7- Laboratory Challenges of Female Infertility Diagnosis - Part 7 (Ovarian Reserve Laboratory Survey)

7- Laboratory Challenges of Female Infertility Diagnosis - Part 7 (Ovarian Reserve Laboratory Survey)

2- Laboratory examination of ovarian storage

The exact number of oocytes available in the ovary is determined by the term ovarian storage.

Decreased ovarian reserve can be seen as a decrease in the quality or quantity of eggs or fertility potential, and no standardized test alone has yet been confirmed for this assessment, as the total number of real and available oocytes cannot be directly measured. Be.

 Determination of ovarian reserve in infertility assessment and treatment, as well as determination of women with a weak under-resppond or over-resppond response to ovulation-stimulating drugs in assisted reproductive protocols (ART). ) Has an important role.

Two types of biomarkers are used to evaluate egg storage:

1- Biomarkers that are produced directly from the growing follicles themselves and as a result their amount in the blood is a sign of the number and quality of eggs such as AMH test, inhibin B.

2. Biomarkers that change indirectly under the influence of follicle function and their level in the blood can reflect the number and quality of eggs such as FSH, estradiol.

- Measurement of FSH level on the third day of the cycle is widely used as a criterion with reasonable price, easy testing and good diagnostic power.

The third day is due to the fact that estradiol is at its lowest level, and as a result, FSH is at its highest level during a negative feedback.

Whenever the ovarian response decreases, FSH levels rise, and generally low levels of ‌ 10 mIU / mL indicate adequate egg storage, and high levels of ‌ 15 mIU / mL‌ decrease with successful pregnancies. Find.

 Note that the amount of this number depends on the laboratory method, but in different reports between 10 and 25 are also mentioned.

With increasing age, FSH levels increase (especially in the early stages of the follicular phase or early follicular phase, which is from the onset of menstruation until the peak of FSH), which is accompanied by a decrease in Inhibin B It can be seen.

 The peak FSH in these women is also earlier in the early stages of the follicular phase (most likely on the second day). This increase in FSH is probably predominant for mobilization and maintenance of normal follicle function

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Note: If the FSH test is performed in a closed method, the effect of biotin should be considered.

If the antibody used in the test is mouse type due to the high titer of heterophilic antibodies (antibodies against animal antigens, here specifically called anti-mouse Ab.‌ Let's go) In Iranian patients, it can interfere with the test and, if necessary, use special tubes that remove this antibody from the patient's serum sample.

It does not require fasting and the sample is stable for up to 48 hours in the refrigerator and up to 6 months in a freezer at minus 20 degrees.

 Interaction of FSH-enhancing drugs such as cimetidine, clomiphene, and levodopa, and FSH-reducing drugs such as phenothiazines and OCPs should be considered.

Factors that inhibit the secretion of FSH (and LH) are growth hormone, estradiol, and testosterone.

- Clomiphene Citrate Challenge Test (CCCT) or Clomid Challenge Test is also one of the good criteria for measuring ovarian stimulation.

The test is based on the fact that if a person's egg storage is sufficient, they seek to use clomiphene citrate (an oral anti-estrogen compound that increases the secretion of FSH and LH by acting on adenohypophysis). This, in turn, increases the maturation and ovulation process by stimulating the antral follicles).

 By producing sufficient amounts of estradiol and inhibin by FSH-sensitive follicles, it inhibits the secretion of FSH from adenohypophysis.

 In this test, first the levels of FSH, LH and estradiol are measured on the third day of the cycle and then from 5 to 9 cycles for 5 days, clomiphene citrate is given at a rate of 100 mg per day.

The FSH level is measured again on the tenth day. Women up to the age of 41 FSH should be below سوم 15 mIU / mL‌ on both the third and tenth days of the cycle, which is a sign of a good ovarian reservation.

Women ‌ 42 years of age and older should have between 12 and 15 mIU / mL‌.

Of course, a normal test does not mean having fertility, but an abnormal test result, and high results of 20 I 20 mIU / mL‌ indicate that the possibility of a normal pregnancy with one's own eggs is unlikely.

The CCCT test is more sensitive than the FSH and estradiol tests alone.

An increase in FSH on the tenth day compared to the third day is also considered as a measure to reduce ovarian reserve.

- During the Clomiphene Citrate Challenge Test (CCCT) or Clomid Challenge Test, the FSH: LH ratio can also be measured.

Normally, this ratio is around one (slightly more than one) during the menstrual cycle (except for the time of ovulation, when this ratio is less than one and reaches about 0.5.

Increasing this ratio is a criterion for reducing egg storage.

At menopausal age, FSH first begins to increase without LH change, so increasing this ratio is a good measure of ovarian age.

Measuring estradiol levels on the third day of the cycle is a not-so-good criterion for ovarian storage.

Normally, estradiol is at its lowest level (nadir level) on the third day, and as a result, negative feedback causes the FSH to have a peak.

High levels of estradiol on the third day can reduce FSH, thus eliminating one of the most important signs of egg storage and misleading the doctor (reducing the false negatives of the FSH test).

This test alone has poor predictive value for IVF outcome.

Estradiol levels should never be used alone as a biomarker to diagnose ovarian storage.

An increase in estradiol at the beginning of the cycle is a sign that follicular growth is inadequate in the final stages of follicle development.

In general, the two main reasons for an increase in estradiol are: an increase in folliculogenesis (instead of 375 days), an unhealthy increase in egg storage (for example, in PCOS). .

• Note: If the estradiol test is performed in a closed method, the effect of biotin should be considered and supplements containing biotin should not be taken for at least 8 hours.

If the antibody used in the test is mouse type, due to the high titer of heterophilic antibodies (antibodies against animal antigens, which are specifically referred to here as anti-mouse Ab. Ab). In Iranian patients, it can interfere with the test and, if necessary, use special tubes that remove the antibody from the patient's serum sample.

 It is preferable for the patient to fast and the sample is stable for up to 48 hours in the refrigerator and up to 6 months in a freezer at minus 20 degrees.

 Any factor that causes a sharp increase in SHBGs (such as androgen transporters - DHT, testosterone and androstenedione - and estrogens - estradiol and estrone -) increases estradiol, such as hyperthyroidism, liver cirrhosis and consumption. OCPs and antiepileptic drugs.

 Any factor that causes a sharp decrease in SHBG also reduces estradiol, such as hypothyroidism, PCO, and obesity.

GAST (Gonadotropin-releasing Hormone Agonist) is also used as one of the stimulant tests for ovarian reserve assessment.

In this test, first the estradiol level is measured on the second or third day of the cycle and then a Gonadotropin-releasing Hormone Agonist ‌ subcutaneous such as Leuprolide acetate (in Iran from two well-known brands Triptorelin such as Microrelin And ‌ Diphereline is used) is injected into the patient at a dose of 1 mg.

 Injections of this substance cause a sharp, temporary, and immediate increase in FSH and LH from adenohypophysis, resulting in an increase in estradiol up to 24 hours.

 This steady-state increase in estradiol is a reflection of the mobilization of follicles in the first stage of the follicular phase and therefore a reflection of good ovarian reserve.

- Because Inhibin B is secreted from small antral follicles and regulates FSH secretion, its level decreases as egg storage decreases and is considered as an complementary test to assess egg storage. It can be used.

Its decrease in older women has nothing to do with the decrease in Inhibin A secreted by the corpus luteum in the luteal phase.

In the early stages of the follicular phase, the marker first decreases and then the FSH increases.

 However, variation of this test is high from one cycle to another and therefore limits its use.

The AMH که hormone, which is produced by the granulosa cells of the antral and small anterior perineal follicles, appears to reflect the growth rate of non-FSH-dependent follicles.

When the follicle undergoes its FSH-dependent developmental stages and matures, the production of this hormone is stopped.

Because its levels are not dependent on other hormones and do not change during the menstrual cycle, it is now used as an attractive and unique test to assess ovarian reserve.

This biomarker is more closely related to the counting of antral follicles (AFC) than other biomarkers.

Numerous studies have shown the use of this biomarker for both the quantity and quality of eggs.

This test does not require fasting and is stable for up to 48 hours in the refrigerator and up to 6 months in a freezer at minus 20 degrees. .

- Use of vaginal ultrasound to count the number of antral follicles (small between 2 and 6 mm in diameter and large antral follicles between 4 and 10 mm in diameter) at the beginning of the cycle and measurement. Ovarian volume is also helpful in determining ovarian reserve as well as menopausal status.

 The number of these follicles should not be less than 8.

However, this criterion changes in women with sub-fertile fertility in different cycles, which is more than in infertile women compared to the ultrasound criteria in different cycles.

The AFC count is used as a very good measure to evaluate the response of the ovaries to ovulatory stimulants.

- Evaluation of ovarian storage by the combination of serum hormones FSH and AMH and pelvic ultrasound is highly sensitive. .

The sensitivity of the AMH test is very high, and even this test can be used alone for primary screening for ovarian storage.