Since the birth of first child conceived using the process of in-vitro fertilization (IVF) in 1978, more than 1 million children have been born world-wide through this fascinating procedure. Although originally developed to treat women with absent or irreparably damaged fallopian tubes, IVF procedure have since been used as a treatment modality for a wide variety of infertility problems. In IVF procedure the physiological process of fertilization takes place outside the body of the female patient. In an artificially constructed environment. For this purpose, one or more oocytes are recovered from preovulatory ovarian follicles and subsequently co incubated in a defined culture medium together with motile spermatozoa previously separated from the seminal plasma, immotile spermatozoa and non-spermatozoal cells. After identification of successful fertilization, either at the pronuclear stage, at an early cleavage stage or at the later blastocyst stage, the embryos are placed in the uterine cavity for nidation in the endometrium and pregnancy.

Introduction
In vitro fertilization (IVF) means “in glass” or “in the laboratory”, hence the term “test-tube baby” is a complex procedure for the treatment of infertility in a wide variety of couples who are unable to conceive naturally. The IVF procedure at its very simplest involves removal of eggs from the ovaries, fertilization with the sperm in the laboratory and transferred of the early embryos into the uterus.The minimum requirements for IVF are that the female has a normal uterine cavity, a source of oocytes and enough sperm in the male to achieve fertilization. Although IVF can be performed with naturally matured oocyte, it has become wide spread practice to carry out this treatment in combination with hormonal stimulation of the ovaries, so that multiple oocyte become available for IVF. The improved pregnancy rate of IVF in hormonally stimulated cycles results mainly from the replacement of several fertilized oocytes. The indications for treatment through IVF have broadened over the years.

Indications For IVF:
As couples undergo evaluation and treatment of infertility, IVF is the first line of therapy in certain situations. Some of these indications are given in table no. 1 given below :

TABLE NO.1
Major Indications for IVF

 

All the indications mentioned in the above table represent either an unfavorable environment for egg – sperm interaction or an inability of an egg and sperm to unite and fertilize in-vivo. The use of in-vitro fertilization technique allows the rescue of eggs and sperms from unfavorable conditions, provides a more favorable environment for egg- sperm interaction, confirms fertilization and ensures delivery of the embryo to the uterus.

An Outline of the IVF-ET Process
The work of the embryologist is the culmination of a series of events that were set in train the day the couple presented to their infertility clinician. The embryologist’s main brief is to identify and take responsibility for the oocytes aspirated by the IVF clinician at the oocyte recovery, with the intention of providing optimal conditions for the creation of embryos which will be returned to the patient or frozen for subsequent use by the patient. The process is summarized below.

Pre OPU Follicular phase tracking Day 0 Sperm Production ---> Sperm Preparation OOCYTE Pick up (OPU) and gamete Intrafallopian transfer (GIFT) Insemination Day 1 Fertilization check Embryo Culture Day 2 or 3 Embryo transfer Embryo cryopreservation

Ovulation Stimulation Protocol
The aim of ovulation stimulation protocol in IVF-ET procedures have always been to produce a sufficient number of oocytes for maximizing the success of treatment. In gonadotropin only stimulation protocols, the existence of elevated basal levels of LH were responsible for cancellation of a large number of treatment cycles. The discovery that the tonic administration of GnRHa induces a state of hypophyseal desensitization helped remove many of the ill effects of previous stimulation regimes. Ovulation stimulation strategies using GnRH analogues are now adopted in ART procedures to control endogenous secretion of LH. GnRHa administration down regulates the pituitary gland and prevents the release of FSH and LH, resulting in ovarian quiescence. Pituitary down regulation can be confirmed by endocrine and ultrasound testing. Complete pituitary suppression shows following endocrine parameters.

1. LH   <   5  IU/L
2. E-2  <  50  pg/ml
3. P    <  1  ng/ml
4. Thereafter adequate superovulation can be achieved by daily administration of 150 / 225 IU of FSH.

The protocol of GnRha together with hMG is the bench mark for ovarian stimulation in all IVF programmes. A number of protocols have been developed by various workers. Brief information about the commonly used protocol called the long down regulation protocol is given step-wise below :

1. In long protocol 500 g GnRHa administration is initiated on day 21 of previous cycle and down regulation is normally complete after 7 days. There after the concentration of GnRHa is reduced to 200 ug and continues till the day of hCG administration.
2. FSH (225 IU daily) started from day 2 of the menstrual cycle and continues till the day of hCG administration when  sufficient number of mature follicles are present. In normo-ovulatory women, 10 days of gonadotropin stimulation is usually sufficient to achieve the criteria for ovulation induction and ensures sufficient estrogen priming for the endometrium
3. Ovulation induction done through administration of hCG  (10,000 units) when leading follicles are >18mm in diameter.
4. Oocyte retrieval done 34-39 hours after hCG administration.

Retrieval Of Oocytes
The retrieval of oocytes for assisted fertilization now-a-days is performed 34 –36 hours following hCG administration by ultrasound guided transvaginal puncture of the follicles. The oocyte retrieval is essentially an outpatient procedure performed under light (neurolept) anesthesia. Usually a combination of Propofol, Midazolam and Fentanyl is administered intravenously. The direct location of ovaries behind the vagina, its plasticity  and insensitivity to pain, makes transvaginal ultrasound guided follicle puncture oocyte retrieval procedure relatively easy and comfortable. Once adequate sedation has been administered, a speculum is used to expose and clean the vagina and cervix of the patient in dorsal lithotomy position in sterile drapes. Thereafter, a transvaginal ultrasound probe with attached fixed needle guide assesses the position of ovaries in relation to blood vessels, bowel and the uterus. Thereafter an appropriate trajectory path for the follicular aspiration needle through the pelvic is selected on the ultrasound screen.
In the next step, the aspirations system is checked to be operational by sucking a small amount of culture medium through the needle into a culture tube. This tube is then discarded. A negative suction pressure of 100 mm Hg is adjusted with the aspiration pump. The needle is then inserted in the vaginal probe guide and the largest ovarian follicle is entered. Using the foot pedal, 100 mm Hg negative suction pressure is applied to the tubing equipped with a test tube trap mechanism and the collapse of the follicle is visualized on the ultrasound screen. The follicular fluid containing oocyte is aspirated into a round bottom test tube containing one ml modified Human Tubal Fluid (HTF). In most cases it is not necessary to withdraw the needle from the ovary but rather to change its position within the ovary to go from one follicle to the next. A continuous negative pressure should be maintained throughout the oocyte aspiralum procedure. After aspiration of all oocytes, the pressure is released and the needle is removed from the ovary and the vagina. This entire process is then repeated on the contralateral ovary. Normally, oocyte retrieval procedure on both ovaries can be completed in about 30 minutes time. Immediately after oocyte retrieval procedure, the vaginal wall puncture sites are visually inspected for hemostasis. If needed, pressure is applied or a suture placed at the bleeding site.

Oocyte With Embryologist
Surgically collected oocytes (collected as aspirated follicular fluid or follicular flushing) in a culture tube in the operation theatre is handed over to the embryologist in the adjacent laboratory which are immediately transferred into a large petri dish and examined under low power magnification zoom stereomicroscope for the presence of oocytes along with surrounding granulosa cells. Based on the appearance of oocyte cumulus mass (OCM) and the granulosa cells and the presence or absence of germinal vesicle and first polar body. The oocyte maturity is classified as shown in Table no.2 below :-

TABLE NO. 2
CLASSIFICATION OF OOCYTE MATURITY

Mature Oocyte Expanded cumulus and corona layer. Presence of first polar body. Germinal vesicle absent. Immature oocyte Compact corona cumulus composed of only a few cell layers. Germinal vesicles present. Atretic oocytes Sparse unexpanded cumulus with a dark and irregularly shaped ooplasm.

Thereafter the embryologist removes the good quality mature oocytes from the follicular fluid using a Pasteur pipette and puts it in buffered culture media vessel or microdrops into the incubator equilibrated with 5% CO2 until the time of insemination some 3 to 6 hours later.

Semen Preparation
Two sperm preparation techniques, that is, swim up and density gradient centrifugation method are currently used in IVF procedure. The goal is to remove both the immotile and morphologically abnormal spermatozoa and to retain as many motile spermatozoa as possible. A brief description of swim up method is given below :

• The semen sample is allowed to liquefied at 37 degree C for 10 – 20 minutes.
• An aliquots of 1ml of semen are placed in 5 ml labeled Falcon tubes and gently overlaid with 2 ml of HTF medium.
• The tubes are incubated at 37 degree C for 45-60 minutes to allow progressively motile sperm to swim into the overlaid medium.
• The medium (up to 90 % of the supernatant) is aspirated and placed into a labeled collection tube and centrifuged at 300-400 g for 10 minutes.
• The supernatant is removed and the pellet is resuspended in 0.3 – 1.0 ml of fresh medium.
• To calculate the insemination volume for IVF, sperm count, motility, and velocity are assessed on a sample in the counting chamber of a standard haemocytometer.
• The sample is allowed to settle (>3 minutes) and motile sperm in a minimum of five squares from the central 25 squares are counted to give a rough estimate of the motile concentration.
• The insemination volume is adjusted to give a total of 1,00,000to 2,00,000 sperm / ml in the medium containing the oocytes.

Insemination Of Oocytes
Approximately 4 – 6 hours after oocyte retrieval, place the sperm sample and the oocytes in the Isolette. Verify the identity of the gametes and record on the patient’s verification form. Have a second biologist verify the gametes prior to insemination to ensure proper identification. Resuspend the spermatozoa, as some settling oocurs while sitting in the incubator. Add the predetermined amount of spermatozoa to each oocyte and observe the culture drops microscopically to confirm the approximate number of spermatozoa per oocyte. Return the oocytes to the incubator until the fertilization check.

Evaluation Of Fertilization
Oocytes are examined between 16 to 20 hours after insemination by sperm to see whether fertilization has occurred or not. The presence of two pronuclei and two polar bodies is indicative of normal fertilization. Once the presence of normal fertilization is confirmed, the fertilized oocytes are returned to the CO2 incubator in a fresh culture medium for another 24 hours.

Selection Of Best Embryos
The oocyte is well equipped with most of the developmental materials in the form of proteins, mRNA etc to take it to 4 to 8 cell stage after fertilization. Thereafter human embryo genome activates to take care of its growing needs. The quality of oocytes is therefore of paramount importance to remain viable and initiate development immediately after fertilization. Many clinics therefore have focussed attention to predict the embryo quality from pronuclear stage. Various parameters have been  employed to identify good quality zygotes and embryos. Zygotes resulting in maximum implantation success rate firstly have nearly equal number of nucleolar precursor bodies (NPB) in both male and female pronuclei and never differed by more than 3 in number. Second, the NPB in both the pronuclei are either polarized or non-polarized but never polarized in one and non-polarized in the other. Embryos showing these morphological criteria at the pronuclear stage when transferred produced pregnancy rates of 50% as compared to pregnancy rates of 9% with embryos not adhering to their criteria. More recently a multiple step scoring system is in use to identify good quality embryos and the following plan could be followed :-

18-19 hours after insemination

• The pronuclei are examined for
  • Symmetry
  • The presence of even number of NPB
  • The positioning of the polar bodies

25-26 hours after insemination

• Embryos that have  already cleaved to the 2 cell stage
• Zygotes that have progressed to nuclear membrane breakdown

42-44 hours after insemination

• Number of blastomeres should be greater than or equal to four
• Fragmentation of blastomeres should be less than 20%
• No multinucleated blastomeres

66-68 hours after insemination

• Number of blastomeres should be greater than or equal to eight
• Fragmentation of blastomeres should be less than 20%
• No multinucleated blastomeres.

Embryo Transfer
Embryo transfer (ET) is normally performed two days after oocyte pick-up or 44 to 48 hours after insemination. Normally, ET procedure is performed without anesthesia. The patient is placed in a slight Trendelenbarg position. After visualization of the cervix with a speculum, the interior portion of the cervix is clamped with a forsal and a catheter is introduced into the cervical canal. Once, the patient is properly prepared for transfer, the embryologist is asked to load 2-3 embryos into the transfer catheter in about 20 ul volume. This fine catheter is then inserted through the previously position broader cathether into the uterine cavity up-to about 6 cm depth. The embryos are released into the uterus by gental pressure on the plunger. It is important to ensure that the embryos are located near the tip of the catheter to minimize the volume of medium injected with the embryos. After the catheter has been withdrawn, the embryologist examines it to ensure that no embryos have been retained.                                    

Luteal Phase Support
As compared to natural cycles, the decline of serum E-2 and P hormone concentrations are more abrupt in ART cycle and needs luteal phase support. Surveillance of the luteal phase may be limited to a serum -hCG measurement two weeks after transfer. Some programs favour a more extensive protocol for luteal monitoring with serial determinations of estradiol, progesterone, and -hCG levels. Luteal phase hormonal support is considered to be beneficial following adjunctive GnRH agonist therapy as the incidence of histological luteal phase defect is increased in IVF cycles. Most  infertility clinics routinely give exogenous luteal support to patients undergoing ART procedures. Progesterone and hCG are most commonly used, although the latter increases the risk of ovarian hyper stimulation syndrome (OHSS) in some patients. More recently micronized P is preferred as luteal support agent due to its improved absorption and can be given through oral or vaginal routes. The latter route however, offers several advantages over the oral route and is thus preferred due to following reasons.

• It is convenient and acceptable to patients.
• It does not hurt or require any special equipment or training to administer.
• It rarely produces allergic reactions.

Intravaginal progesterone pessaries are also advised during the luteal phase of stimulated cycles after IVF-ET procedure to overcome the undesirable ill effects of excessive estrogen action on the uterine endometrium. Progesterone also increases endometrial receptivity and thus improve the implantation rate of the embryos.

Complications
Although IVF is a safe procedure, some complication related to the stimulation, the procedure and the outcome of the treatment are not uncommon. Some of the complications are given in the table no.3 given below :

TABLE NO.3
COMPLICATIONS OF IVF-ET TREATMENT

 

Stimulated related complications Ovarian hyper stimulation syndrome (OHSS). Procedure related complications  Anesthesia reactions (nausea, vomiting, adverse drug reactions, airway obstruction, airway injury & aspiration  pneumonia. Tromatic organ injury (bowel perforation, blood vessel laceration & ovarian hemorrhage. Post procedure complications (ovarian abscess, pyosalpinges, endomyometritis, ovarian tortion, hydrosalpinges & pelvic hematome. Outcome related complications Multiple gestations Spontaneous abortion Premature labour Preeclampsia Ectopic gestation

Clinical Focus

• In order to maximize the chances of a successful pregnancy through IVF, ovaries are stimulated using gonadotropins hormones to produce multiple eggs for fertilization.
• Transvaginal ultrasound guided oocytes retrieval is nearly an outpatient procedures which can normally be performed without intensive anesthetics.
• Proper oocyte scoring for identification and selection of mature oocytes significant enhances fertilization and pregnancy rates.
• Exposure of sperm, oocytes and embryos for temperature, pH and CO2 concentration and changes causes irrepareable damage and needs to be strictly monitored for providing optimal conditions. 
• The embryology laboratory is a key component of any IVF programme where vital steps of oocyte culture are undertaken, so the laboratory must be appropriately located, sized and equipped.

Conclusion
The evolution of IVF has been rapid and significant. Once available to only a few couples around the world, IVF is now a viable therapeutic option for thousands of couples. No longer considered experimental, IVF has become a routine part of infertility treatment protocols. As advances in technology continue to simplify IVF procedures, IVF is rapidly becoming a routine part of infertility treatment in the office. Hormonal stimulation protocols are designed to mimic the natural events that lead to production of many oocytes. These oocytes are aspirated from the ovarian follicles few hours prior to ovulation and fertilized by sperm cells in the laboratory. Success rates continue to improve, and costs continue to decrease.

 

Male Infertility:- Total Motile Sperm Count (TMSC) between 1-10 million if duration of infertility is of >2years. If >10million treat as unexplained infertility. Tubal Pathology Bilateral tubal block but tubal surgery is not a practical option. Tubal function impaired but no occlusion present. Infertility duration of 2 years after tubal surgery. Unexpalined Infertility Idiopathic infertility for more than 3 years. Hormonal disturbances Failed treatment of anovulatory women for 12 cycles with ovulation induction. Cervical/Immunological factor infertility Infertility duration cervical/ immunological factors for more than 2 years. Endometriosis Mild and moderate endometriosis is considered as unexplained infertility. Severe endometriosis is considered as tubal pathology.