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In Vitro Fertilization (IVF)

Introduction

Conventional IVF is the name given to the original ‘test tube baby’ treatment method and involves mixing prepared sperm and eggs in a dish in the laboratory. The first test tube baby, Louise Brown, was born in 1978. It is now 32 years since her birth and dramatic developments have occurred in IVF.

Who will benefit from IVF treatment?

  • Women with tubal problem
  • Woman with unexplained infertility
  • Women with severe endometriosis
  • Couples with male factor infertility*

*If there are less than 1 million motile sperm in the sample after preparation intra-cytoplasmic sperm injection (ICSI) is the treatment of choice. IVF can be only performed if there are more than 1 million motile sperm.

What is involved in IVF treatment?

The main strategy in IVF treatment is to stimulate the ovaries with hormones to produce multiple follicles containing eggs. These eggs are retrieved and mixed with sperm and embryos are generated.

 

Ovarian stimulation

  • The first step is the shutting down of the pituitary-ovarian hormones called down regulation. This is done with a drug known as Gonadotrophin releasing hormone agonist (GnRHa). This is available in the form of a nasal spray, daily subcutaneous injections or depot injections (the effect of one injection lasts for 4-6 weeks). This suppression can be started on day two or day 21 of a period depending on the type of protocol (long or short). Please see protocols. It takes approximately two weeks for complete suppression. This is confirmed by an ultrasound scan which will show a thin uterine lining and quiescent ovaries without any cyst. A blood test is also performed to check the hormonal level (Oestradiol) for the confirmation of down regulation or suppression.
  • Once down regulation is confirmed the daily injections of FSH or menotrophin will be started to stimulate the ovaries. These injections need to be administered daily for 12-16 days. The GnRH agonist is also continued during this period of ovarian stimulation.

 

Monitoring the cycle

The ovarian response to the stimulation is monitored by serial ultrasound scanning and blood test. Usually, three to four scans are necessary during this two week period of injections. In addition, blood tests are carried out to check the level of Oestradiol along with scans in all patients. The scans are usually internal scans which give a better picture of the uterus and the ovaries. During the scans the number and the size of the follicles are measured and the thickness and the texture of the lining of the womb are assessed. When the leading follicles reach a diameter of 18 mm or over, the lining of the womb is at least 8 mm in thickness and the Oestradiol levels correspond to the number of growing follicles, it is time to give the trigger injection (HCG injection). At this stage, the GnRH agonist and FSH or menotrophin injections will be stopped. The trigger injection is the Human Chorionic Gonadotrophin (HCG) which starts the maturation process of the eggs. This is usually given in the late evening and the egg collection will be performed 36 hours later.

 

Natural cycle IVF

This term refers to IVF treatment using one’s natural cycle i.e. without any stimulation with drugs. Normally, only one egg is produced and released every month. This egg is collected and mixed with sperm. If fertilization takes place, there is one embryo which is replaced after 48 hours.

The main advantage is that there is no injection or use of any other drugs. This means there are no side effects of drugs. Usually, the injections used in the treatment of IVF are expensive. Hence, natural cycle IVF is also cheaper.

The main disadvantage is that the chance of success is rather low because there is only one egg and one embryo.

 

Egg collection procedure

Egg collection is usually performed in the morning, approximately 36 hours after the HCG injection.

Route: the egg collection is performed vaginally under ultrasound guidance. When IVF was started the egg collection was routinely done laparoscopically. Because of the invasive nature of this method, it is used only in selected cases. For example, if one or both ovaries are not accessible vaginally, laparoscopic egg collection is performed.

Anaesthesia: the vaginal egg collection is done under intravenous sedation (conscious sedation). Some centres use general anaesthesia for this procedure.

Procedure: a needle is attached to the vaginal probe of the ultrasound. Under ultrasound guidance, the needle is passed through vagina and into the ovary, as shown in the diagram above. The fluid from an ovarian follicle is aspirated and the fluid is passed to the embryologist who identifies the egg in this fluid under a microscope. If there is no egg, in the aspirated fluid the follicle is flushed with culture medium and sucked out. Once the egg is identified, the needle is moved to the next follicle and the procedure is repeated. Once all the follicles are emptied, the needle is taken out of the ovary and passed into the other ovary and the same procedure is carried out.

The number of follicles does not correspond to the number of eggs collected. Some follicles may not contain an egg.

 

After the procedure

Transvaginal egg collection is a relatively safe procedure. It takes an average of 20-30 minutes. The woman is allowed to go home after two hours. She must be taken home by her partner or friend. She should not drive a vehicle for 24 hours.

After the egg collection procedure, there may be some discomfort or soreness in the tummy. This might be more if the number of eggs collected is more (more than 12). Pain-killers can be used to control the symptom. This will not interfere with the fertility treatment.

There may be some spotting. This is usually minimal and dark brown in colour. This may be due to the oozing from the needle puncture site in the vagina.

There may be nausea and vomiting. This can be due to the anaesthetic drugs or mild hyper-stimulation. When the number of eggs is more than 15, there may the bloating of the abdomen.

If any of the symptoms becomes severe, one should consult the doctor.

 

Semen sample

The husband or the patient’s partner is required to produce a semen sample on the day of egg collection. The man is advised to abstain for 2-3 days prior to producing sample. If the period of abstinence is more than 7-10 days, the quality of the semen sample may become poor.

The semen sample is generally produced by masturbation. It is recommended that the man washes his hands and genitals with soap, rinses with clean water and dries with a clean towel. No lubricant such as petroleum jelly, should be used during masturbation to avoid problems of toxicity to sperms. The sample is collected into sterile plastic containers which are non-toxic to the sperm.

The partner usually produces the sample in a separate room in the clinic. This is to ensure that the sample is as fresh as possible. If some men find it difficult to produce samples in the clinic’s environment, they can produce sample at home and bring it to the clinic, provided it is delivered within one hour. Also, it is important to keep the sample warm by carrying it in the inner pocket of the jacket. If some men find it difficult to produce into a small plastic container, they can use a special condom provided by the clinic. This condom is a special one and does not have the spermicidal agents present in the normal condoms.

If the partner is not going to be available on the day of egg collection, he could produce a sample before and have it frozen. The frozen sample can be thawed and used for the treatment.

If a partner has or is likely to have difficulty in producing a sample, it is better for him to produce a sample before the commencement of the treatment and freeze it as a backup arrangement.

 

Progesterone supplementation

After the egg collection, the patient is advised to take the hormone progesterone as a support for the implantation. This is usually given in the form of a pessary or suppository. It is given in the dose of 400 mg twice daily rectally until embryo transfer and rectally or vaginally after embryo transfer. This is continued until the day of pregnancy test which is performed 12 days from the embryo transfer. In the pleasant event of the test being positive, it is continued for another two months (till 12 weeks of gestation). If the pregnancy test is negative, the medication is stopped on that day.

 

Fertilization

  • The eggs and the prepared sperm are mixed in a cultured medium in the laboratory. This is kept in a labelled dish in an incubator.
  • The dish is checked next morning for fertilization. The sign of fertilization is the presence of two pronuclei (one from the sperm and one from the egg), as shown in the picture on the right. Normally about 60% of the eggs fertilise.
  • The embryos are checked for further cell division the next day. There should be 2-4 cells the next day (day 2) and 6-8 cells on the third day, as shown in the pictures on the right. Sometimes the embryos may be rather slow in their division. The embryos which have divided and show regular outline and minimal or no fragments are usually transferred. The grading of the embryos is based on the regularity of cellular outline and the presence of fragment.
  • The number of embryos to be transferred will be discussed with the patient/couple. In general, it is recommended to have one embryo transferred if the patient is under 35years of age. This is to reduce the risk of twin pregnancy. In the UK, the HFEA allows clinics to transfer a maximum of two embryos in women under the age of 40 years and 3 embryos in women of 40 years and above.
  • Depending on the number of embryos available, after choosing the best embryo for transfer, the other embryos will be frozen for future use if they are of good quality.

 

Embryo transfer

The embryo transfer is a simple procedure. Normally no anaesthesia is used for this procedure. However, in some difficult cases, mild sedation may be used. The couple may be able to see the embryos through a monitor before the procedure.

Procedure: The woman lies on the couch with her legs in stirrups. The doctor introduces a speculum to visualise the cervix and then cleans the cervix. Meanwhile, the embryos are loaded on to a catheter by the embryologist. Finally, the doctor introduces the catheter gently into the uterine cavity and deposits the embryos.

Embryo transfer is carried out under ultrasound guidance with a full bladder. This is to visualise the position of the catheter and the placement of the embryos. This is reassuring to the doctor and the patient. In addition the full bladder may correct the angle between the cervix and the body of the uterus and make the transfer easier. This is to reduce the trauma to the embryos during the procedure. After transferring the embryos, the catheter is withdrawn slowly and handed over to the embryologist. The embryologist checks the catheter to make sure that no embryo is left in the catheter. If one or more embryos are retained in the catheter, they are loaded again and replaced. This does not reduce the chance of success.

If the transfer is difficult, a tenaculum is applied to the cervix and a different catheter which would negotiate the angle between the cervix and the uterus is used.

After the embryo transfer, the patient is advised to lie down for 15 minutes. She is then allowed to get up and empty her bladder.

The question that is present in every patients mind: “Will the embryos fall out when I get up?” Luckily, the answer is: no.

Dos and don’ts after embryo transfer:

  • Take it easy for the next few days. This does not mean bed rest. One can take leisurely walks.
  • It is better to avoid strenuous activities, lifting heavy weights, jogging, riding and gym activities.
  • Avoid swimming.

 

Pregnancy test

During IVF treatment, a blood test is performed 14 days after the egg collection. This period of waiting is very stressful for couples. This is said to be the longest two weeks in one’s life. There is nothing one can do to improve the chances.

The pregnancy test is a quick test and the result should be ready in one or two hours. If the test is positive it is usually repeated within 2-5days to check the rising level of the hormone β-HCG.

If the hormone level does not rise in the usual pattern, it indicates problems with the pregnancy, such as miscarriage or ectopic pregnancy. This is followed up with serial blood test. Some women may start spotting or bleeding before the pregnancy test is due. They may assume that they have started period and not attend the hospital for the blood test. It is important that a blood test is carried out even under these circumstances to establish whether there is a pregnancy or not.

  • Positive outcome: if the pregnancy test is positive, the woman is advised to continue with the progesterone supplementation. An ultrasound scan is arranged 2-3 weeks after the test. This is to confirm that the pregnancy is in the right place (uterus), check the number of pregnancy sacs and viability of foetuses. If the findings are normal the progesterone is continued for another 5-6 weeks (until 12 weeks of pregnancy).
  • Negative outcome: this is the worst fear of every woman who goes through IVF treatment. If the pregnancy test is negative, the progesterone supplementation is stopped. The negative pregnancy test is very upsetting to the couple. Some couples may benefit from seeing a counsellor. They need time to recover from the shock. When they are in a position to discuss the matter they can make an appointment to see the doctor in hospital/clinic to plan for future treatment.

Ovarian hyperstimulation syndrome (OHSS)

This is one of the most important complications of IVF treatment. The ovarian response to stimulation may be excessive with the ovaries becoming enlarged and fluid accumulation in the abdominal cavity. This can be mild, moderate or severe.

  • Mild: this occurs in some 10-20% of the cases. The usual symptoms are abdominal distension and discomfort, nausea, vomiting, and diarrhoea. Ultrasound scans shows ovarian enlargement of less than 5 cms.
  • Moderate: this occurs in some 5% of the cases. The usual symptoms are as described for mild OHSS. Ultrasound scan shows accumulation of fluid in the abdominal cavity and ovarian enlargement between 5-12 cms.
  • Severe: this occurs in some 1-2 % of the cases. The ovarian enlargement is over 12 cms. There is breathing difficulty and clinical ascites. Other features like haemoconcentration, coagulation disorders, oliguria and liver and renal failure may be present.

Treatment

  • Mild: all that is needed for mild OHSS is an ultrasound scan to confirm the diagnosis and for reassurance. Bed rest, increased intake of oral fluids and mild analgesics such as Paracetamol are helpful.
  • Moderate: patients with moderate OHSS need close monitoring with ultrasound scans, blood tests and intravenous fluids if necessary.
  • Severe: severe cases of OHSS may require hospital admission. Drainage of the fluid in the abdomen by vaginal route under ultrasound guidance will relieve some of the symptoms markedly and help the patients recover dramatically.

 

Multiple pregnancy

The incidence of multiple pregnancy is high as more than one embryo is transferred to improve the chance of success. If 100 women conceive after three embryo transfers 75 of them will have singleton and 25 twins. These figures represent approximately 20 and 50 times increased frequency for twins compared with spontaneous conception. These twins are non-identical (different eggs)twins. Sometimes, the single embryo may split and give rise to identical twins (monozygotic twins). This is the reason why a patient who has only one embryo transferred can still have twins and the woman who has two transferred can have triplets. This risk is particularly higher with blastocyst transfer and assisted hatching. The incidence of monozygotic twins is 4.9% of IVF treatment, 12 times higher than the 0.4% rate observed in natural conception.

With multiple pregnancy, the pregnancy is more complicated than singleton pregnancy. The risk to the mother is increased due to complications of pregnancy such as pre-eclampsia, diabetes, placenta praevia and postpartum haemorrhage. The babies have an increased risk due to pre-term labour, intra-uterine growth restrictions and malformations. The perinatal mortality rate is higher in twins compared to singleton pregnancies.

 

Ectopic pregnancy

This is more common after IVF treatment than in the general population. The incidence is 3-5% after IVF treatment. When a woman has high risk factor for ectopic pregnancy, such as previous ectopic, damaged tubes, previous tubal surgery and past history of PID, serial measurements of quantitative β-HCG is carried out. If the β-HCG does not rise in the usual way, an early ultrasound scan is arranged to exclude ectopic pregnancy. Laparoscopy may be needed to exclude ectopic pregnancy if there is no gestational sac seen in the scan.

 

Heterotopic pregnancy

This refers to a combination of intrauterine and extra uterine pregnancy. This is rare in spontaneous conception with an incidence of 1 in 20,000 to 30,000; but it increases dramatically after IVF treatment.

 

Miscarriage

There is a 20-25% risk of miscarriage with IVF treatment. The risk of miscarriage with spontaneous conception is 15%. The higher incidence of miscarriage in IVF may be due to polycystic ovaries in these women.

 

Risk of ovarian cancer following ovarian stimulation

A lot of research has been carried out to study the association between the ovarian stimulation and ovarian cancer. So far, no increased risk has been observed.

Protocols for ovarian stimulation in IVF treatment

There are different protocols for ovarian stimulation. In the initial days of IVF treatment, there was no hormonal suppression as the drug GnRH agonist was not available. Clomiphene citrate tablets and Gonadotrophin injections were used for ovarian stimulation for approximately 12-14 days and when the follicles reached the mature size (>=18 mm), the egg collection was planned. With the introduction of GnRH agonists, other protocols emerged.

 

Long

Long Luteal: this is the standard protocol and is used when the woman has regular 28-30 day cycle. The hormonal suppression (down regulation) is commenced on day 21 of the period before treatment. It can be in the form of daily subcutaneous injection, nasal spray or a single injection of depot preparation (the effect of which lasts for 4-6 weeks). The next period usually starts 7-10 days after the commencement of the spray or injection. After confirmation of down regulation with an ultrasound scan and blood test on day 5 of the period, the FSH injections are started to stimulate the ovaries. These injections are administered daily by the subcutaneous route for approximately 12-16 days till the leading follicles reach the size of >= 18 mm, when the egg collection is planned. At this stage, the suppression spray or injection is stopped and a trigger injection of Human Chorionic Gonadotrophin (HCG) is administered 36 hours before egg collection.

Pill: this protocol is used in women with long or irregular cycles. The purpose of the pill is to regulate the cycle. It is started on day 1 or 2 of the period. The downregulation is commenced on day 17 of the pill. The course of pill will finish on day 21 which will be followed by a period after a few days.The rest of the steps are the same as above.

 

GnRH antagonist protocol

As GnRH inhibits the premature LH surge selectively, there is no need for the down regulation of all the hormones with this protocol. This is a “soft stimulation” protocol as the number of days of FSH injections and the number of ampoules needed is lower, thereby reducing the cost. Therefore it is a “patient friendly” protocol.

The main advantage of this protocol is to reduce the risk of OHSS in women with polycystic ovaries. Poor responders produce more eggs with this protocol.

The stimulation starts on day 2 of the period with FSH or HMG injections. This is started after a baseline check scan. In women with previously raised FSH or poor response hormone levels are checked before commencing treatment. Serial scans are performed from day 6 onwards. GnRH antagonist (Orgalutran, Cetrorelix or Antagon) injection is started on day 6 and administered daily in the form of a subcutaneous injection till the day of HCG injection. The patient will be having 2 injections daily (FSH and GnRH antagonist injection).

Advantages of IVF

  • Key information about fertilization between sperm and egg is obtained only in IVF treatment
  • One can assess egg and embryo quality
  • Surplus embryos can be frozen

 

Success rate with IVF treatment

This varies from country to country (due to varying restrictions in different countries) and in the same country, from one clinic to another. The clinical pregnancy rate varies from 30-50%. The presence of intra-uterine gestation with foetal heart movement is defined as clinical pregnancy. Some of these pregnancies end in miscarriages. The live birth rate or “take home baby rate” varies from 25-40%.

 

Factors affecting the success rate

  • Age of the woman: this is the most important factor affecting the success rate with assisted conception treatment. The younger the woman, the higher is the success rate. The success rate declines considerably in women over the age of 40 years.
  • Duration of infertility: there is some evidence to say that the longer the duration of infertility, the lower the success rate with IVF treatment.
  • Type of infertility: women who have had children or who have been pregnant in the past do better with IVF treatment. Those who have had a baby from previous IVF treatment have a higher chance of success.
  • Cause of infertility: women with tubal factor, particularly hydrosalpinx have a reduced chance of success compared to other female causes of infertility. Couples with male factor infertility that is treated by intra-cytoplasmic sperm injection (ICSI) have a higher chance of success.
  • Number of previous IVF treatments: live birth rate is highest in the first cycle and it becomes lower after three attempts. The cumulative pregnancy rate after three attempts of IVF is in the region of 70-80% which is encouraging. Not everybody is lucky to become pregnant in the first attempt. The important thing is to keep trying.
  • Quality of embryos: this is one of the important criteria for success. The higher the quality of embryos, the better is the chance of success. The quality of embryos depend on the quality of eggs which is related to female age, and quality of sperm. This is the reason for higher success rate with egg donation programme where the eggs are obtained from young and fertile women.
  • Number of embryos transferred: transferring a higher number of embryos leads to a greater chance of success. However, this is associated with a higher risk of multiple pregnancy. In the UK, according to HFEA regulations, only one or two embryos are allowed to be transferred in women under the age of 40 years; women aged 40 and above are allowed to have up to 3 embryos. Younger women (under 35 years) are advised to have single embryo transferred if their embryo grade is excellent, to reduce the risk of twin pregnancy.

Assisted hatching

Introduction

The shell or protective layer of the embryo is called zona pellucida. This may become hard due to the laboratory techniques involved in IVF and due to freezing techniques.

The assisted hatching procedure involves thinning or making a hole in the zona pellucida to help the hatching process of the embryos. There is some evidence that assisted hatching might improve the implantation rate.

 

Techniques

A hole can be made in the zona by a micro-needle or with the use of a chemical called acid Tyrode’s solution or with a laser. Of these, the laser technique is the best as it has a greater degree of control and more precision during the procedure. A laser can be used to create a full thickness breach or thinning of zona pellucida.

Assisted hatching is performed immediately prior to transferring the embryos into the womb.

 

Effect on treatment outcome

Some studies have reported improvement in the outcome following assisted hatching. However, some clinicians have reported no difference in outcome.

 

Who will benefit from assisted hatching?
  • Women older than 38 years
  • Women with repeated unsuccessful treatments with IVF
  • Women with high FSH
  • Embryos with a thick zona pellucida
  • Embryos that have been frozen and thawed

 

Risks

Assisted hatching increases the risk of monozygotic twinning.

Blastocyst transfer

Introduction

This is a new technique developed to maximise the chance of success with IVF treatment. For IVF treatment, the embryos are normally replaced on the second or third day of fertilization, but in blastocyst transfer they are replaced on the fifth day.

 

Technique

With this technique, the embryos are kept in the culture medium in the laboratory till the fifth day of fertilization. A day 2-3 embryo has 4-8 cells whereas blastocyst, or day 5 embryo, has more than 100 cells. It has differentiated into thin outer layers of cells which will develop into placenta and an inner cell mass which will develop into the foetus. There is a cavity inside and hence the name blastocyst, as shown in the picture. The metabolic requirement of the embryos changes after the third day and therefore they have to be placed in different culture media, which is called sequential media for this technique.

 

How does it work?

Usually one has to have 8-10 embryos on the day of fertilization. Everyday the scientist will look at the embryos to see how many of them have divided and place them in sequential media. Some embryos may stop dividing and on the third day there may be only six embryos which have divided. Out of the six embryos only three or four may develop into blastocysts. Others may not have divided. If one started with eight embryos on the first day, and by the third day, if there are less than four embryos, it is better to have these embryos replaced on that day rather than continue with the original plan of blastocyst transfer. As far as the transfer procedure is concerned, it is the same as the conventional day two or day three embryo transfer.

As the implantation rate is high with blastocysts it is advisable to have only one or two blastocysts for transfer to avoid multiple births. Supernumerary blastocysts can be frozen for future use.

Blastocyst transfer can be used with frozen-thawed embryo transfer treatment also. A minimum of at least eight embryos should be in storage for this. All of them can be thawed and embryos transferred at blastocyst stage.

 

Who will benefit from blastocyst transfer?
  • Women who have had two or more failed IVF attempts following the transfer of good quality embryos and who produce good number of eggs.
  • Women who would like to avoid multiple pregnancy without reducing their chances.
  • Women who do not wish to have the surplus embryos frozen for whatever reason may benefit from this technique.

 

Advantages

This technique gives the clinicians the opportunity to choose the best embryos for the transfer. If embryo has divided and developed into blastocyst stage, it is more likely to continue to grow and get implanted. With day 2-3 transfers, the embryos may stop growing inside after transfer. In a natural cycle, this is the stage at which the embryo reaches the uterine cavity.

The uterine endometrium is said to be more receptive on day 5 rather than on day 2-3 and quiescent without any substantial myometrial contractions.

All of these contribute to a success rate of 50-60% with blastocyst transfer in IVF treatment.

 

Disadvantages

The risk of monozygotic twins is higher.

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