Image of assisted reproductive technology
Image of assisted reproductive technology
The role of cryoprotectants with elevated degrees is essential for the safe cryopreservation or recovery of embryos. The commonly used method currently is the slow freezing and rapid rewarming method. The mechanism of sperm cryopreservation and the principle of freeze-thaw are similar to the above.
2.8 After embryo transfer, monitoring the urine HCG positive test on the 14th day after transfer indicates biochemical pregnancy, indicating normal embryo implantation and development. Abdominal ultrasound examination of fetal sac, embryo, and cardiac pulsation at 4-6 weeks after transplantation indicates a clinical pregnancy.
2.9 The main complications of embryo transfer include miscarriage, ectopic pregnancy, multiple pregnancies, and ovarian hyperstimulation syndrome (OHSS).
The pregnancy rate is currently between 25% and 35%. With in-depth research on factors affecting pregnancy success and improvements in corresponding technical aspects, it is expected that the pregnancy rate of IVF-ET will also increase.
。
Intrafollicular intracytoplasmic sperm injection (ICSI)
This technology is developed on the basis of in vitro fertilization methods such as micro drop method, zona pellucida partial excision method, and zona pellucida insemination, which are used to address the insufficient number of male sperm and fertilization disorders caused by functional abnormalities. In 1992, Palerme et al. reported the first case of in vitro fertilization using this technology. This technology, also known as the second generation in vitro fertilization (IVF), operates by selecting a sperm with normal morphology and slow movement to immobilize without undergoing sperm induction and capacitation treatment. Method: Squeeze the tail of the sperm with an injection needle, slightly scratch the cell plasma membrane, and induce the sperm to release sperm cytoplasmic factors from the scratch point to activate the egg cell. The activation of the egg cell is crucial for the normal fertilization of ICSI [6]. Then, suck the sperm in the order of tail to tail and insert it into the injection needle. Through microscopic operation, inject the sperm into the egg cytoplasm to complete fertilization. Other technical aspects are the same as conventional IVF-ET. Patients with obstructed sperm ducts can undergo epididymal puncture. If there is no sperm in the aspirate, sperm can be separated from live testicular tissue or used after activation of sperm cells. Indications include severe oligozoospermia, oligozoospermia, obstruction of the vas deferens, congenital bilateral absence of the vas deferens, child injury or death after vas deferens ligation, failure or inability to anastomose the vas deferens. ICSI is an effective method for treating male infertility, but current research has revealed that severe oligozoospermia and asthenozoospermia are caused by chromosomal mosaic abnormalities. Congenital bilateral absence of the vas deferens is often associated with mutations in cystic fibrous membrane conduction genes, both of which are genetic diseases [2]. For these two types of patients, genetic risks should be informed before treatment, and detailed prenatal diagnosis should be conducted after pregnancy. If there are abnormalities, pregnancy should be terminated, Alternatively, genetic diagnosis (PGD) can be performed before embryo transfer to screen for high-quality embryo transfer.
Preimplantation Genetic Diagnosis (PGD)
This method, also known as the third generation in vitro fertilization, refers to the method of analyzing the genetic material of embryos before IVF-ET embryo transfer, diagnosing any abnormalities, screening healthy embryo transfers, and preventing the transmission of genetic diseases. The test substance is taken from one cell of 4-8 cell stage embryos or the first and second polar bodies of eggs before and after fertilization. Sampling does not affect embryonic development. Single cell DNA analysis method is used for detection. Firstly, polymerase chain reaction (PCR) is used to detect male and female gender and single gene genetic diseases; Another method is fluorescence in situ hybridization (FISH), which detects gender and chromosomal diseases. As early as 1964, Edwards proposed the idea of PGD. In 1989, Handyside AH first successfully applied PGD in clinical practice [7], using PCR technology to amplify Y-chromosome specific genes in vitro, and successfully transplanted embryos diagnosed as female into the uterus to achieve pregnancy. At the beginning, PGD was tested for gender using PCR or FISH, and female embryo transfer was selected to help couples at risk of having X-linked genetic diseases such as hemophilia A and progressive muscular dystrophy conceive and deliver a normal female baby. However, according to genetic laws, this method undoubtedly denies the birth of healthy boys and allows carrier girls to reproduce, but does not cut off the transmission of pathogenic genes. In 1992, the United States first reported the successful detection of cystic fibers using PCR, and through embryo screening, a healthy baby was born. Afterwards, α- 1- Anti insulin deficiency, family black? The establishment of PGD detection methods for various monogenic genetic diseases such as sexual dementia and pigmented retinitis, and the entry of PGD into the detection and prevention class for monogenic genetic diseases. After 1993, due to late marriage and childbirth, the number of elderly mothers increased, and women over 45 years old had a high rate of chromosomal abnormalities and were prone to giving birth to 18-3 and 21-3 body fools during natural pregnancy. Therefore, the focus of PGD's work shifted to the detection and prevention of chromosomal diseases, using FISH for testing. Due to the use of the first polar body for sampling, the selected egg is an unfertilized egg, which requires intracytoplasmic injection of a single sperm to be cultured and developed into an embryo before transplantation. According to statistics, embryos that stop developing midway have a chromosomal abnormality rate of 70% [8], so choosing embryos with normal chromosomes for transfer can also improve the success rate of IVF-ET [9]. In theory, any genetic disease that can be diagnosed should be able to prevent its transmission through PGD, but due to current technical conditions, the indications for PGD still have certain limitations. Eugenics is a common concern in the world. With the development of molecular biotechnology and the identification of more genetic disease genes, it is believed that some accurate and safe genetic diagnosis technologies will continue to emerge, and PGD technology will become increasingly perfect, better benefiting humanity.
Egg donation IVF-ET
This technology was first reported by Trounson et al. in 1983 as the first successful pregnancy. The indications are: (1) ovarian failure or absence of ovaries. (2) The female has genetic diseases or chromosomal abnormalities. The selection of egg donors should comply with: (1) those under the age of 35 who have given birth to one child. (2) There is no family history of hereditary disease, mental illness, excluding hepatitis B, hepatitis C and sexually transmitted diseases, especially HIV positive people. (3) Good intelligence and appearance. (4) The blood type matches the recipient. Compared with conventional IVF-ET, technically, there are two important steps that need to be added to the donor IVF-ET: (1) the menstrual cycle of the recipient and the donor must be synchronized. The method is to perform steroid hormone replacement therapy (HRT) on patients without ovarian function 3-5 days before the donor's expected menstrual cycle, to promote cyclic changes in the patient's endometrium, possess the ability to accept embryo implantation, and induce the production of endogenous LH peaks and endometrial estrogen receptors, thereby improving the endocrine environment of the body. For patients with ovarian function, first downregulate with gonadotropin releasing hormone analogues or enhancers GnRH-A, and then treat with HRT. During the HRT treatment cycle, serum hormone measurement, endometrial biopsy, B-ultrasound Doppler monitoring, and understanding of endometrial receptivity are necessary to adjust the dosage and ensure good endometrial receptivity. (2) Maintain pregnancy. After in vitro fertilization and embryo transfer with the sperm of the donor egg and the patient's husband, the patient must gradually increase HRT administration. To maintain pregnancy until the recipient's own placental function is established. The pregnancy rate of IVF-ET with egg donation is relatively high, with some reports reaching 44.9% [11]. In addition to treating patients with indications, it can also provide a human model for studying the interaction between embryos, endometrium, and steroids, with broad clinical application prospects.
Surrogacy
Used for patients with uterine rupture or severe adhesions after hysterectomy. The principle of surrogacy technology is the same as that of IVF-ET, but the donor is a patient and the recipient is a surrogate. Due to conceptual reasons, this technology has not yet been recognized in China.
Main methods
artificial insemination
(AI) is a method of inserting sperm into the female reproductive tract through non sexual intercourse, allowing them to naturally combine with the egg and achieve conception. The earliest successful AI treatment in humans was John Hunter's diagnosis of severe hypospadias in 1790的妻子进行的配偶间artificial fertilization。Although it has been more than 200 years now, it is still a commonly used and effective pregnancy aid technique. Due to different sources of semen, AI is divided into artificial insemination with husband sperm (AIH) and artificial insemination with donor sperm (non spouse) (AID). The two have different indications, and AIH treatment includes: (1) sexual intercourse disorders; (2) Obstruction of sperm movement in the female reproductive tract;(3)Oligozoospermia and asthenozoospermia. AID treatment: (1) azoospermia; (2) The man has a genetic disease; (3) Special blood type or immune incompatibility between spouses. When implementing AID treatment, donors must choose young adults who are physically healthy, have good intellectual development, and have no family history of genetic diseases. Chromosome variation, hepatitis B, hepatitis C, gonorrhea, syphilis, especially AIDS must be excluded. The blood type should be the same as the recipient's husband. Sperm donors should be frozen for 6 months and tested HIV negative before use. Due to the incubation period of about 6 months for HIV infection, diagnosis is difficult to determine, so donor sperm should generally be obtained from a sperm bank.
Whether AIH or AID treatment is implemented, sperm must undergo optimal induction and capacitation treatment before fertilization, which is an important routine technique for intrauterine or in vitro fertilization. Its function is to remove seminal plasma containing inhibitory and affecting fertilization components, and activate and induce sperm capacitation. In natural fertilization, sperm undergo these changes during the process of passing through cervical mucus and staying in the fallopian tube waiting for the egg. In clinical treatment, a combination of ion washing and fertilization culture medium with components similar to fallopian tube fluid is used, including sperm upstream method and Percoll gradient centrifugation method. The former method is relatively simple, but the sperm recovery rate is low. For those with few or weak sperm, the latter method should be used. The insemination time should be selected between 48 hours before ovulation and 12 hours after ovulation based on preoperative monitoring of the female's ovulation. The commonly used method for insemination is to inject sperm into the cervix or into the uterine cavity under strict aseptic measures.
Oligozoospermia and asthenozoospermia. AID treatment: (1) azoospermia; (2) The man has a genetic disease; (3) Special blood type or immune incompatibility between spouses. When implementing AID treatment, donors must choose young adults who are physically healthy, have good intellectual development, and have no family history of genetic diseases. Chromosome variation, hepatitis B, hepatitis C, gonorrhea, syphilis, especially AIDS must be excluded. The blood type should be the same as the recipient's husband. Sperm donors should be frozen for 6 months and tested HIV negative before use. Due to the incubation period of about 6 months for HIV infection, diagnosis is difficult to determine, so donor sperm should generally be obtained from a sperm bank.
2.3 Controlled superovulation and follicle monitoring: Eggs are collected according to natural cycles, and only one egg can be obtained in one cycle. In order to improve pregnancy, currently in IVF-ET technology, controlled ovarian hyperstimulation is commonly used, which uses human gonadotropins to enhance and improve ovarian function, allowing multiple follicles to develop in one cycle and retrieving multiple eggs for fertilization. To obtain more embryos for transplantation. But sometimes, the method of promoting superovulation may result in premature maturation and poor quality of follicles. In 1991, the First Affiliated Hospital of Zhongshan Medical University used Buserelin, a nasal spray of hypothalamic gonadotropin agonist (GnRH-a), for downregulation. Combined with the use of gonadotropins, the number of dominant follicles significantly increased [3]. During the implementation of ovarian hyperstimulation, it is necessary to use vaginal ultrasound scans to monitor the number and size of follicular development, as well as changes in urine LH and blood hormones, in order to adjust medication in a timely manner, accurately estimate the time of egg retrieval, and try to match the function of the corpus luteum and endometrium, pregnancy occurrence, and pregnancy maintenance.
2.4 Egg retrieval: Our hospital uses B-ultrasound guided transvaginal puncture for egg retrieval. This method does not require anesthesia or incision, nor does it pass through the bladder, avoiding the harm of urine to sperm, and is superior to the method of using laparoscopy or B-ultrasound to guide transabdominal egg retrieval.
2.5 In vitro fertilization: Inject the obtained follicular fluid into a culture dish, and quickly identify with the naked eye the oocytes and their surrounding zona pellucida, as well as the coronal complex of the radiating corona. After confirming the presence of egg cells under the dissecting microscope, they are placed in a CO 2 incubator for 4-8 hours. Mature egg cells are selected based on the morphological changes of the complex. According to the ratio of 100000 to 200000 sperm per egg, washed and selected induced sperm are added. After 16 to 18 hours of fertilization, the situation is observed, and the fertilized egg is transferred to a culture tube/dish for cultivation.
2.6 Embryo transfer occurs 48 hours after egg retrieval, when the embryo develops into 2-8 cell stages, or when the embryo develops to 8-16 cells 72 hours after egg retrieval, it is implanted into the uterus. The latter is more in line with the time when naturally fertilized embryos enter the uterus, and under traditional in vitro culture conditions, only the healthiest embryos can survive to 3 days, so the success rate of transplantation is high. It has been reported that the application of co culture can enable embryo culture to the blastocyst stage before transplantation, with a pregnancy rate of up to 50% [4]. It is advisable to transfer 2-3 embryos at a time. Due to the increase in the number of embryo transfers, although the pregnancy rate does not increase proportionally, the multiple pregnancy rate will also increase accordingly. After comparing the data of several groups of 1-6 embryo transfers [5], the pregnancy rate of 3 embryos transferred is relatively high, while the multiple pregnancy rate is relatively low.
2.7 Embryo freeze-thaw is not only necessary for AID treated sperm, but also for IVF-ET due to the application of superovulation promotion. Follicles recovered in one cycle and embryos developed after fertilization also need to be frozen and stored after transfer. If the transfer fails, it can be transferred in the next natural cycle or HRT cycle to improve the pregnancy rate of one egg retrieval. In addition, when encountering ovarian stimulation syndrome caused by ovarian hyperstimulation, in order to prevent pregnancy from worsening the condition, embryos can also be frozen and left for future transplantation. The mechanism of embryo cryopreservation is that ultra-low temperature can inhibit cell metabolism, causing life to enter a dormant state and be preserved. The storage temperature is -196 ℃, and the storage device is a liquid nitrogen tank using liquid nitrogen as the cooling source. However, during the freezing and heating recovery process of embryos, if the temperature drops too fast within the temperature range of 0 ℃ to 60 ℃, the water in the intracellular fluid will quickly freeze again, causing cell death due to volume expansion and breaking of the cell membrane; If the temperature drops too slowly, the water in the extracellular fluid will first form small ice crystals, causing the osmotic pressure of the extracellular fluid to increase, causing the water in the intracellular fluid to penetrate outward and the solute concentration to increase relatively, leading to the decomposition and denaturation of cell proteins, making it difficult for cells to escape the fate of death.Therefore, in the freezing and thawing of embryos, it is necessary to choose appropriate cooling and heating rates, and use certain techniques that can reduce the formation of ice crystals within cells while also delaying the concentration of solutes in extracellular fluids Image of assisted reproductive technology Image of assisted reproductive technology The role of cryoprotectants with elevated degrees is essential for the safe cryopreservation or recovery of embryos. The commonly used method currently is the slow freezing and rapid rewarming method. The mechanism of sperm cryopreservation and the principle of freeze-thaw are similar to the above.
2.8 After embryo transfer, monitoring the urine HCG positive test on the 14th day after transfer indicates biochemical pregnancy, indicating normal embryo implantation and development. Abdominal ultrasound examination of fetal sac, embryo, and cardiac pulsation at 4-6 weeks after transplantation indicates a clinical pregnancy.
2.9 The main complications of embryo transfer include miscarriage, ectopic pregnancy, multiple pregnancies, and ovarian hyperstimulation syndrome (OHSS).
The pregnancy rate is currently between 25% and 35%. With in-depth research on factors affecting pregnancy success and improvements in corresponding technical aspects, it is expected that the pregnancy rate of IVF-ET will also increase.
。
Intrafollicular intracytoplasmic sperm injection (ICSI)
This technology is developed on the basis of in vitro fertilization methods such as micro drop method, zona pellucida partial excision method, and zona pellucida insemination, which are used to address the insufficient number of male sperm and fertilization disorders caused by functional abnormalities. In 1992, Palerme et al. reported the first case of in vitro fertilization using this technology. This technology, also known as the second generation in vitro fertilization (IVF), operates by selecting a sperm with normal morphology and slow movement to immobilize without undergoing sperm induction and capacitation treatment. Method: Squeeze the tail of the sperm with an injection needle, slightly scratch the cell plasma membrane, and induce the sperm to release sperm cytoplasmic factors from the scratch point to activate the egg cell. The activation of the egg cell is crucial for the normal fertilization of ICSI [6]. Then, suck the sperm in the order of tail to tail and insert it into the injection needle. Through microscopic operation, inject the sperm into the egg cytoplasm to complete fertilization. Other technical aspects are the same as conventional IVF-ET. Patients with obstructed sperm ducts can undergo epididymal puncture. If there is no sperm in the aspirate, sperm can be separated from live testicular tissue or used after activation of sperm cells. Indications include severe oligozoospermia, oligozoospermia, obstruction of the vas deferens, congenital bilateral absence of the vas deferens, child injury or death after vas deferens ligation, failure or inability to anastomose the vas deferens. ICSI is an effective method for treating male infertility, but current research has revealed that severe oligozoospermia and asthenozoospermia are caused by chromosomal mosaic abnormalities. Congenital bilateral absence of the vas deferens is often associated with mutations in cystic fibrous membrane conduction genes, both of which are genetic diseases [2]. For these two types of patients, genetic risks should be informed before treatment, and detailed prenatal diagnosis should be conducted after pregnancy. If there are abnormalities, pregnancy should be terminated, Alternatively, genetic diagnosis (PGD) can be performed before embryo transfer to screen for high-quality embryo transfer.
Preimplantation Genetic Diagnosis (PGD)
This method, also known as the third generation in vitro fertilization, refers to the method of analyzing the genetic material of embryos before IVF-ET embryo transfer, diagnosing any abnormalities, screening healthy embryo transfers, and preventing the transmission of genetic diseases. The test substance is taken from one cell of 4-8 cell stage embryos or the first and second polar bodies of eggs before and after fertilization. Sampling does not affect embryonic development. Single cell DNA analysis method is used for detection. Firstly, polymerase chain reaction (PCR) is used to detect male and female gender and single gene genetic diseases; Another method is fluorescence in situ hybridization (FISH), which detects gender and chromosomal diseases. As early as 1964, Edwards proposed the idea of PGD. In 1989, Handyside AH first successfully applied PGD in clinical practice [7], using PCR technology to amplify Y-chromosome specific genes in vitro, and successfully transplanted embryos diagnosed as female into the uterus to achieve pregnancy. At the beginning, PGD was tested for gender using PCR or FISH, and female embryo transfer was selected to help couples at risk of having X-linked genetic diseases such as hemophilia A and progressive muscular dystrophy conceive and deliver a normal female baby. However, according to genetic laws, this method undoubtedly denies the birth of healthy boys and allows carrier girls to reproduce, but does not cut off the transmission of pathogenic genes. In 1992, the United States first reported the successful detection of cystic fibers using PCR, and through embryo screening, a healthy baby was born. Afterwards, α- 1- Anti insulin deficiency, family black? The establishment of PGD detection methods for various monogenic genetic diseases such as sexual dementia and pigmented retinitis, and the entry of PGD into the detection and prevention class for monogenic genetic diseases. After 1993, due to late marriage and childbirth, the number of elderly mothers increased, and women over 45 years old had a high rate of chromosomal abnormalities and were prone to giving birth to 18-3 and 21-3 body fools during natural pregnancy. Therefore, the focus of PGD's work shifted to the detection and prevention of chromosomal diseases, using FISH for testing. Due to the use of the first polar body for sampling, the selected egg is an unfertilized egg, which requires intracytoplasmic injection of a single sperm to be cultured and developed into an embryo before transplantation. According to statistics, embryos that stop developing midway have a chromosomal abnormality rate of 70% [8], so choosing embryos with normal chromosomes for transfer can also improve the success rate of IVF-ET [9]. In theory, any genetic disease that can be diagnosed should be able to prevent its transmission through PGD, but due to current technical conditions, the indications for PGD still have certain limitations. Eugenics is a common concern in the world. With the development of molecular biotechnology and the identification of more genetic disease genes, it is believed that some accurate and safe genetic diagnosis technologies will continue to emerge, and PGD technology will become increasingly perfect, better benefiting humanity.
Egg donation IVF-ET
This technology was first reported by Trounson et al. in 1983 as the first successful pregnancy. The indications are: (1) ovarian failure or absence of ovaries. (2) The female has genetic diseases or chromosomal abnormalities. The selection of egg donors should comply with: (1) those under the age of 35 who have given birth to one child. (2) There is no family history of hereditary disease, mental illness, excluding hepatitis B, hepatitis C and sexually transmitted diseases, especially HIV positive people. (3) Good intelligence and appearance. (4) The blood type matches the recipient. Compared with conventional IVF-ET, technically, there are two important steps that need to be added to the donor IVF-ET: (1) the menstrual cycle of the recipient and the donor must be synchronized. The method is to perform steroid hormone replacement therapy (HRT) on patients without ovarian function 3-5 days before the donor's expected menstrual cycle, to promote cyclic changes in the patient's endometrium, possess the ability to accept embryo implantation, and induce the production of endogenous LH peaks and endometrial estrogen receptors, thereby improving the endocrine environment of the body. For patients with ovarian function, first downregulate with gonadotropin releasing hormone analogues or enhancers GnRH-A, and then treat with HRT. During the HRT treatment cycle, serum hormone measurement, endometrial biopsy, B-ultrasound Doppler monitoring, and understanding of endometrial receptivity are necessary to adjust the dosage and ensure good endometrial receptivity. (2) Maintain pregnancy. After in vitro fertilization and embryo transfer with the sperm of the donor egg and the patient's husband, the patient must gradually increase HRT administration. To maintain pregnancy until the recipient's own placental function is established. The pregnancy rate of IVF-ET with egg donation is relatively high, with some reports reaching 44.9% [11]. In addition to treating patients with indications, it can also provide a human model for studying the interaction between embryos, endometrium, and steroids, with broad clinical application prospects.
Surrogacy
Used for patients with uterine rupture or severe adhesions after hysterectomy. The principle of surrogacy technology is the same as that of IVF-ET, but the donor is a patient and the recipient is a surrogate. Due to conceptual reasons, this technology has not yet been recognized in China.