Lecture of assisted reproductive methods

2021-03-19 12:00 AM

Normally, fertilization occurs in 1/3 out of the ovary and travels into the uterus after 7 days and implants in the uterus.

General perception

According to the World Health Organization, the infertility rate accounts for 8-10%. Infertility treatment is an urgent need for infertile patients to ensure family happiness, especially for women.

Assisted fertility is a general term encompassing new medical techniques, used in the treatment of infertility of a variety of causes. Reproductive support is one of the miraculous advances in medicine over the past 30 years.

In 1969, the first in vitro fertilization of the human ovule was performed. In 1970, the first division in human embryo culture was reported, but it was not until 1978 that professors Steptoe and Edvvards performed in vitro fertilization and transfer the embryo into the uterus for a woman with Two oviductal obstruction and the result was that Louis Brovvn was born. In the early 1980's, Alain Trouson used ovarian stimulation to increase the number of oocytes and having more mature follicles in in vitro fertilization increased the success rate of this approach.

More and more new technologies developed such as transfer of the uterus to the oviduct via abdominal smear, and sperm injection in the treatment of male infertility have increased success rates.

The general rule of fertility treatments is to support natural pregnancy by:

Prepare the ovule and sperm.

Create conditions for oocytes and sperms to meet to form embryos.

Create conditions for the embryo to implant in the lining of the uterus.

From there, depending on the cause of infertility, the physician chooses the appropriate method for each infertility pair.

In Vietnam, the incorporation of world medical advances into infertility treatment has made significant progress with the introduction of hundreds of in vitro fertilized babies.

Natural conditions required for conception

Fertilization is the result of an adult ovule meeting an embryo-producing sperm, an embryo implantation in the uterine lining. Normally, fertilization occurs in 1/3 out of the ovary and travels into the uterus after 7 days and implants in the uterus.

Conditions to have normal conception:


The testicles must produce a sufficient number of normal spermatozoa.

The sperm must pass through the male genital tract, where it reaches maturity and mobility.


The cervical mucus must be suitable for sperm life.

The female genitals allow sperm to reach the oviduct, while the oviduct must capture the ovule.

The development and maturity of the ovule depends on the function of the ovary and pituitary gland (sources of FSH and LH secretions). The rupture of a follicle and the release of an adult, called an oocyte release, usually occurs on day 14 of the normal menstrual cycle (28 days).

The uterine cavity and uterine lining are qualified to accept embryos and develop.

Methods of assisted reproduction


Artificial insemination.

The sperm wash (percoll - swim up).

Inject sperm into the uterus (IUI).

In vitro fertilization (IVF).

Pump sperm into the egg (ICSI).


Ovarian tube: surgical endoscopy (detachable speaker tube remover).

Stimulates ovulation.

Intra-abdominal injection (DIPI).

Tubal transfer (GIFT).

Transfer of zygote to the oviduct (ZIFT).

Direct egg transfer (DOT).

Fill the abdomen with eggs and sperm (POST).

Transplantation of the embryo into the oviduct (TUFT).

Surgical transfer of embryos (SET).

Vaginal culture (IVC).

Transfer the eggs into the tap and pump post-fertilized sperm (FREDI).

In vitro fertilization (IVF).


Here are some commonly used methods:

Ovarian stimulation

The goal is to stimulate the ovaries not to ovulate or to release normally to have one or more mature oocytes for normal conception or in vitro fertilization, pumping sperm into the uterus.

To adjust the dose and determine the time of oocyte release during drug therapy, always combine ultrasound to measure the size of the follicles and quantify the endocrine estradiol, LH.

Medicines that stimulate ovulation:

Estrogen inhibitors: clomiphen citrate, tamoxiíen. These drugs have a structure similar to estrogen, inhibiting competition with estrogen receptors in the hypothalamus, inhibiting the reverse control of estrogen leading to increased secretion of FSH, LH (mainly FSH) stimulates the release process. follicle proliferation.

Gonadotrophin: hMG/SFH, hCG.

hMG (1: 1 ratio FSH and LH) stimulates the development of follicles.

hCG is similar to LH, but is stronger and longer lasting, maturing to mature follicles and causing oocyte release.

GnRH: injection of GnRFI in normal physiological rhythm will stimulate the pituitary gland to secrete more FSF ON, LH stimulates the maturation of follicles and oocysts.

Commonly used regimens

Clomiphen citrate (CC) tablets 50mg :

Dosage 50 - 250mg / day X 5 days, starting from day 2 to day 5 of menstruation.

Initial dose 50mg / day, if not respond, increase with each cycle after, most have a response to doses of 150mg / day or less. If used early on the second or third day of the menstrual cycle, the number of mature oocysts will increase.

Can be combined with normal sex or injecting filtered sperm into the uterus for a higher success rate.

Can be used in combination with hMG. Also combine with hCG when assessing matured oocysts.

hMG is used at a dose of 1 ampoule of 75 units daily after administration of clomiphen citrate. hCG: 5,000 - 10,000 units injected once.

Due to the anti-estrogenic effect of clomiphen citrate can reduce the amount of cervical and endometrial mucus, corrections can be made with the use of additional estrogen.

This regimen typically has a high incidence of LH peak occurrence, soon before hCG injection.

Before hCG injection, if LH is elevated, the treatment cycle can be stopped.

Clomipheri citrate is the first drug for polycystic ovaries with an initial dose of 50mg / day. The success rate will be reduced by about 50% if there is endometriosis or ovarian abnormality. Should an ultrasound before giving the drug, if there are follicles, it should be treated with oral contraceptives or aspirated with ultrasound of the vaginal transducer. These follicles, if not treated, will affect the development of follicles or will increase in size very quickly, causing dangerous complications.

The number of cycles of clomiphen citrate treatment is usually limited to 6 cycles. If used more than the success rate usually does not increase significantly. Besides, some studies have found that ovarian cancer risk may increase with multiple cycles of clomiphen citrate (over 12 cycles).

hMG/FSH + hCG:

hMG is 75 units used at the beginning of the menstrual cycle, usually from day two. Dose . 1-3 ampoules / day, can increase or decrease the dose depending on the response of each patient. This regimen also has an increased incidence of LH.

GnRH + hMG/FSH + hCG:

The inhibitory effect of GnRI when continuous injection should be used to inhibit the early LH peak. The follicles only begin to develop maturation and oocysts when injected with HCG.

GnRH comes in two forms: low-dose subcutaneous daily or inhaled by the nose and high-dose subcutaneously with long-acting effects.

The combination regimen of GnRH + hMG / FSH + hCG is used the most today in many different forms:

Extremely short regimen: GnRH is usually used only for the first 3 days of the cycle, from day two, as a low dose form. This regimen used the stimulant effects of GnRH. The incidence of early, high LH peak appearance is high.

Short Regimen: GnRH is taken from day 2 of the cycle and extended to the time of hCG. This regimen uses both stimulant and inhibitory effects.

Long Regimen: GnRIT can be used from the luteal phase or from the beginning of the previous menstrual cycle. Can use low dose or high dose, long acting. This regimen uses only the inhibitory effect of GnRH. The effect of inhibiting LH peak is better than that of short-term regimen

Extremely long-lasting regimen: long-acting high-dose GnRH use, can be used 3-6 months before starting hMG / FSH. The most commonly used regimen in the world today is the long one. Compared with other regimens, the long-term regimen used 1.5-2 times higher hMG / FSH. However, this is the regimen with the highest success rate.

Problems to watch out for when stimulating ovaries

The distance between the response is good and the overstatement is very short.

The response of each person is different, need to be closely monitored.

When to use gonadotropin.

Dosage used by gonadotropin.

Increase or decrease the dose of gonadotropin.

When to use hCG to induce ovulation.

Filter and wash sperm


Choosing portable normal sperm is good for treatments, and concentrates sperm in a small volume, which is convenient for injecting semen into the uterus.

Removing dead cells, most microorganisms, most toxins to the sperm thus greatly reducing the risk of infection from the semen.

Most prostaglandins are removed in semen, avoiding uterine contractions in the intrauterine injection method (IUI).

Stimulates the activation of the sperm's head, facilitates fertilization with the ovule.

Avoid the risk of anaphylaxis that sometimes occurs when semen is inserted into the uterus.

The two most widely used and effective methods available are the swim-up method and the percoll method.

The Swim-up method: how to do it :

Add to test tubes of lOml 1-1.5ml culture medium.

Put 1-1.5 ml of the dissolved semen gently into the layer under the medium (ratio 1: 1).

Place the test tube in the incubator for about 45-60 minutes.

Place 0.7-l ml of the upper portion of the medium column into a 5 ml test tube.

Add 2 ml of medium and mix well.

Centrifuge 1500 rpm for 15 minutes.

Absorb the housing medium, to remove about 0.4-0.5ml.

Mix well, take 1 drop for the night chamber to check again.

The rest is to do IUI.


Only well-motivated sperm will swim upwards by itself, leaving the bottom semen layer.

The simple method is easy to do.

Selected sperm are of high quality.

Only applies to semen samples with relatively good quality sperm (with over 10 million sperm motions coming first).

Implementation techniques vary depending on the experience of each center.

Percoll concentration scale method:

Equipment needed:

Inoculation medium HAM F10 IX and 10X.

Percoll solvent.

Test tube 5ml, 10ml sterilized.

Pasteur pipette, centrifuge, microscope, Neubauer blood count chamber.

C02 5%, 37 ° c incubator, sterile operation cabinet.


Mix isotonic percoll solvents, usually 95% and 47.5% two types.

Create a medium scale: add two layers of solvent to the test tube, 'each lml tire, 95% layer below.

Gently add l, 5 ml of semen on top of the two percolls.

Centrifuge 1500 rpm for 15 minutes.

Absorb the upper layers, retain 0.5 ml of the sediment from the bottom and place in a new tube with 2 ml of new medium.

Centrifuge 1500 rpm for 15 minutes.

Discard the medium above, and retain 0.5 ml of sediment and transfer it to a new tube containing 2 ml of the medium.

Centrifuge 1500 rpm for 15 minutes.

Absorb the layers above, retain 0.5 ml of residue there, put into a new tube with 2 ml of new medium.

Centrifuge 1500 rpm for 15 minutes.

Discard the medium above, retain 0.3-0.5ml of residue, mix well, take a drop to check. + The rest do IUI or IVF.


Percoll solvents at different concentrations filter and remove dead sperm, deformed, poor mobility and other components in semen. The filtered sperm fraction was washed twice with the culture medium to remove the percoll.

Simple, easy method to do with a weaker sperm sample (over 5 million mobile spermatozoa).

Exercise time is longer than swim-up.

Implementation techniques may vary depending on the experience of each center, especially the number of layers and the concentration of isotonic percoll solvents used.

Intra Uterine Insemination (IUI)


Infertility of unknown cause.

Mild endometriosis.

Disorders of oocyte.

Cervical mucus is not convenient for sperm to move.

Antimicrobial antibodies.

Sperm abnormality.

Steps to be taken :

Stimulates the ovaries to get some follicles to develop (about 2-3 follicles).

Follow up the development of follicles by ultrasonic transvaginal probe, in combination with "quantitative serum estradiol" to evaluate the maturation of oocytes.

Inject 5,000-10,000 HCG units to stimulate the final stage of development of the follicle and stimulate oocyte release.

Filter and wash sperm and pump into the uterus 36 hours after hCG injection. The time to pump spermatozoa should not be more than 2 hours after semen sample collection. In some cases, it is recommended to pump sperm 2 times 18 hours and 42 hours after hCG injection to increase fertility. However, the effectiveness of the sperm pump 2 times has not been clearly proven.

Invitro Fertilization: IVF


In vitro fertilization involves aspiration of one or more mature ovules from the ovary to fertilize with (filtered) overlapping sperm in the laboratory. After the egg has developed into an embryo, transfer the good embryo into the uterus for the embryo to implant and develop into a fetus. Pregnancy rates vary from center to center.


Infertility due to obstruction of the two ovaries.

Unexplained infertility after two years of laparoscopy and no results with IUI3-6 times.

Steps to be taken:

Using ovarian stimulant drugs for many follicles to develop and mature.

Follow and measure oocyte size and size by ultrasound and combination with serum estradiol. Adjust the dose to avoid side effects. When ultrasound, the size of the follicles is about> 18mm, E2 250 pg / ml per adult follicle, and hCG is injected with 5,000 - 10,000 units.

Vaginal aspiration of follicles 32-36 hours after hCG injection.

The process of oocyte collection:

Place the aspirated solution into Petri dishes placed on a microscope with a 37 ° C thermostatic device to find ovule.

Rinse the oocyte to remove the surrounding blood and granulosa cells.

Transfer of oocytes into the culture system.

Evaluate the ovules for quality and maturity and record the intended time of fertilization.

Place in an incubator at 37 ° C and 5% C02 to keep the pH stable.

After 3-4 hours each oocyte will transfer to a drop containing 100,000 sperm / ml of medium. If that is the case, then do ICSI at this point.

Filter and wash sperm on the same day with egg aspiration.

Follow up fertilization and embryo development in the following days.

Transfer of good embryos into the uterus 2-3 days after fertilization. Usually transfer 3 embryos at a time in patients younger than 35 years, can transfer up to 4 embryos in older patients. Before embryo transfer, the uterus must be wiped with a culture medium. Exploration of the uterus in the direction of the catheter from the day before.

Follow up and test to diagnose pregnancy 2 weeks after embryo transfer.

Evaluation of embryo quality:

Good: uniform size of embryonic cells, no debris, or very little.

Fair: relatively uniform embryonic cell size, some debris, 20-30% volume of embryo.

Bad: the sizes of embryonic cells are not uniform. There are debris,> 50% by volume of the embryo.

Gamete Intra Fallopian Transfer: GIFT


Men have a low sperm count.

Infertility of unknown cause.

Two normal oviducts.


Performed with laparoscopy.

The oocyte spread after aspiration will be immediately returned to the oviduct with a small amount of filtered sperm, the ovule and sperm will be fertilized in the natural environment at the oviduct. Take 2-3 oocytes and 50,000 - 100,000 sperm and place it about 3 cm from the speaker of the oviduct.

This method is sometimes combined with IVF, since the excess oocytes will be used for in vitro fertilization, the embryo will be frozen and transferred to the uterus in future births.

Insemination in vitro with sperm injection technique (Intracytoplasmic Sperm Injection: ICSI)

Treatment of male infertility has made more and more progress with the technique of injecting sperm into the ovule.

There are three methods:

Inject a sperm into the cytoplasm.

Inject 1-2 sperms through the transparent membrane but outside the cytoplasm.

Puncture the transparent membrane to let the sperm through the hole into the fertilized cytoplasm.

The technique of injecting sperm into the cytoplasm is now widely used.


Men who cannot obtain sperm or do not have sperm due to congenital vasectomy, constriction of the vas deferens. Sperm can be obtained from the epididymis through a crest biopsy. Sperm collected this way can be very rare and immature. Such sperm, when inserted into the ovule, also leads to fertilization.


The oocyte sucks out the surrounding seed cell layer.

Choose the best mobile sperm, fix them by pressing the sperm's tail and suck in a glass needle to pump into the ovule. There is another glass needle used to immobilize the oocyte when sperm is injected into the cytoplasm.

Some other methods

Zygote Intra Fallopian Transfer (ZIFT). When the ovule is newly fertilized in the second stage, the progenitor is transferred via laparoscopy.