Circular No. 12/2012/TT-BYT Issuing the Technical Procedure for Artificial Insemination and In Vitro Fertilization

 

CIRCULAR

Issuing the Artificial Insemination and In Vitro Fertilization Techniques Procedure

and in vitro fertilization

Pursuant to Decree No. 188/2007/NĐ-CP dated December 21, 2007 of the Government on the functions, tasks, powers, and organizational structure of the Ministry of Health;

Pursuant to Decree No. 12/2003/NĐ-CP dated February 12, 2003 of the Government on childbirth using scientific methods;

Considering the proposal of the Director of the Maternal and Child Health Department;

The Minister of Health hereby promulgates the Artificial Insemination and In Vitro Fertilization Techniques Procedure as follows:

Section I
GENERAL PROVISIONS

Article 1. Scope of Regulation

This Circular stipulates the procedures for examination, diagnosis, and artificial insemination techniques (intrauterine insemination), in vitro fertilization, and sperm storage, egg storage, embryo storage, and testicular tissue storage.

Article 2. Interpretation of Terms

1. Pregnancy is the product of the fertilization process, starting from week 09 post-fertilization until birth or miscarriage.

2. Biochemical pregnancy refers to cases where βhCG in serum (≥25 IU/L) or in urine is detected without the use of hCG to support corpus luteum.

3. Clinical pregnancy refers to cases where ultrasound shows a gestational sac.

PART II
PROCEDURE FOR EXAMINATION AND DIAGNOSIS OF INFERTILITY

Article 3. Examination and diagnostic tests for infertility for couples

1. Principle: Conducting medical history inquiries and physical examinations concurrently for both spouses.

2. For the wife:

a) Clinical examination:

- General examination;

- Internal medicine examination;

- Gynecological examination, breast examination.

b) Laboratory tests:

- Hysterosalpingography with contrast medium;

- Hormonal tests conducted at the beginning of the menstrual cycle to assess ovarian reserve (days 2-4): Estradiol (E2), FSH, LH, and AMH (on any day of the menstrual cycle), quantitative Progesterone during the luteal phase;

- Ultrasound: detecting abnormalities in the uterus and ovaries, counting secondary follicles at the beginning of the menstrual cycle (days 2-4);

- Blood cell count and biochemical blood tests;

- Hepatitis B, syphilis, tuberculosis, HIV testing (note to provide counseling before testing according to current regulations on HIV/AIDS prevention);

- Chlamydia testing;

- Other special tests (depending on the patient): Cytomegalovirus, Anti-phospholipid antibodies, mammography, vaginal and cervical cytology, genetic testing.

3. For the husband:

a) Clinical examination:

- General examination;

- Internal medicine examination;

- Genital examination;

b) Laboratory tests:

- Hepatitis B, syphilis, tuberculosis, HIV testing (note to provide counseling before testing according to current regulations on HIV/AIDS prevention);

- Semen analysis test according to World Health Organization standards.

Article 4. Examination and diagnostic tests for infertility for single women

Single women shall undergo examination and testing as provided in Clause 2 of Article 3 of this Circular. For sperm donors for single women, the tests specified in Point b of Clause 3 of Article 3 of this Circular shall be performed.

Section III
ARTIFICIAL INSEMINATION TECHNIQUES PROCEDURE

Article 5. Counseling for cases of infertility treated with artificial insemination methods

1. Requirements:

a) Counselors are specialists, midwives, or nurses who have general knowledge about infertility, counseling skills, understanding of patients' needs, knowledge of policies and laws on childbirth using scientific methods, and related issues;

b) There should be a private counseling room equipped with pictures, posters, and other aids to assist in counseling on infertility.

2. Content of counseling:

a) Explain the treatment procedure for couples including steps such as examination, laboratory tests, use of ovulation-stimulating drugs (if applicable), monitoring during ovulation stimulation, and handling of sperm samples;

b) Predict the timing of intrauterine insemination, the technique of intrauterine insemination;

c) Support for luteal phase support after intrauterine insemination, monitoring after intrauterine insemination, and methods of monitoring if pregnancy occurs;

d) Success rates of the method and child development;

e) Possible complications;

f) Treatment costs.

3. For cases requesting sperm samples: perform the counseling content as stipulated in Clause 2 of this Article.

Article 6. Sperm Washing and Preparation for Intrauterine Insemination Procedure

1. Overview: Sperm washing is a technique aimed at removing dead sperm and seminal plasma to obtain a sample of healthy sperm for intrauterine insemination or in vitro fertilization.

2. Indications:

a) Cases where intrauterine insemination is indicated;

b) Sperm washing for cases of in vitro fertilization.

3. Preparation:

a) Preparing the husband: advising abstinence from sexual intercourse for 02 to 07 days;

b) Preparing equipment and tools: centrifuge machine, microscope with 10x and 40x objectives, 10x eyepiece, operating table, warming cabinet for semen samples;

c) Preparing consumables: sharp-bottomed test tubes, syringes, pipettes, slides, slide covers, labeled jars for husband and wife's samples, sperm washing and rinsing medium.

4. Procedure:

a) Collecting Semen Sample:

- The husband abstains from sexual intercourse for 02 to 07 days;

- Prepare all equipment used for handling the sperm sample, each person has their own set of equipment labeled with the husband and wife's name or coded number;

- Collect semen through masturbation, wash hands and genital area thoroughly before collecting the sample.

b) Sperm Washing:

- Allow the semen to liquefy completely in a warming cabinet at 37°C or room temperature, approximately 30 minutes;

- Evaluate semen parameters: volume, liquefaction time, pH, sperm concentration;

- Select the washing method (gradient or swim-up);

- Collect the washed portion about 0.3-0.5 ml for intrauterine insemination;

- Re-evaluate the sperm concentration and motility post-washing.

Article 7. Intrauterine Insemination Technique Procedure

1. Overview: Intrauterine insemination is a technique where a catheter is inserted through the cervix to directly inject washed sperm into the uterus. The purpose of this method is to increase pregnancy rates for infertile couples.

2. Indications:

a) Applied to couples suffering from infertility due to the following reasons:

- Male factor infertility due to weak or abnormal sperm but still eligible for intrauterine insemination treatment;

- Female factor infertility due to ovulation disorders;

- Unexplained infertility;

- Endometriosis-related infertility;

- Cervical factor infertility;

- Azoospermia in the male partner, requiring sperm donation for intrauterine insemination in the female partner;

- Cases of sperm storage.

b) Applied to single women who wish to conceive using this method.

3. Conditions: At least one patent fallopian tube is required.

4. Preparation:

a) Preparing the wife/single woman wishing to conceive: ovarian stimulation may be performed on the wife/single woman, followed by monitoring follicular development until ovulation or during a natural cycle without stimulation, the husband abstains from sexual intercourse with his wife for 02 to 07 days to collect the semen sample, which is then washed before insemination;

b) Preparing equipment and tools for washing the semen sample: as specified in Point b Clause 3 Article 6 of this Circular. Additionally, forceps, two sterilized clamps, and a cup of physiological saline solution for cleaning the vagina and cervix must be prepared;

c) Preparing consumables: 1 ml syringe, catheter for intrauterine insemination, gauze for cleaning the vagina and cervix, physiological saline solution.

5. Procedure (for the wife or single woman wishing to conceive using this method):

a) Timing of insemination: once 36 hours after hCG injection or twice 24 hours and 48 hours after hCG injection;

b) The woman lies in a gynecological position, sterile drapes are placed over her abdomen and thighs;

c) Clean the vulva with physiological saline solution;

d) Insert forceps, expose the cervix;

e) Clean the vagina and cervix with physiological saline solution, then dry with sterile gauze;

f) Draw the washed sperm sample into the syringe attached to the catheter;

g) Gently insert the catheter until it passes through the cervical canal;

h) Slowly inject the sperm into the uterus;

i) Carefully remove the catheter from the uterus;

j) Remove the forceps and have the patient rest for 30 minutes;

k) Support the corpus luteum: administer progesterone to support the luteal phase after intrauterine insemination;

l) Pregnancy assessment: conduct a pregnancy test 14 days after intrauterine insemination.

Chapter IV
IN VITRO FERTILIZATION TECHNICAL PROCEDURE

Article 8. Counseling for a couple undergoing in vitro fertilization

1. Requirements: to be implemented according to the provisions of Clause 1, Article 5 of this Circular.

2. Content:

a) Explain the treatment process for the couple including clinical examination, laboratory tests, ovarian stimulation regimen, duration of medication use, and monitoring during medication use;

b) The expected time for egg retrieval and explain the need to collect sperm at this time;

c) The expected time for embryo transfer;

d) Support for corpus luteum phase and follow-up after embryo transfer;

đ) Success rate of in vitro fertilization method;

e) Possible complications;

g) Treatment costs.

Article 9. Counseling for special cases

1. Counseling for couples seeking egg donation in in vitro fertilization:

a) Cases of in vitro fertilization with egg donation include older individuals, those suffering from premature ovarian failure, poor ovarian response, and genetic abnormalities.

b) In addition to the information required to be provided to the couple as stipulated in Clause 2, Article 8 of this Circular, additional counseling should include:

- Written commitment from both the egg donor and recipient spouses;

- While the egg donor uses ovarian stimulation medications, the recipient needs to use medications to prepare the endometrium;

- Success rates depend on the age of the egg donor;

- Genetic characteristics of the child born;

- Complications of egg retrieval.

2. Counseling for cases of in vitro fertilization due to the husband's lack of sperm:

a) The husband will undergo testicular biopsy or epididymal aspiration to determine if there is sperm present, and if so, in vitro fertilization can proceed using the husband's sperm;

b) Explain the process of sperm collection through procedures (testicular or epididymal biopsy) and in vitro fertilization using intracytoplasmic sperm injection (ICSI);

c) Provide counseling on success rates and costs;

d) Provide counseling on possible complications;

đ) In cases where sperm cannot be retrieved, donor sperm may need to be used;

e) Genetic characteristics of the child born when donor sperm is used.

3. Counseling for cases of in vitro fertilization with donated embryos: for couples wishing to use donated embryos, follow the provisions of Clause 1 of this Article.

Article 10. In Vitro Fertilization (IVF) Process

1. Overview: in vitro fertilization (IVF) is a reproductive technology in which sperm fertilizes an egg in a culture dish (Petri dish). The resulting embryo is transferred to the uterus to implant or frozen for later use.

2. Indications:

a) Cases of infertility due to blocked fallopian tubes;

b) Infertility due to endometriosis;

c) Infertility due to ovulation disorders (no ovulation, poor ovulation, polycystic ovaries, advanced age);

d) Infertility due to abnormal semen analysis;

đ) Unexplained infertility;

e) Previously attempted intrauterine insemination without success.

3. Contraindications: cases of infertility due to uterine factors.

4. Preparation:

a) Patient preparation (examination, counseling, ovarian stimulation);

b) Preparation of sperm sample (see section on sperm washing);

c) Preparation of equipment and tools: vaginal ultrasound probe, operating table, CO incubator;₂;

d) Preparation of consumables: egg retrieval needles, culture dishes, egg picking dishes, pipettes, media;

5. Procedure:

a) Examine the couple;

b) Conduct necessary basic tests;

c) Assess ovarian reserve (hormone testing, early cycle follicle count via ultrasound);

d) Ovarian stimulation (short agonist protocol, antagonist protocol, long protocol);

đ) Monitor follicular development by ultrasound and hormone levels of estradiol, progesterone, LH;

e) Administer hCG to mature the follicles when conditions are met;

g) Retrieve eggs transvaginally under ultrasound guidance 34 to 36 hours post-hCG injection;

h) Use progesterone to support the luteal phase immediately after egg retrieval;

i) Simultaneously collect sperm samples and prepare them through washing;

k) Allow sperm to fertilize the egg using conventional IVF or intracytoplasmic sperm injection (ICSI);

l) Culture in a CO incubator at 37°C and 5% CO or 6% CO depending on the required medium;₂ or 6% CO₂ as required by the type of environment;

m) Check for fertilization 16 to 18 hours later;

n) Continue culturing the embryo in the incubator until day 2, day 3, or day 5;

o) Transfer the embryo on day 2, day 3, or day 5 (blastocyst);

p) Continue using progesterone to support the luteal phase;

q) Test βhCG 14 days after embryo transfer or 12 days after blastocyst transfer, biochemical pregnancy confirmed when βhCG ≥ 25 IU/L;

r) Perform vaginal ultrasound 28 days after embryo transfer if biochemical pregnancy is confirmed.

Article 11. Sperm Washing Procedure for In Vitro Fertilization

1. General: carried out in accordance with the provisions of Clause 1, Article 6 of this Circular.

2. Indications: Cases requiring sperm washing to perform in vitro fertilization.

3. Preparation:

a) Preparation of the husband: abstain from sexual intercourse for 02 to 07 days, collect sperm sample on the day of egg retrieval.

b) Equipment preparation: microscope, centrifuge machine, incubator, operating table, counting chamber.

c) Consumables preparation: sharp-bottomed test tubes, cover slips, various washing media, pipettes, syringes.

4. Procedure:

a) Collection of semen sample: carried out according to the provisions of Point a, Clause 4, Article 6 of this Circular.

b) Sperm Washing:

- Allow the semen sample to completely liquefy in the incubator for 30 minutes, record the complete liquefaction time;

- Take a small amount of semen to evaluate indicators such as sperm density, measure pH, stain sperm according to WHO recommendations to assess abnormality rate and viability;

- Wash sperm using the "gradient and swim-up" method;

- The obtained sediment is used for in vitro fertilization (used for insemination or intracytoplasmic sperm injection).

Article 12. Oocyte Retrieval Procedure for In Vitro Fertilization

1. General: oocyte retrieval is a technique where oocytes are extracted through the vagina under ultrasound guidance and then fertilized with sperm in a culture dish.

2. Indications: all cases after ovarian stimulation that meet the conditions for oocyte retrieval.

3. Preparation:

a) Preparation of the wife/single woman: stimulate ovaries until mature follicles develop.

b) Equipment preparation: ultrasound machine with vaginal probe, inverted microscope, incubator;

c) Consumables preparation: vaginal cleaning solution, cotton swabs, cups, sterilizing forceps, speculum, gloves, test tubes, aspiration needle, syringe (or suction machine), oocyte picking dish, two-well dish, four-well dish, various media.

4. Procedure:

a) Timing of retrieval: 34 to 36 hours after hCG injection;

b) Pain relief by general anesthesia or local anesthesia, premedication may be combined;

c) The wife/single woman must fast before oocyte retrieval and urinate before the procedure;

d) Clean the vulva, vagina, and cervix with physiological saline;

e) Cover the patient's legs and abdomen with a sterile sheet;

Rinse the syringe and oocyte aspiration needle with the medium used for oocyte retrieval before aspiration;

f) Perform oocyte retrieval under ultrasound guidance, aspirating each follicle sequentially and each ovary separately, the entire process should be gentle and performed under dim light;

g) Immediately transfer the aspirated follicular fluid to the laboratory to find and pick up the oocytes;

h) Rinse the syringe and needle again to avoid oocytes remaining in the needle and syringe.

5. Post-retrieval monitoring:

a) The wife/single woman rests in the post-retrieval room;

b) Monitor pulse, blood pressure, breathing rate, abdominal pain, vaginal bleeding for 01 to 02 hours after retrieval;

c) Guide the wife/single woman after retrieval: medication use, schedule for embryo transfer.

6. Complications:

a) Internal bleeding due to puncturing large abdominal blood vessels, causing ovarian damage;

b) Infection due to puncturing the intestines or colon;

c) Bladder bleeding due to the needle puncturing the bladder.

Article 13. Embryo Transfer Procedure

1. Overview: embryo transfer is a technique in which one or more embryos are transferred into the uterus of the recipient to implant.

2. Indications: all cases of in vitro fertilization with available embryos will proceed with embryo transfer on day 2, day 3, or day 5 after egg retrieval.

3. Contraindications: there are no contraindications, but embryo transfer may be delayed in certain cases such as ovarian hyperstimulation syndrome or when the endometrium has not been adequately prepared.

4. Preparation:

a) Preparation of the wife/single woman: use progesterone after egg retrieval or prepare the endometrium using estrogen and progesterone if transferring frozen-thawed embryos or for egg/oocyte donation.

b) Preparation of equipment and tools: ultrasound machine for guided embryo transfer, speculum, sterilized forceps, cervical clamp, cervical dilator (if necessary), cup for water, inverted microscope.

c) Preparation of disposable materials: vaginal swabs, cotton swabs for the cervix, cervical cleaning solution, embryo transfer catheter, preparation dish for embryo transfer.

5. Procedure:

a) Timing of embryo transfer: day 2, day 3, or day 5.

b) Prepare the embryos for transfer in the dish, carefully check the name and file number.

c) The wife/single woman must refrain from urination to ensure bladder fullness.

d) Assume the gynecological position.

đ) Clean the vulvar area.

e) Open the speculum, clean the cervix thoroughly with the cervical cleaning solution.

g) Gently insert the outer catheter through the cervical canal to the uterine fundus while observing under abdominal ultrasound guidance.

h) Inform the embryologist to aspirate the embryo into the inner catheter once the outer catheter is inserted into the uterine fundus.

i) Gently insert the inner catheter containing the embryo into the uterine cavity, with the catheter tip approximately 2 cm away from the fundus.

k) Gently place the embryo into the uterine cavity, do not transfer more than five embryos.

l) Gently remove the catheter from the uterine cavity.

m) Check the catheter for cleanliness and whether any embryos remain.

n) Remove the speculum.

o) The wife/single woman should rest for at least thirty minutes before leaving.

p) Support luteal phase.

Article 14. Intracytoplasmic Sperm Injection (ICSI) Procedure

1. Overview: intracytoplasmic sperm injection is a micro-manipulation technique that involves directly injecting a single sperm into the cytoplasm of an egg to achieve fertilization.

2. Indications:

a) Cases of infertility due to severe semen abnormalities (low count, weak motility, abnormal morphology).

b) Retrograde ejaculation.

c) Sperm samples obtained from the epididymis or testes.

d) History of poor fertilization in previous in vitro fertilization cycles.

đ) In vitro maturation (IVM) cycles.

e) Cycles using thawed eggs.

g) Women over forty years old.

3. Preparation:

a) Preparation of the egg and sperm.

b) Preparation of equipment and tools: microscope and micro-manipulation set, holding pipette and injection needle, microscope adjustment, incubator.

c) Preparation of disposable materials: ICSI dish, medium, oil overlay.

4. Procedure:

a) Prepare the sperm sample for ICSI using the "density gradient swim-up" method or the "density gradient" method.

b) Eggs retrieved and stored in a warm incubator for one to three hours prior to the procedure.

c) Prepare the ICSI dish.

d) Adjust the microscope and micro-manipulation set.

đ) Separate the polar body from the egg, incubate the egg for one hour.

e) Perform intracytoplasmic sperm injection.

g) Incubate the injected egg in a 37°C, 5% CO2 incubator.₂;

h) Check for fertilization after sixteen to eighteen hours.

Article 15. Procedure for sperm retrieval by surgical technique

1. Overview: Sperm retrieval by surgical technique is a procedure involving aspiration of sperm from the epididymis or testicle to fertilize an egg using intracytoplasmic sperm injection (ICSI) technology.

2. Indications: Cases of infertility due to azoospermia (absence of sperm due to blockage) or ejaculation disorders.

3. Contraindications: Cases where there is no sperm after diagnostic biopsy.

4. Counseling: To be carried out in accordance with the provisions of Clause 2, Article 9 of this Circular.

5. Examination:

a) Taking medical history;

b) General examination;

c) Genital examination: measuring the volume of the testicles, epididymis, and vas deferens.

6. Testing:

a) Measuring sex hormone levels;

b) Basic tests: blood, coagulation, hepatitis B, syphilis, tuberculosis, HIV.

7. Preparation:

a) Preparing the patient: consulting with the husband;

b) Preparing equipment: sterilizing forceps, gauze, antiseptic solution, physiological saline, container for antiseptic solution, centrifuge, microscope;

c) Preparing consumables: needles and syringes for aspiration, various media for washing and filtering, Petri dishes.

8. Procedure:

a) Administering general anesthesia or local anesthesia;

b) Cleaning the genital area and surrounding areas with physiological saline;

c) Fixing the epididymis (if aspirating from the epididymis), or fixing the testicle;

d) Aspirating from the epididymis or testicle (multiple aspirations may be required to obtain a sperm sample);

đ) Sterilizing the aspiration site, checking for bleeding or hematoma;

e) Searching for sperm in the aspirated sample (sperm is easier to find in samples taken from the epididymis than from the testicle);

g) Filtering and washing sperm from the sample for intracytoplasmic sperm injection;

h) Post-procedure care instructions.

Article 16. Procedure for cryopreservation of sperm

1. Overview: Cryopreservation of sperm is a technique in which a sperm sample is frozen and stored in a cold preservation environment. When necessary, it can be thawed for use.

2. Indications:

a) Cases where patients with cancer undergo radiation therapy before treatment to avoid affecting sperm quality;

b) Cases before undergoing vasectomy, to prevent future desire for children;

c) Cases of spinal cord injury that cannot ejaculate naturally, where sperm can be obtained through electrical stimulation and then frozen;

d) Cases where sperm samples taken from the epididymis or testicle can be frozen to avoid multiple biopsies;

đ) Cases preparing for assisted reproductive techniques but the husband must be absent;

e) Difficult cases for sperm retrieval: cryopreservation will be indicated to avoid situations where the wife has already undergone egg retrieval or prepared eggs without sperm available for use;

g) Donor sperm cases will be cryopreserved in a sperm bank to provide for those seeking sperm.

3. Preparation:

a) Preparing the patient: obtaining a sperm sample from the husband for freezing;

b) Preparing equipment: sperm storage tubes, storage containers; freezing machine (in cases of temperature reduction by machine);

c) Preparing consumables: liquid nitrogen, various media.

4. Slow freezing procedure:

a) Evaluating sperm quality before cryopreservation according to World Health Organization standards;

b) Balancing with the cold preservation medium by adding semen to the storage tube followed by the cryopreservation medium at a specific ratio (01 ml of semen mixed with 0.5 - 1 ml of cryopreservation medium), mixing thoroughly (avoiding air bubbles), and leaving at room temperature for 12-15 minutes;

c) Packaging: sealing the storage tube, labeling the patient's name, code, and date of cryopreservation;

d) Reducing temperature according to the machine program or reducing temperature manually (by hand);

đ) Storing in liquid nitrogen: after reducing temperature, attaching the tube to an aluminum rod and storing in a liquid nitrogen container.

Article 17. Sperm Thawing Procedure

1. Overview: Sperm thawing is a technique where frozen sperm samples stored in storage containers are thawed, then filtered and washed for use.

2. Indications:

a) Thawing sperm for intrauterine insemination (IUI);

b) Thawing sperm for in vitro fertilization (IVF).

3. Preparation:

a) Preparing the sperm sample to be thawed;

b) Preparing equipment: warm water container at 37°C, tools and equipment for filtering and washing the sperm sample (refer to the sperm washing section);

c) Preparing consumables: tools and equipment for filtering and washing the sperm sample.

4. Procedure:

a) Remove the storage tube containing the sperm sample from liquid nitrogen, leave it at room temperature for 01 to 03 seconds until the outer ice layer melts;

b) Place the storage tube in warm water at 37°C for 10 to 20 minutes;

c) Evaluate the quality of the sperm post-thaw according to World Health Organization standards;

d) The thawed sperm sample will be filtered and washed for IUI or IVF.

Article 18. Cold Storage of Testicular Tissue Procedure

1. Overview: Cold storage of testicular tissue is a technique where testicular tissue obtained through biopsy is frozen and stored in a cold preservation environment. When necessary, the tissue can be thawed to extract sperm for use.

2. Indications: Cases of infertility without sperm, testicular biopsies with sperm are performed for cold storage to avoid multiple biopsies.

3. Preparation:

a) Patient preparation: examination, counseling;

b) Preparation of equipment and tools: sterilized forceps, container for sterilizing solution, scalpel, scissors, cryopreservation machine, testicular tissue storage tubes, storage vessel;

c) Preparation of consumables: petri dishes, cryopreservation medium, liquid nitrogen, sterilizing solution.

4. Procedure:

a) Conduct examination, testing, and counseling for the patient similar to cases of sperm retrieval procedures;

b) Perform testicular biopsy, place the tissue in a dish containing medium to wash clean;

c) Cut the testicular tissue into small pieces using specialized forceps, determine the presence of sperm, evaluate motility under an inverted microscope;

d) Separate each seminiferous tubule to proceed with freezing;

đ) Add and mix cryoprotectant evenly into the separated seminiferous tubules, shake well and place in cryostorage tubes;

e) Leave the tubes at room temperature, then lower the temperature according to the program;

g) Place the sample in liquid nitrogen and store.

Article 19. Testicular Tissue Thawing Procedure

1. Overview: Testicular tissue thawing is a technique where frozen testicular tissue stored in storage vessels is thawed to extract sperm.

2. Indications: Cases of cold-stored testicular tissue that need to be thawed to obtain sperm for intracytoplasmic sperm injection (ICSI).

3. Preparation:

a) Prepare the testicular tissue storage tube;

b) Prepare equipment and tools: microscope;

c) Prepare consumables: various media for filtering and washing sperm, petri dishes.

4. Procedure:

a) Remove the testicular tissue storage tube from the liquid nitrogen container, leave it at room temperature for 15 to 30 minutes;

b) Wash the testicular tissue with washing medium;

c) Place the testicular tissue sample in a petri dish containing IVF medium, proceed to cut the tissue to find sperm;

d) Evaluate sperm motility;

đ) Culture sperm at 37°C, 5% CO2 for 24 hours;₂ e) Re-evaluate sperm motility and use for ICSI.

e) Re-evaluate the motility of sperm and use for ICSI.

Article 20. Cryopreservation Process for Oocytes

1. Overview: Cryopreservation of oocytes involves the extraction of oocytes from the ovaries, their freezing, and storage in a cold preservation environment. When a woman is ready to conceive, the oocytes will be thawed, fertilized with sperm using intracytoplasmic sperm injection (ICSI) technique, and transferred to the uterus.

2. Indications:

a) Cases requiring surgical removal of the ovaries due to pathological conditions;

b) Cases where sperm samples cannot be obtained after oocyte retrieval.

3. Preparation:

a) Patient preparation: ovarian stimulation, oocyte retrieval, separation of cumulus cells;

b) Preparation of equipment: petri dish, styrofoam container with liquid nitrogen, oocyte cryopreservation straw, oocyte cryopreservation vials, phase contrast microscope;

c) Consumables preparation: pipette, syringe, liquid nitrogen, various media used for oocyte freezing.

4. Procedure:

Similar to the cryopreservation process for embryos, there are various methods for oocyte cryopreservation, but vitrification is currently preferred due to its advantages and is widely adopted by most assisted reproductive centers.

Oocytes should be vitrified within two to six hours after retrieval and immediately after separating the cumulus cells. The quality of the oocytes should be assessed and all parameters recorded before proceeding with freezing. Specific steps of the process may vary depending on the type of medium used.

a) Preparation:

- Washing solution (WS) medium, vitrification solution (VS) medium, equilibration solution (ES) medium kept at 25°C - 27°C;

- Oocyte cryopreservation straws labeled with patient's name, age, date, month, year of freezing;

- Styrofoam container containing liquid nitrogen;

- Pre-freezing oocyte quality assessment;

- Dish preparation: place one drop of WS and three drops of ES on the lid of the petri dish. Place a drop of VS on the petri dish.

b) Oocyte cryopreservation:

- Equilibration: use a pipette to place the oocyte on a drop of WS, then move the oocyte from the WS drop to the ES drops using a pipette to combine the drops, typically taking about three minutes. Transfer the oocyte from the drops to the final ES drop, the oocyte will return to its original shape in approximately nine minutes, additional waiting time can be given if the oocyte has not yet returned to its original form;

- Vitrification: use a pasteur pipette to transfer the oocyte from the ES medium to the VS medium, rinse the ES medium off the oocyte by sucking up and down, then use a pipette to transfer the oocyte onto the cryopreservation straw. Dip the straw into liquid nitrogen, lay the straw horizontally to achieve rapid cooling. Use forceps to attach a plastic tube to the straw.

(Note: the operation time should be within one minute and minimize the amount of medium when transferring the oocyte into the cryopreservation tube).

Article 21. Thawing Process for Oocytes

1. Overview: Thawing of oocytes involves retrieving frozen oocytes stored in cryopreservation vials and thawing them for fertilization with sperm using intracytoplasmic sperm injection (ICSI). Oocytes frozen by a specific method should be thawed using that same method.

2. Indications: cases requiring thawing of oocytes for fertilization with sperm after preparing the endometrium for the recipient.

3. Preparation:

a) Patient preparation: prepare the endometrium for embryo implantation, sperm sample for ICSI after thawing oocytes, prepare the straw containing the oocytes to be thawed;

b) Prepare equipment and tools: microscope;

c) Consumables preparation: various media, petri dishes, pipettes.

4. Procedure:

a) Preparation:

- Thawing solution (TS) placed in a petri dish and kept in a 37°C incubator for at least 30 minutes;

- Dishes containing dilution solution (DS), washing solution (WS1) and WS2, label the patient's name on the thawing dish, and label the types of media on the dish compartments.

b) Thawing:

- Check the name, file number, storage date, and patient's name on the cryopreservation straw;

- Place the prepared petri dish with TS medium on the phase contrast microscope, immediately immerse the cryopreserved oocyte straw taken out of liquid nitrogen into the TS medium for 60 seconds;

- Suck the oocyte along with a small amount of TS medium into a pipette and transfer it to the DS medium dish for three minutes;

- Continue to suck the oocyte and transfer it to the WS1 medium dish for five minutes;

- Transfer the oocyte to the WS2 medium dish for five minutes;

- Finally, transfer the oocyte to a dish containing pre-prepared culture medium, assess the morphology and quality of the oocyte. ICSI can be performed two hours later.

Article 22. Embryo Cryopreservation Procedure

1. Overview: Embryo cryopreservation is a technique where embryos are frozen and stored in a cold preservation environment. When a woman is ready to become pregnant, the embryo will be thawed and transferred to the uterus.

2. Indications:

a) Cases with surplus embryos after embryo transfer;

b) Cases at risk of ovarian hyperstimulation syndrome or experiencing ovarian hyperstimulation;

c) Cases where endometrial preparation for receiving oocytes has not been adequately prepared;

d) Cases with unfavorable endometrium.

3. Contraindications: There are no contraindications, but poor quality embryos are usually not cryopreserved because the degeneration rate after thawing these embryos is often high.

4. Preparation:

a) Preparing embryos for cryopreservation;

b) Preparing equipment: embryo storage straws, embryo storage tanks, pipettes, syringes, inverted microscopes;

c) Preparing consumables: petri dishes, foam boxes containing liquid nitrogen, liquid nitrogen, various media used in embryo freezing.

5. Procedure:

There are many different embryo cryopreservation methods, but vitrification has many advantages and is currently applied by most reproductive assistance centers.

Evaluate embryo quality and record all parameters before proceeding with embryo cryopreservation.

a) Preparation:

- Vitrification Solution (VS) medium, Equilibration Solution (ES) medium at room temperature 25°C - 27°C;

- Embryo storage straw (Cryotop) labeled with patient name, age, date, month, year of freezing;

- Styrofoam container containing liquid nitrogen;

- Evaluating embryo quality before freezing;

- Preparing the dish: 300 µl ES in the first well of a 4-well dish, 300 µl VS in the second well.

b) Embryo cryopreservation:

- Equilibration: using a pipette to draw the embryo from the culture medium onto a droplet of ES medium for 15 minutes, observing under an inverted microscope to see if the embryo size returns to its original state;

- Vitrification: using a Pasteur pipette to transfer the embryo from ES medium to VS medium, sucking up and down to change the embryo's position, then placing the embryo on the straw using a Pasteur pipette, removing some medium, and immersing the straw in liquid nitrogen. This process takes place within 30 seconds, maximum not exceeding 01 minute.

Article 23. Embryo Thawing Procedure

1. Overview: Embryo thawing is a technique where previously frozen and stored embryos are taken out to be thawed, then transferred to the recipient's uterus. The method of freezing the embryo determines the method of thawing it.

2. Indications: Cases of transferring frozen embryos after adequate endometrial preparation.

3. Preparation:

a) Preparing the patient: preparing the recipient's endometrium;

b) Preparing equipment: microscope, incubator;

c) Preparing consumables: petri dishes, 4-well dishes, various types of pipettes, media used for embryo thawing.

4. Procedure:

a) Preparation:

- Thawing Solution (TS) medium in a petri dish placed in an incubator at 37°C for at least 30 minutes;

- Diluent Solution (DS) medium in the first well of a 4-well dish, Washing Solution 1 (WS1) medium in the second well and WS2 in the third well, labeling the patient's name on the thawing dish, and placing the media in the respective wells.

b) Thawing:

- Checking the patient's name, file number, and storage date on the straw;

- Placing the petri dish with the prepared TS medium on an inverted microscope, immediately immersing the embryo or oocyte straw removed from liquid nitrogen into the TS medium for 01 minute;

- Transferring the embryo to the well containing DS medium for 03 minutes;

- Transferring the embryo to WS1 medium for 05 minutes;

- Transferring the embryo to WS2 medium for washing;

- Transferring the embryo into a pre-prepared IVF culture dish, placed in a 37°C CO₂incubator, evaluating embryo quality post-thawing.

c) Embryos can be transferred 02 to 03 hours after thawing or cultured overnight and transferred the next day.

Article 24. Frozen Embryo Transfer (FET) Procedure

1. Overview: Frozen embryo transfer is a technique in which one or more frozen embryos that have been thawed are transferred into the prepared endometrium of the recipient's uterus.

2. Indications: Cases with frozen embryos.

3. Contraindications: There are no contraindications, however, the embryo transfer cycle may be cancelled if the endometrium has not been adequately prepared.

4. Preparation:

a) Patient preparation: Prepare the endometrium;

b) Preparation of equipment, tools, and consumables: Follow the provisions set out in Point b, c Clause 4 Article 13 of this Circular.

5. Procedure:

a) Hormonal tests and other necessary tests at the beginning of the menstrual cycle;

b) Ultrasound evaluation of the uterus and both ovaries;

c) Prepare the endometrium with estrogen at the beginning of the menstrual cycle;

d) Monitor the development of the endometrium through ultrasound;

đ) When conditions are met for embryo transfer, begin using progesterone from 48 to 72 hours before embryo transfer;

e) Notify the embryologist about the thawing of the embryo on the following day;

g) Thaw the embryo, depending on the freezing method to select the appropriate thawing method;

h) Culture the embryo post-thaw in a culture medium and incubator;

i) Evaluate the development and quality of the embryo prior to transfer;

k) Transfer frozen embryos after 03 hours or the next day;

l) Embryo transfer under ultrasound guidance;

m) Continue using estrogen and progesterone at similar doses before embryo transfer;

n) Quantify βhCG 14 days after embryo transfer;

o) Perform ultrasound 28 days after embryo transfer if there is biochemical pregnancy;

p) If pregnant, continue using estrogen and progesterone until the end of 12 weeks.

Article 25. In Vitro Maturation (IVM) Procedure for Unfertilized Oocytes in In Vitro Fertilization (IVF)

1. Overview: In vitro maturation in IVF is a technique where oocytes are retrieved from the ovary at an early stage, then matured in a culture dish and fertilized with sperm via intracytoplasmic sperm injection (ICSI). The resulting embryos can be transferred to the recipient's uterus or cryopreserved.

2. Indications: Cases where patients are at risk of ovarian hyperstimulation syndrome (polycystic ovaries, history of ovarian hyperstimulation) require immature oocyte maturation to avoid ovarian hyperstimulation.

3. Preparation: Similar to IVF but differs in some aspects:

a) The needle used for aspiration is an IVM needle;

b) Medium for culturing immature oocytes;

c) A filter to separate oocytes during retrieval.

4. Procedure:

a) The patient receives supplemental FSH;

b) Monitor the development of follicles;

c) Use hCG;

d) Aspiration of immature oocytes;

đ) In vitro maturation of immature oocytes in IVM medium for an average time of 24 to 26 hours;

e) Perform separation of cumulus cells from the oocytes;

g) Intracytoplasmic sperm injection into the oocyte;

h) Incubate the oocytes post-fertilization in an incubator;

i) Check for fertilization 16 to 18 hours later;

k) Transfer embryos on day 2, day 3, or day 5;

l) Quantify βhCG 14 days after embryo transfer;

m) Perform ultrasound 28 days after embryo transfer if there is biochemical pregnancy.

Article 26. Oocyte Donation In Vitro Fertilization Procedure

1. Overview: Oocyte donation in vitro fertilization is a technique where sperm is fertilized with donated oocytes in a culture dish. The resulting embryos are transferred into the prepared endometrium of the recipient's uterus to implant.

2. Indications:

a) For the oocyte recipient:

- Premature ovarian failure;

- Poor oocyte quality;

- Poor ovarian response (increased FSH levels at the beginning of the menstrual cycle, few secondary follicles);

- Advanced age (over 40 years old);

- Genetic abnormalities.

b) For the oocyte donor:

- Age as prescribed;

- Has had at least one healthy child;

- If currently nursing, the child must be at least 12 months old and not breastfeeding;

- Does not suffer from chronic diseases or genetic disorders.

3. Counseling for couples seeking oocyte donation: Follow the provisions set out in Clause 1 Article 9 of this Circular.

4. Preparation: Follow the provisions set out in Clause 4 Article 10 of this Circular but also prepare the oocyte donor and prepare the endometrium of the recipient.

5. Procedure:

a) Comprehensive examination, gynecological examination, and testing of the oocyte donor;

b) Sign a commitment agreement between the donating and receiving couple;

c) Adjust menstrual cycles between the donor and recipient;

d) Stimulate the ovaries of the oocyte donor while preparing the endometrium of the recipient with estrogen;

đ) Monitor the development of follicles in the oocyte donor through ultrasound and hormone quantification;

e) Monitor the endometrium of the recipient through ultrasound;

g) When the follicles reach maturity, inject hCG into the oocyte donor;

h) Aspirate oocytes from the oocyte donor, simultaneously administer additional progesterone to the recipient;

i) Collect sperm, wash and purify the husband's sperm;

k) Fertilize the oocytes with the husband's sperm using conventional in vitro fertilization (IVF) or intracytoplasmic sperm injection (ICSI) techniques based on sperm quality;

l) Check for fertilization 16 to 18 hours later;

m) Transfer embryos on day 2, day 3, or day 5 to the recipient (wife);

n) Continue using estrogen and progesterone after embryo transfer;

o) Quantify βhCG 14 days after embryo transfer;

p) Perform ultrasound 28 days after embryo transfer if there is biochemical pregnancy;

q) Continue using estrogen and progesterone until 12 weeks.

Article 27. In Vitro Fertilization Using Donor Sperm

1. Overview: In vitro fertilization using donor sperm is a technique in which the sperm of the donor is fertilized with the oocyte of the recipient in a culture dish. The resulting embryo will be transferred to the uterus for implantation or frozen for future use.

2. Indications:

a) The husband has no sperm.

b) Genetic abnormalities.

3. Conditions for the sperm donor:

a) Not suffering from infectious diseases;

b) Not suffering from mental illnesses or genetic diseases;

c) The sperm sample meets the World Health Organization standards.

4. Counseling for couples requesting donor sperm: carried out according to the provisions of Clause 2, Article 9 of this Circular.

5. Preparation: preparation is similar to the case of in vitro fertilization, but the sperm sample in this case will be taken from the donor.

6. Procedure:

a) Examination and testing of the sperm donor;

b) The sperm sample was previously collected for cryopreservation (see cryopreservation procedure);

c) Stimulation of the ovaries according to the ovarian stimulation protocol;

d) Monitoring the development of follicles and endometrium by ultrasound and hormone quantification;

đ) When the follicle develops to maturity, inject hCG;

e) Ovum retrieval, using progesterone to support corpus luteum;

g) Filtering and washing the donor's sperm sample;

h) Fertilizing the oocyte with the donor's sperm using conventional in vitro fertilization (IVF) or intracytoplasmic sperm injection (ICSI) techniques depending on the quality of the sperm sample;

i) Placing the oocyte and sperm or the oocyte after ICSI in the incubator;

k) Checking fertilization after 16 to 18 hours;

l) Embryo transfer on day 2, day 3, or day 5;

m) Continuing progesterone after embryo transfer;

o) Quantify βhCG 14 days after embryo transfer;

p) Perform ultrasound 28 days after embryo transfer if there is biochemical pregnancy;

q) Continuing progesterone to support corpus luteum until week 12.

Article 28. Selective Embryo Reduction Procedure

1. Overview: selective embryo reduction is a procedure using a needle under ultrasound guidance to reduce the number of embryos in cases of multiple pregnancies.

2. Indications:

a) Cases with three or more embryos after embryo transfer, usually two embryos or one embryo are left depending on specific circumstances;

b) For twin pregnancies, one embryo may be reduced depending on specific circumstances.

3. Timing of embryo reduction: the ideal timing for embryo reduction is at 7 weeks plus 3 days.

4. Counseling:

a) Reasons for embryo reduction;

b) Embryo reduction procedure;

c) Potential complications.

5. Pain management: general anesthesia or local anesthesia, premedication may be combined.

6. Preparation:

a) Preparing the pregnant woman: blood test, blood type, and coagulation factors; the pregnant woman must fast and urinate before the procedure;

b) Preparing equipment: sterilized forceps, ultrasound machine;

c) Consumables preparation; sterile solution container, sterilizing solution, aspiration needle, syringe.

7. Procedure:

a) Pre-procedure examination and anesthesia;

b) Cleaning the vulva, vagina, and cervix with physiological saline;

c) Laying down a sterile sheet covering the legs and abdomen of the pregnant woman;

d) Ultrasound re-evaluation of the number of embryos, embryo status, and the embryo to be reduced. The embryo to be reduced is the one closest to the needle path and closest to the cervix;

đ) Proceeding to puncture the needle accurately at the position of the embryo to be reduced under ultrasound guidance, once the needle tip touches the embryo, proceed with aspiration;

e) For small embryos, all can be aspirated, for larger embryos that cannot be completely aspirated, ensure fetal heart activity has ceased;

g) For larger embryos, potassium chloride can be injected into the fetal heart cavity;

h) Prophylactic antibiotics during the procedure.

8. Post-procedure monitoring:

a) The pregnant woman rests in bed;

b) Monitoring pulse, blood pressure, breathing rate, abdominal pain, vaginal bleeding 2 hours post-aspiration;

c) Follow-up appointment scheduled for 2 days later.

9. Complications:

a) Bleeding;

b) Puncture of blood vessels, bladder;

c) Infection;

d) Miscarriage, fetal demise.

Section 5
IMPLEMENTING PROVISIONS

Article 29. Effective Date

This Circular takes effect from August 20, 2012.

Article 30. Responsibility for organizing implementation

The Maternal and Child Health Department shall be responsible for leading and coordinating with the Medical Examination and Treatment Management Department, the Legal Affairs Department, and related units to organize guidance, direction, inspection, and supervision of the implementation of this Circular.

During the implementation process, if there are difficulties or obstacles, they are requested to report in writing to the Ministry of Health (Maternal and Child Health Department) for study and resolution./.