Basic theory of follicle development; 2. Characteristics of ovulation-promoting drugs: CC, Letrozole, FSH, etc.; 3. Common protocols in assisted conception technology: long protocol, short protocol, antagonist protocol, ultra-long protocol, microstimulation protocol, etc.; 4. Choice of conventional ovulation-promoting protocols in infertility: normal response, low response, high response; 5. The oocyte is the largest single cell in the human body, and its developmental process is roughly the migration and differentiation of primordial germ cells to form the primordial follicle, then initiating growth to form the primary follicle, secondary follicle, and finally developing into mature ovulation. The number of primary follicles at birth is about 300,000 to 500,000, of which only about 400, or 0.1% of the total, are able to develop and ovulate, and will not increase later. Most of the oocytes undergo atresia and atresia during the growth process. The oogenic cells in the follicle gradually develop into primary oocytes. They then begin their first maturation division and remain in the bilinear phase until just before ovulation, a period that can last 12 to 40 years. Normally, the first maturation division is completed only 36 to 48 hours before ovulation, producing two daughter cells, one of which receives most of the cytoplasm, the secondary oocyte, and the other, a very small first polar body, which soon degenerates. The secondary oocyte immediately undergoes a second maturation division and stops at midphase. This division is identical to the general mitotic process. If the expelled egg is fertilized, the secondary oocyte only completes its second maturation division, producing a mature oocyte and a second polar body, which soon degenerates. If the egg is not fertilized, the oocyte will degenerate and be absorbed. Two maturation divisions from the primary oocyte, one of which is meiotic, produce haploid oocytes, i.e., the chromosomes are reduced from 23 pairs to 23, of which 22 are autosomes and 1 is a sex chromosome X The oocyte must undergo two important stages of initiating growth and selective growth before it can finally develop into a dominant follicle, both of which are strictly regulated by various factors in vivo. The role of FSH in follicular development is mainly manifested in the following aspects: promoting the division and proliferation of granulosa cells, thus increasing the total number of FSH receptors; inducing the production of luteinizing hormone (LH) receptors in interstitial cells; activating the aromatase activity in granulosa cells; and converting androgens to estrogens. FSH has a threshold for selection of follicles for growth, and only when the follicle has a sufficient number of FSH receptors to be sensitive to this threshold can it be recruited from the follicular pool to develop into a dominant follicle. Two factors are at work throughout follicular growth and development, namely stimulators and inhibitors, which can be either hormonal or cytokines. For example, some common stimulators of follicular growth and development are low levels of FSH, low levels of LH androgens, and cytokines such as growth differentiation factor (GDF9), Kit ligand (KL), cytokinetic gain factor (KGF), and epidermal growth factor (EGF), while inhibitors of follicular growth include high levels of LH, high androgens, anti-mullerian hormone (AMH), and insulin growth factor binding. and insulin growth factor binding protein (IGFBP). In normal follicular development, the stimulating and inhibiting factors are in balance, and if the stimulating factors are dominant, the follicles will be over-recruited and overgrown, as in the case of PCOS. On the contrary, if the inhibitory factor is predominant, the follicle growth and development will be impaired, which may manifest clinically as anovulation and amenorrhea. Although there are many types of ovulation-promoting drugs, they can be broadly classified into two categories, one is to promote ovulation by acting directly on the hypothalamus2pituitary2ovary axis, and the other is to regulate the endocrine status of the body to provide a good environment for follicle development and growth, thus indirectly promoting ovulation. CC is a non-steroidal agent with two isomers: en2CC and zu2CC, the latter has a longer half-life and greater estrogenic activity than the former, and has a half-life of up to 5 d. The mechanism of action of CC is that because CC is structurally similar to estrogen, it can bind to the estrogen receptor (ER), but unlike estrogen, CC can occupy the ER for weeks rather than hours, with the end result of inhibiting the negative feedback effect of estrogen on the hypothalamus. The end result is to inhibit the negative feedback effect of estrogen on the hypothalamus, which increases circulating levels of follicle-stimulating hormone (FSH) and luteinizing hormone (LH), thereby stimulating follicle growth. The hypothalamus is the main site of action of CC, which acts directly on the ovaries and, in the absence of estrogen, acts as an estrogen analogue, directly increasing FSH stimulation of L H receptor synthesis in granulosa cells.CC’s action in the uterus, cervix and vagina is mainly anti-estrogenic, thus affecting fertilization, sperm transport and early embryonic development. Conventional dosage of CC: A cluster analysis of 13 reports showed that 46% of patients responded to a daily dose of 50 mg, 21% responded to 100 mg, and 8% responded to 150 mg. Therefore, in order to save time, the starting dose of 100 mg/d was chosen. 50 mg/d remains the usual starting dose for CC treatment, usually starting on day 5 of the menstrual cycle for 5 d. If ovulation does not occur in the first cycle, the dose can be increased in the next cycle, usually by 50 mg each time. CC resistance should be considered. Doses above 150 mg/d do not increase ovulation rates. Aromatase, a cytochrome P450 enzyme complex, is a CYP19 gene product that catalyzes the conversion of androstenedione and testosterone to estrone and estradiol, the rate-limiting enzymes of estrogen synthesis. Aromatase inhibitors are divided into steroidal and non-steroidal classes. Letrozole (LE) is a 3rd generation non-steroidal aromatase inhibitor. LE was first used in 1999 by Mitwally et al. for ovulation control in infertile women. LE can be used to promote ovulation through two mechanisms of action: central and peripheral. On the one hand, aromatase inhibitors inhibit the activity of aromatase and reduce the biosynthesis of estrogen, which decreases the serum estrogen level and increases the secretion of FSH by the pituitary gland through negative feedback, and FSH acts on the ovary to promote follicle development; on the other hand, increased androgen level in the ovary can promote the expression of follicular FSH receptors and increase the sensitivity of follicles to FSH. In addition, increased levels of androgens in the ovary can also promote the secretion of insulin-like growth factor (IGF-1), which synergizes with FSH to promote follicular growth and development. There are single-dose regimens and multi-dose regimens. The single-dose regimen is 20 mg of oral LE on d 3 of menstruation, and the multi-dose regimen is 2.5-5.0 mg/d of oral LE on d 3-7 or d 5-9 of menstruation, although the optimal dose of LE is still not known, but 5 mg/d × 5 d seems to be the most effective. The advantages of LE over CC are as follows: ① short half-life (45 h), rapid clearance by the body, no adverse effects of residual anti-estrogenic effects; ② high specificity, no adverse effects on the endometrium, although the estrogen level at ovulation is 2-3 times lower than that of CC, but because the endometrial ER is not depleted, the endometrium can respond well to the high level of estrogen in the late follicular phase, and sufficient thickness of the endometrium for fertilized egg implantation can be achieved at ovulation; ③ the effects on the endometrium are as follows (3) There is no adverse effect on the cervical mucus status, which facilitates the passage of sperm. Therefore, LE provides a better intrauterine environment for embryo implantation. In 2007, Rachel et al. compared 112 infants in the LE treatment group, 271 infants in the CC treatment group, and 94 infants in the natural delivery group. The mean fetal weight and length in the CC group were lower than those in the natural delivery group and the LE group; the difference in mean fetal weight between the LE and natural delivery groups was not statistically significant. Since LE has been used for ovulation treatment for more than 10 years, there is no epidemiological data on the reproduction/genetics of this drug; based on the above data, it can be tentatively concluded that short-term LE ovulation treatment in the early follicular phase should not increase the rate of fetal malformation and will not affect embryonic growth and development. Another drug commonly used for direct ovarian stimulation is HMG, each containing 75 U of FSH and 75 U of LH, which is often used for hypothalamic and pituitary dysfunctional anovulation. In addition, recombinant FSH, which is synthesized by biotechnology, is commonly used to promote superovulation in patients with IUI and IVF2ET because of its high purity and complete absence of LH and impurities. GnRHa and chorionic gonadotropin (HCG) are also used as ovulation-inducing drugs at specific times. GnRH agonists work by replacing the 6th glycine of the natural decapeptide of GnRHa with a D-type amino acid and the 10th glycamide with an ethylamine, which improves the affinity for receptor binding and resistance to enzymatic cleavage. The agonist initially causes a large release of LH and FSH from the pituitary, leading to a 5-fold rise in FSH, a 10-fold rise in LH and a 4-fold rise in estrogen after about 12 hours, and thereafter, a more sustained binding to the GnRH receptor due to the higher affinity of GnRH-a for the GnRH receptor, and when GnRH-a persists, most of the receptor is occupied and moves inward into the cell, causing the pituitary When GnRH-a persists, most of the receptors are occupied and moved intracellularly, leaving the pituitary gland significantly deprived of GnRH receptors on the surface and unsupplemented and lacking in GnRH receptors, preventing further responses to endogenous or exogenous GnRH. In addition, in addition to the use of drugs to directly stimulate ovulation, there is an increasing emphasis on improving the endocrine environment in ovulatory disorders. For example, in patients with PCOS, who often have hyperandrogenism and hyperinsulinemia, adjusting the endocrine environment before ovulation stimulation can not only achieve a satisfactory ovulation rate, but also reduce the abortion rate. For example, in patients with hyperprolactinemia and amenorrhea, bromocriptine can be used to restore ovulation, which is also one of the indirect ovulation methods. 6. Common regimens in assisted conception techniques: long regimen, short regimen, antagonist regimen, ultra-long regimen, microstimulation regimen, etc. Long regimen: starting with GnRH-a on day 21 of menstruation and using follicle stimulating hormone on day 2 of menstruation; the extensive experience of using GnRH agonists provides doctors with strong confidence that the GnRH agonist long regimen has become the worldwide “gold standard”. The long protocol allows flexibility in the timing of egg retrieval. Lower risk of cancelled cycles (the LH peak drops from an average of 20% to 2% all thanks to GnRH agonists). Disadvantages of GnRH agonists: early “flame effect” leading to disruption of the normal menstrual cycle; side effects such as flushing, lack of libido, fatigue or weight gain; long treatment time (weeks to obtain desensitization of the pituitary gland); long regimen requiring multiple injections making it extremely inconvenient for the patient; slow recovery of the pituitary gland after treatment. Short regimen: GnRH-a from day 2 of menstruation and folliculopoietin on day 3 of menstruation; antagonist regimen: folliculopoietin from day 2 of menstruation with additional antagonist when appropriate. Specific antagonist regimen: Cetrotide injection starting on day 5-7 of Gn injection, 0.25 mg daily until HCG injection day. Antagonists act by competitive, dose-dependent blockade of pituitary GnRH receptors. Antagonist characteristics: rapid onset of action, causing a decrease in LH levels within a few hours; GnRH antagonists do not produce any stimulatory effect and therefore have no flame effect. Microstimulation regimen: Gn 150-225 IU/d, CC 50 mg/d and short-acting daphylline 0.1 mg/d are applied simultaneously on d 3 of the menstrual cycle, and Gn and CC are discontinued after 3-5 d of application. This microstimulation regimen uses CC to achieve ovulation with the synergistic effect of GnRHa. With the combined clomiphene regimen, the clinical pregnancy rate (25.0%) was significantly higher than that of the conventional regimen (12.5%); the embryo implantation rate (14%) was significantly higher than that of the conventional regimen (5%). In some patients, the endometrium of the clomiphene-stimulated cycle is very thin and does not appear to be suitable for embryo implantation. In some cases, stimulation can be performed with a regimen of micro-Gn. 75 U of FSH or HMG is administered every other day starting on day 3 of the cycle, and follicles are monitored by ultrasound on day 10. After a few dominant follicles have matured, eggs are retrieved for in vitro fertilization. Low-dose Gn microstimulation regimens are also particularly suitable for patients with polycystic ovary syndrome (PCOS), which is prone to two extreme outcomes: persistent ovarian non-response with numerous small follicles resistant to both clomiphene and Gn, delayed follicular growth and slow rise in estradiol levels; and ovarian over-response with the risk of ovarian hyperstimulation syndrome. The more popular microstimulation regimen is initiated on day 2 to 3 of the FSH 75 U cycle with daily or alternate day injections and continued at 50% dose increments every 3 days until the dominant follicle matures under ultrasound monitoring starting on day 7. This stimulation regimen is effective in improving the prognosis of OHSS and also reduces the number of eggs obtained at one time, but the pregnancy rate does not appear to be low. The disadvantage is that patients and physicians may not be able to tolerate such long periods of dosing and monitoring, and the cycle termination rate is high. Natural cycle protocol: The first public report of a pregnancy obtained with natural cycle IVF (NC-IVF) was made by Foulot et al. in 1989. Urinary LH peak is measured when the dominant follicle reaches 14 mm in diameter, and hCG is injected intramuscularly in the presence of a urinary LH peak or when the follicle reaches 18 mm in diameter. Options for ovarian hyperresponsiveness: CC, LE, GnRH-a long regimen, OC+GnRH-a overlapping long regimen, GnRH antagonist regimen, etc. Definition of ovarian hyporesponsiveness (varies): number of follicles >12 mm in diameter on the day of HCG injection.