The pursuit of advanced education and participation in social activities by modern women has caused many changes in modern society, and the rates of late marriage and divorce are gradually increasing. With the delayed age of women at marriage and childbirth, the incidence of infertility is increasing, and there is a growing concern about increasing age and thus the impact on the female reproductive system. In-depth studies addressing different levels and aspects of reproductive physiology and pathology in advanced age are important not only to deepen the understanding of the etiology of infertility, but also to continuously improve the management options for infertility-related disorders.
This chapter focuses on the main effects of age change on female reproductive endocrinology and fertility and the role of in vitro fertilization embryo transfer techniques in the fertility of older women.
I. The female reproductive endocrine regulatory axis
The female endocrine system includes all endocrine glands in the body, including the hypothalamus, pituitary, pineal, adrenal, thyroid, ovary, etc. Each regulatory system interacts and influences each other to form the complete female endocrine regulatory system. The hypothalamic-pituitary-ovarian axis (H-P-O axis), as the main functional axis in women, plays a crucial role in the regulation of reproduction and endocrine.
Under the regulation of hypothalamic gonadotropin-releasing hormone (GnRH), the pituitary gland secretes folliculopoietin (FSH) and luteinizing hormone (LH), ovarian sex hormones depend on the action of FSH and LH, and endometrial cycle changes are regulated by ovarian sex hormones.
The neurosecretory cells of the hypothalamus secrete follicle-stimulating hormone releasing hormone (FSH-RH) and luteinizing hormone releasing hormone (LH-RH), which enter the pituitary gland through the portal venous system between the hypothalamus and pituitary gland. The pituitary gland secretes FSH and LH under the control of hormones produced by the hypothalamus, which stimulate ovulation of mature follicles, induce the ovulated follicles to become luteal, and produce progesterone and estrogen The function of the gonadal axis is regulated through the production of hormones by the hypothalamus. The function of the gonadal axis is regulated by neural regulation and hormonal feedback.
A large amount of estrogen inhibits the secretion of FSH-RH in the hypothalamus through negative feedback; at the same time, it stimulates the secretion of LH-RH in the hypothalamus through positive feedback, while a large amount of progesterone inhibits the secretion of LH-RH through negative feedback. When the hypothalamus is affected by the negative feedback of ovarian sex hormones and the secretion of GnRH decreases, the release of pituitary gonadotropins also decreases, the corpus luteum loses the support of Gn and atrophies, and the two ovarian hormones produced by it also decrease. The endometrium atrophies, necroses, bleeds and exfoliates due to the loss of ovarian sex hormone support, resulting in menstrual flow.
At the same time as the ovarian sex hormones decrease, the inhibition of the hypothalamus is lifted and the hypothalamus is able to secrete the relevant releasing hormones again, so another new cycle begins, and so on and so forth. The hypothalamic-pituitary-ovarian axis is a complete and coordinated neuroendocrine system, with each link having its own unique neuroendocrine function, and regulating and influencing each other.
Inhibin, a glycoprotein polymer, is involved in the regulation of FSH secretion by the pituitary gland as a paracrine regulator in the ovary and is composed of two subunits, α and β. It can be divided into two types, inhibin A and B, depending on their composition. Inhibin B is produced by granulosa cells of the ovary in women and by supporting cells of the testis in men, whereas inhibin A is produced by flavinized granulosa cells in the corpus luteum. During the normal menstrual cycle, either inhibin A or inhibin B has the effect of inhibiting the release of FSH from the pituitary gland.
Activin, a similar substance to inhibin, acts in the opposite way to inhibin, promoting both the secretion of FSH from pituitary cells and the development and continued growth of the primordial follicle. Leptin activates the hypothalamic-pituitary-ovarian axis, and fluctuations in plasma leptin concentration are synchronized with LH and E2.
Androgens are important for maintaining normal ovarian function and follicle development.
Androgens are synthetic substrates of estrogen, which can promote the growth of small follicles and the proliferation of granulosa and membrane cells, and stimulate the expression of FSH receptors, IGF-I, and IGF-I receptors; androgens have dual effects on follicular growth and development; androgen supplementation in patients with low ovarian function can enhance ovarian response and improve pregnancy outcome; persistent hyperandrogenemia in patients with PCOS can cause However, further research is needed on the regulatory mechanisms of androgens on follicular development and their safe and effective application in clinical settings.
Endocrine characteristics and fertility changes in older women
The changes in the endocrine system in older women are mainly in the gonadal axis due to the decline of ovarian function, and other changes in the adrenal axis and growth hormone axis have occurred before and afterwards. The first endocrine gland to change as women age is the ovary, and changes in ovarian function decline begin about 10 years before menopause, such as an increase in the number of non-ovulatory menstrual cycles, a mild decrease in estrogen levels, a rise in FSH, a shortening of the follicular phase or insufficient luteal function, and a decrease in fertility, resulting in a series of physiological and pathological changes leading to menopause.
Human fertility is related to the number of follicles in the ovary. Age-related changes in endocrine hormones and menstrual cycle are the result of progressive decrease in the number of follicles in the ovary, which can be confirmed in the early follicular stage by guided ultrasound examination showing a decrease in ovarian volume and number of sinus follicles. The so-called reproductive age is actually the age of the ovaries. The number of follicles and the quality of eggs are best between the ages of 18 and 31, decreasing between the ages of 31 and 37, and decreasing sharply between the ages of 37 and 45 to almost “0” at the age of 51, which is the famous Faddy curve.
The 2001 Reproductive Aging Staging Workshop divided female reproductive function into 6 stages (-5 to +2) based on cycle regularity, endocrine changes (FSH), presence or absence of ovulation, and relative fertility of oocyte function, Stage -5: Irregular menstrual cycles from post menarche to regular with normal FSH, also known as early reproductive stage, Stage -4: Regular cycles with normal FSH, also known as peak reproductive stage, Stage -3. Regular menstrual cycle.
However, FSH is elevated, also known as late reproductive phase, the first 3 phases are also collectively referred to as reproductive phase, because there is only a subtle decrease in fertility over several years without obvious boundaries; when the menstrual cycle is still regular but shortened (>7 days difference from normal) and FSH is elevated begins to enter phase -2 that is, early menopausal transition, when there are ≥2 shedding cycles and cycle interval ≥60 days that is, enter phase -1 that is, late menopausal transition; amenorrhea within 4 The late menopause is defined as 4 years after menopause until death.
Since the actual age does not really reflect the age of the ovaries, there is no clear cut-off for the actual age of each stage. However, some studies have also concluded that women present shortened menstrual cycles, rising FSH, accelerated follicular atresia, and declining menstrual function after the age of 38 years, so the stage between 40 and 45 years is also referred to as the period of reproductive decline.
Endocrine and menstrual cycle changes.
1. Endocrine changes during the reproductive phase
The first increase in serum FSH level is usually seen in the late reproductive phase, with 10miu/ml as the threshold value, and different laboratories should set the reference value according to their own situation. Estradiol (E2) levels are normal or mildly elevated during the late reproductive period. If E2 is elevated, it will suppress the increase in FSH, so FSH levels should be tested along with E2 levels.
A single elevated FSH level is sufficient to be classified as late reproductive, but approximately 30% of women with normal FSH and regular menstrual cycles between the ages of 40 and 45 have elevated FSH again, and women in this age group should be retested if their initial FSH test is normal.
Serum androgen levels decline sharply with age in early reproductive age and do not change significantly with menopause. The ovaries of postmenopausal women still produce testosterone, but the role of androgens in female reproduction is still poorly understood.
In women over 35 years of age with regular ovulation and normal follicular FSH levels, FSH levels rise only marginally but significantly during the early follicular phase, and inhibin B decreases throughout the follicular phase. In older women with menstrual cycles, inhibin A, inhibin B, and luteinizing hormone levels decrease during the luteal phase, while E2 remains unchanged. Thus, the earliest decline in inhibin B becomes evident precisely at the time of rapid follicular decline, which signals the onset of menopause, suggesting that the declining inhibin B levels respond to the decrease in follicular number with age.
The menstrual cycle in women of advanced reproductive age is characterized by a selective increase in FSH associated with early development of the dominant follicle and ovulation, and increased levels of activin expression in follicular fluid, which may be associated with earlier ovulation in advanced women.
2. Endocrine changes during the menopausal transition and menopause
Although FSH rises gradually throughout the menopausal transition, a meaningful threshold difference from -3 to +1 phase cannot be determined by the wide variation. In addition to the hallmark FSH elevation during the menopausal transition, there is a gradual decrease to cessation of E2 secretion due to progressive follicular atrophy. The interstitial cells of the ovary, still have some function in the secretion of androgens. The lack of aromatase in the ovary prevents the conversion of androstenedione to estrogen in the ovary.
Androstenedione, which mainly comes from the adrenal cortex, is converted into estrone (E1) in extra-glandular adipose tissue. Estrone and estradiol can also be converted into each other, and the conversion sites are mainly in fat, muscle, brain, liver, kidney, skin and other tissues, so estrogen in postmenopausal women is mainly estrone, unlike in women of childbearing age.
LH changes later than FSH but eventually rises gradually during this period, while progesterone levels drop due to the cessation of ovulation. It has also been suggested that the decline in inhibin B underlies the rise in FSH, and that its changes with ovarian function are earlier than those of FSH and more accurately reflect changes in ovarian function, but no consensus has been reached. In early menopause, ovarian hormone secretion further decreases, but it can still secrete androgens, and FSH and LH levels gradually rise. In late menopause, estrogen levels are low and gonadotropins are slightly decreased.
Changes in serum leptin levels with increasing age are somewhat controversial and may be correlated with body mass index, which generally decreases with increasing age. Many studies in recent years have found a significant correlation between decreased anti-mullerian hormone levels and advancing age, but it has not been widely tested in the clinic as a marker of ovarian aging.
Fertility changes in older women.
Female fertility undergoes a gradual decline from the peak of reproduction to menopause (-4 to -1 phase), with decreased fertility being the first sign indicating reproductive aging that precedes elevated FSH and cyclic changes in menstruation. Age is an important factor that independently affects female fertility, mainly by influencing egg quality and uterus, and can be confirmed by comparing fertility with that of unrestricted couples.
Data from the United States in 1989 showed that the birth rate was 28% at age <25 years, 18% at age 25-29 years, 16% at age 30-40 years, 14% at age 35-49 years, and 6% at age >40 years. Generally, from the age of 35 years, the number and quality of eggs begin to decline, the oocytes do not separate chromosomes during meiosis, or the oocytes lose their mitochondrial DNA. Thereafter, the embryo implantation rate decreases by about 2.77% for each additional year of age.
Recent data from the United States and Europe over the last 20 years indicate a significant decrease in fertility in women over 35 years of age, mainly due to.
1. reduced ovulation and low egg quality.
2. Decrease in fertility rate.
3. increased rate of pregnancy loss.
4. Decreased local and systemic function of the uterus, which makes it difficult to adapt to pregnancy.
5. Genetic abnormalities affect the maintenance of fertilization and pregnancy, and can also cause offspring abnormalities.
(1) Effects on the ovaries
The rate of follicle depletion in the primordial follicular pool accelerates significantly after the age of 38 years, suggesting a decrease in the number of remaining follicles, which may be related to a decrease in factors that inhibit the natural growth of the primordial follicles. Why does the number of follicles in the ovary decrease with age, especially after 38 years of age when it decreases rapidly? Ovarian stimulation cycles have observed a progressive decrease in the sensitivity of atrophic follicles to gonadotropins. Both the total dose of gonadotropins and the duration of treatment need to increase with age to induce multiple follicle growth and development.
The rate and peak of estrogen elevation also decreases, reflecting the fact that only a small number of follicles are recruited. The concentration of estrogen secreted from these follicles and follicular maturation can still reach the same levels as in younger women. exogenous hCG treatment before the age of 30 years can cause a decrease in androgen production but serum estradiol concentrations remain normal except during the reproductive years, probably related to the compensatory effect of FSH. Monitoring of follicular development and measurement of follicular fluid sex hormones in both older and younger women did not reveal age-related follicular hypofunction, and follicular growth and development were normal.
Follicular size, volume, and inhibin concentration were similar between older and younger women at ovulation, while follicular fluid progesterone concentration and estrogen/androgen ratio were even higher in older women than in younger women. As women age, follicles in the ovaries continue to deplete and granulosa cells become less sensitive to gonadotropins.
The success rate of assisted reproductive techniques decreases with age. The rate of oocyte acquisition and embryo availability decreases, while the rate of embryo fragmentation increases and transfer success decreases. annual reports of ART success rates registered by the American Society for Assisted Reproduction and the Centers for Disease Control and Prevention since 1989 show that age is the most important factor affecting success rates.
During the performance of ART, for women ≤32 years of age, age had little effect on the application of fresh, non-donor eggs and embryo transfer pregnancy and delivery rates, whereas for women >32 years of age, pregnancy and delivery rates decreased linearly with increasing age.
Regular ovulatory menses are more frequent in older women than in younger women, and the former have elevated serum FSH concentrations as a compensatory response to reduced sensitivity to gonadotropins. Pre-ovulatory follicles appear earlier in older women but still develop and mature within a normal time frame, and follicular fluid characteristics are indicative of healthy follicles. Why, then, does a woman’s fertility decrease with age? Studies have shown that age-related decreases in fertility and increased spontaneous abortion rates in women can contribute significantly to follicle loss and increase the incidence of abnormal oocytes.
Genetic studies of unfertilized oocytes in IVF have found that the incidence of oocyte aneuploidy increases with age. The age-related decrease in live birth rate not only reflects reduced fertility but also is a consequence of increased rates of pregnancy miscarriage and preterm birth. The rate of spontaneous abortion increases with increasing age. The spontaneous abortion rate for natural pregnancies is low at 7-15% in women younger than 30 years old, increases mildly to 8-21% at 30-34 years old, rises significantly to 17-28% at 35-39 years old, and rises to 34-52% at older than 44 years old.
2.Impact on the uterus
With the increase of age, in addition to the decline of ovarian function, the uterus and endometrium are also affected by ovarian hormone changes. Animal experiments have shown that the rate of implantation and pregnancy decreases with age, even when embryos from younger animals are transferred to older animals, indicating that the aging of the uterus has some effect on implantation. Studies of human embryo implantation rates in relation to age have shown that the success rate of single embryo transfer in women younger than 30 years is about 20%, decreasing to 9% over 35 years and 5% over 40 years.
There are many adverse factors affecting uterine function in older women, including decreased blood flow, reduced receptor volume, and fibrosis of the uterine wall. Uterine smooth muscle tumors, endometrial polyps and adenomyosis, which increase with age, may also be factors that affect fertility in older women.
However, age does not affect endometrial growth and function, nor does it affect hormonal responsiveness. 2000 U.S. statistical data on ART success rates showed that the live birth rate in older women using their own oocytes for ART was reduced, whereas the live birth rate per cycle after IVF using donor eggs was 43% and was independent of the recipient’s age, indicating that egg quality is the most critical factor in determining conception.
III. Assisted reproduction in older women
It is believed that “reproductive menopause” is about 10 years earlier than “endocrine menopause”, that is, the reproductive function of women has already declined 10 years before menopause. The spontaneous abortion rate is greater than 50%. The main reasons for this are attributed to the uterus and ovaries, and the necessary preparations should be made prior to assisted conception.
1. Ovarian function assessment
Despite the low pregnancy rate in older women, some patients can still have a spontaneous pregnancy, how to assess their fertility reserve? Currently, it is usually determined by measuring the serum FSH level on the third day of menstruation. The reference value varies from test to test, but an increased FSH level in women with normal menstrual cycles usually indicates the beginning of a decline in fertility. Clomiphene excitation test or GnRHa stimulation test, monitoring of FSH, LH, FSH/LH, E2 and inhibin, and AMH testing are of great value in predicting ovarian reserve function and IVF prognosis.
It is important to note that the specific age at which ovarian reserve decreases varies greatly for each individual, and some women may already have reduced ovarian reserve in their 30s or even 20s. Therefore, treatment of infertile patients should be started as early as possible after adequate preparation, provided that the indications for IVF are clear, in order to improve pregnancy rates.
2. Estrogen and progestin therapy
For these women with high gonadotropins, estrogen and progestin cycle therapy can be used clinically. After about 3 cycles of treatment, some patients can resume natural ovulation and conceive. However, the application of hormone replacement therapy in senior women may increase the risk of breast cancer, endometrial cancer, ovarian cancer, thrombophilia, diabetes mellitus, hypertension, cholelithiasis, etc. It should be used under the condition of proficiency in its indications, contraindications and cautionary principles.
3. Superovulation stimulation and and IVF-ET
Since natural fertility declines in women over 40 years of age, the chances of obtaining a pregnancy after that age are small. Studies have shown that combining superovulation stimulation and intrauterine insemination for infertility can improve the conception rate, however, the efficacy of this method remains suboptimal in women older than 40 years of age. For older women, IVF-ET is more effective than the former, but the results are still poor for women older than 42 years, with only 9.8% IVF-ET pregnancy rates reported in the literature for women older than 40 years.
There are more current superovulatory stimulation protocols, mainly including clomiphene microstimulation protocol, long protocol, short protocol, and ultra-short protocol. Countermeasures for ovarian hyporesponsiveness.
(1) Decrease the dosage of GnRHa.
(2) Increase the starting dose of FSH.
(3) Combine GH to modulate the effect of FSH on granulosa cells and follicular membranes by upregulating the local synthesis of IGF-1, which amplifies the effect of FSH on corymbs and follicular membrane cells.
4. Egg gift and surrogacy
In 1984, the world’s first egg donation IVF was born, bringing hope to couples lacking normal eggs to have children.
In recent years, egg donation IVF-ET has been used as a proven pregnancy aid, but egg donation must have clear indications for.
(1) Ovarian failure or absence of ovaries, such as premature ovarian failure, congenital absence of ovaries (Tuener’s syndrome) or removal of both ovaries due to tumor.
(2) The female partner has a genetic disease or chromosomal nuclear abnormality.
(3) Death of the child after menopause. This condition often occurs in older women (e.g., 38 years or older) with poor ovarian reserve capacity, and occasionally in younger women. The reason may be related to several previous pelvic surgeries that affected the blood supply to the ovaries even though large amounts of gonadotropins cannot enter the ovaries to function. IVF-ET with egg donation can increase the rate of fertilization and pregnancy, as well as reduce the rate of miscarriage and the incidence of chromosomal abnormalities.
Surrogacy was initially used for those with ovarian function who were unable to conceive due to uterine factors. Surrogacy is available for women of advanced age who are menopausal and have no eggs or patients with diseases that cannot tolerate pregnancy (such as severe heart disease and high blood pressure) but expect to obtain a child from their own husband. Some countries allow surrogacy, which must first have a medical indication. After the patient couple is examined, ovulation is routinely promoted, in vitro fertilization is performed, embryos are frozen and stored for 6 months, and after a negative HIV test, the frozen-thawed embryos are then transferred to the surrogate mother.