Semen Analysis | Hysterosalpingogram | Laparoscopy | Postcoital Test |
Transvaginal Ultrasonography | Artificial Insemination | Fertility Drug Stimulation |
Antisperm Antibody Determinations | Hormonal Evaluation | IVF | Embryo Transfer | ICSI


Semen Analysis
A semen analysis is performed on a freshly collected specimen following a requested three-day continence period. The numerical evaluation includes volume, pH, motility and morphology. Except for specimens with extremely low sperm counts and compromised parameters, it is unusual for just seminal fluid to be responsible for infertility. With the availability of highly sophisticated in vitro techniques, even a severely oligospermic specimen can be successfully managed.

Improving the sperm count by using fertility drugs is a notoriously frustrating experience. Rather than using Clomiphene, Pergonal or HCG for a lengthy period of time we turn to the IVF procedure. In selected cases, varicocele repair may improve semen quality. The single, most effective therapy for improving semen quality, in our experience, is antibiotic therapy.
Semen Analysis Normal Ranges (WHO Criteria, 1992)

Hysterosalpingogram
The structural evaluation of the female pelvis starts with a Hysterosalpingogram. Between day 5 and day 10 of the cycle, just prior to ovulation, dye is injected through the cervical canal and the uterine cavity and the fallopian tubes are photographed as they fill with the dye. There is no immediate need for a laparoscopy with completely normal findings. If any adhesions, tubal blockage, intrauterine pathology with congenital abnormalities are documented, we proceed to laparoscopy.

Laparoscopy
Laparoscopy is performed at The K.J Hospital under general anesthesia. The abdominal cavity is inflated with carbon dioxide and a fiberoptic instrument is placed through the navel and through entry sites in the lower quadrants. Small instruments are introduced into the abdominal cavity to accomplish surgical repair of pelvic pathology.


Postcoital Test
This test is performed during the time of ovulation. An ovulation predictor kit is used to predict the exact timing. When the color turns, the couple is instructed to have intercourse that night and the female partner is asked to come to the office the next morning. The test results are considered good if, after twelve hours, highly active sperm are present in a clear, cervical mucous. We do not perform the postcoital test one or two hours after intercourse since conditions, such as anti-sperm antibodies or bacterial contamination in the cervical mucous allow sperm to survive for a few hours, thus giving a falsely favorable reading.

Transvaginal Ultrasonography
The routine use of vaginal ultrasound has practically replaced bi-manual examination of the female pelvis. Uterine and ovarian pathology and functional changes in the ovaries and in the uterine lining can be carefully assessed with vaginal ultrasound. Vaginal ultrasound is part of the monitoring protocol during stimulation with fertility drugs, including Clomid, Metrodin, Pergonal and HCG. Prior to intrauterine insemination or postcoital testing, a vaginal sonogram is performed to document the presence of the ovulatory follicle. Vaginal sonography is part of the routine GYN examination and essential in the serial follow-up visits for benign ovarian cysts. Sonographic measurement of the uterine lining during ovulation makes endometrial biopsy for staging obsolete, since the lining measurement of 10mm or more excludes a luteal phase defect.


Artificial Insemination
Through this procedure, washed or unwashed spermatozoa are injected either in the cervical canal or into the uterine cavity. A clear cut indication for the procedure is sexual dysfunction when spontaneous intercourse fails to deposit the sperm. There is much uncertainty about the actual benefit of artificial insemination with oligospermia or when it is coupled with fertility drug stimulation. To overcome a poor cervical factor, using artificial insemination is legitimate only if all other efforts have been exhausted to eradicate a cervical infection that prevents spontaneous sperm migration. We do not see any rationale behind performing artificial insemination if a postcoital test is favorable. In a case of primary infertility, when a poor cervical factor is the only documented obstacle to achieve a pregnancy, there is always a danger of creating a secondary infertility condition if an infectious cervical factor is overlooked. The infection can be introduced into the previously clear uterine cavity by the procedure. Even though a single pregnancy may be achieved, subsequent pregnancies fail to occur due to this contamination process.

Fertility Drug Stimulation
In clear-cut cases of ovulatory disorders, with associated polycystic ovarian disease, there is a definite place for Clomiphene, Pergonal, Metrodin and HCG stimulation. When all other fertility procedures are exhausted and patients must turn to an IVF procedure, fertility drugs are the only option. It is by far less certain, however, when to resort to fertility drugs in an apparently normally functioning female. Due to published studies, implicating fertility drugs in association with increased ovarian cancer risks, we disagree with the commonly practiced approach of prescribing Clominphene and subsequently, Pergonal and Metrodin therapies, following an incomplete fertility evaluation. Clomiphene in low doses has relatively few side-effects and there is only a remote chance that ovarian cysts will form. Pergonal, HCG and Metrodin, however, need close monitoring with serial vaginal ultrasound examinations and with blood estradiol determinations.


Antisperm Antibody Determinations
A great uncertainty exists about the exact significance of antisperm antibodies either in the male or in the female genital canal. Unquestionably, very high concentrations interfere with sperm migration or with the fertilization process. These cases are clear candidates for IVF procedures. With lower antibody concentrations, however, we try to exhaust all other remedies before resorting to IVF.



Hormone Evaluation
Day-2 or day-3 determination of estradiol, FSH and LH values are probably the most important predicators of good ovarian function for any cycle. Elevated levels of the pituitary hormones, FSH and LH with variable estrogen levels, suggest resistant ovaries. If genital-tract infections are documented through culture studies, especially Chlamydia trachomatis, there is an excellent chance that the levels will normalize after adequate antibiotic therapy. When Chlamydia trachomatis infects the ovaries, causing an amenorrheic state, the condition can be reversed with antibiotic therapy. When the sonogram shows noraml uterine lining, we find serum estradiol and progesterone determination during the luteal phase unneccesary. To complete the workup, serum prolactin, thyroid hormone and adrenal hormone determination are performed.


IVF [In Vitro Fertilization]
CRAFT offers either non-stimulated, natural cycle IVF procedures or Pergonal/Metrodin/HCG stimulated IVF cycles. With a natural cycle, IVF, a single egg is monitored during the woman’s normal cycle and when ovulation is imminent, using vaginal ultrasound, the egg is retrieved. The fertilization occurs in the laboratory and two to three days later, the embryo is transferred into the uterine cavity. The advantage of this procedure is that no drugs are introduced and there is essentially no limit to how many times the patient can repeat the procedure. Working with one egg, however, gives a considerably smaller success rate when compared to stimulated IVF cycles.
The stimulated IVF cycles use a combination of Lupron, Metrodin, Pergonal, and HCG to stimulate several eggs during the first part of the menstrual cycle, which are subsequently retrieved through vaginal aspiration. The eggs are matured in the laboratory and once embryos form, the best three or four are replaced two to three days after retrieval. The disadvantages of this method include the expense, the potential side effects of the powerful drugs used and the anesthesia needed for the retrieval. Due to the availability of multiple eggs, however, the pregnancy rate is much improved compared to natural cycle IVF. At CRAFT, assisted reproductive technology is offered only if other remedies are exhausted in reversing the infertility condition.

Special IVF Techniques
The standard IVF procedure may not prove successful in producing a pregnancy for some couples. In those cases, the physician may determine that special IVF techniques should be employed.

ICSI (Intracytoplasmic Sperm Injection)
1. Indications for ICSI
2. Technique of ICSI
3. Results of ICSI
4. Factors Affecting Results of ICSI
5. Risk of ICSI
6. Birth Defects After Assisted Reproduction
7. Associated Procedures
8. Summary

If the male's sperm count is low, or exhibits poor motility (ability to move about), fertilization may fail to occur with standard IVF techniques. For these patients, and for patients who have poor or absent fertilization In Vitro, Intracytoplasmic Sperm Injection (ICSI) is a significant breakthrough. In ICSI, a single sperm is picked up in a microscopic needle and injected directly into the center of the egg. Since ICSI can be employed even when the semen contains only a very few motile sperm, ICSI may offer an otherwise infertile male a very real chance of becoming a biological parent.

In some cases, in men who ejaculate no sperm at all, sperm cells can be directly removed from the testicles to be used via ICSI to fertilize the wife's eggs. With the advent of ICSI, many couples now have the possibly of achieving pregnancy without resorting to the use of donor semen (AID).

Assisted Hatching
Certain patients produce embryos which, although they appear to be viable, are unable to hatch out of the zona pellucida (outer covering) to implant in the uterus. This seems to be especially true of women over 38 years of age. These patients may benefit from Assisted Hatching, in which the zona is artificially breached by creating a small opening. This is performed after fertilization occurs, but prior to the embryo transfer. Assisted Hatching may be employed in patients who have failed to achieve pregnancy despite being able to produce good embryos.

Indications for ICSI
Until recently, the clinical application of direct injection of a single sperm into the cytoplasm of an oocyte during IVF was not shown to be feasible. The demonstration of fertilization and live births by Palermo et al. in 1993 was the first successful application of ICSI. Since that time, ICSI has been performed extensively in multiple centers to treat patients with severe male factor infertility. To date, the success of ICSI procedures has been related to several factors: (1) the viability of the spermatozoon, (2) the quality of the oocyte, (3) effective activation of the oocyte, and (4) ability of the oocyte to tolerate intracytoplasmic manipulation. Application of this treatment is described below. To date, rigorous indications for ICSI have not yet been defined. Most clinical series report on using ICSI in cases where standard IVF is highly unlikely to succeed, that is, in patients with less than 500,000 motile sperm present in the ejaculate, or less than 4% normal forms with strict criteria evaluation. In addition, couples who have failed to fertilize any oocytes in a prior IVF cycle are considered appropriate candidates for IVF-ICSI. We have proposed the following indications for ICSI:

a) sperm concentration < 2 x 106
b) sperm motility < 5 %
c) strict criteria normal morphology < 4 %
d) use of surgically retrieved spermatozoa
e) failure of fertilization in a previous IVF cycle

Although fertilization and pregnancy rates with ICSI are similar or better than those achieved with normal sperm in other couples undergoing IVF concurrently at the same center, couples with only minor semen abnormalities have not been routinely treated with IVF-ICSI. Given the relatively brief history of ICSI, and its potential effects on progeny, it would seem prudent to avoid over-application of this new technology. Therefore, ICSI should not be recommended to couples for whom there is no documented benefit, since unknown risk may exist.


Technique of ICSI
1. Oocyte processing: Oocytes are prepared by removing the cumulus mass and corona radiata with hyaluronidase. The oocytes are then examined under the inverted microscope to assess the maturation stage by observing the presence of a germinal vesicle, germinal vesicle breakdown, and the extruded first polar body. Metaphase II oocytes are identified by the presence of the extruded first polar body. Intracytoplasmic sperm injection is performed on all metaphase II oocytes. Metaphase II oocytes have their diploid complement of chromosomes delicately arranged on the metaphase plate near the polar body. Mechanical disruption of the metaphase plate can occur by injury from the injection pipette or the presence of a motile sperm in the oocyte cytoplasm. Each oocyte is placed in a droplet of medium surrounding the central droplet which contains the spermatozoa. The droplets are covered with lightweight paraffin oil and the petri dish is placed on a heated stage of the microscope. The microscope is equipped with two hydraulic micromanipulators which are fitted to two tool holders for the micropipettes. During the injection procedure oocytes are stabilized with a holding micropipette, and injected with an injection pipette.

2. Microinjection: Details of the preparation of microtools and protocols for ICSI are described in detail elsewhere. The holding and injection pipettes are made by drawing glass capillary tubes with a pipette puller and further processed on microgrinder and microforges. The outer and inner diameters of the holding and injection pipettes are, respectively, 60 and 20 µm, and 7 and 5 µm. The injection pipette has a bevel angle of 50º and a sharp spike to assist penetration through the oolemma. Washed sperm are prepared on a discontinuous mini-Percoll gradient. The sperm h-action is washed with T6 medium containing 5 mM CaCl2 just prior to the injection procedure. The sperm pellet is resuspended and transferred with a polyvinylpyrrolidone solution in HEPESbuffered Earle's medium. From a 3-5 µl sperm-PVP droplet covered with lightweight paraffm oil, a single sperm is aspirated into an injection micropipette. A metaphase II oocyte is immobilized with slight negative pressure on the holding pipette. The polar body is held at the 12 or 6 o'clock position and the injection micropipette containing the single sperm is pushed through the zona pellucida and oolemma into the cytoplasm of the oocyte at the 3 o'clock position. A single sperm is injected head first into the ooplasm with 1-2 pl of medium. The injection pipette is withdrawn gently and the oocyte is released from the holding pipette. Further handling of injected oocytes is similar to that for oocytes in standard IVF.

Embryo Transfer
Timing

Embryos are generally transferred back to the woman's uterus at the 2 - 8 cell stage, which occurs 48 - 72 hours after the retrieval.

The Procedure
When she arrives for the embryo transfer, the patient (and her partner) will return to the Procedure Room. The patient is required to have a mildly full bladder so that her uterus can be visualized by abdominal sonogram during the procedure. She will undress, don an examining smock, assume the usual examining position, and be placed under abdominal sonographic guidance. The physician will then insert a speculum into the vagina and clean the cervix. The patient may feel one cramp as an outer catheter is placed through the cervix into the lower segment of the uterus. A fine plastic catheter, into which the Embryologist has transferred the embryos, is then placed through the outer transfer catheter and advanced near the top of the uterus. The sonographer will visualize the lining of the uterus and guide the physician in the placement of the catheter. Once the placement is correct, the embryos will be expelled from the catheter and inserted into the uterus.

The Rest Period
After the transfer, the patient will be wheeled to the recovery area where she will rest on her back for two hours. Her husband is welcome to remain with her for the entire rest period.

Post-Transfer Medications
The patient may receive an injection of HCG after the embryo transfer to help the ovaries produce more progesterone during the embryonic implantation phase. In some cases, she will also be instructed to self-administer two additional HCG injections on specific days following the transfer. She may also be asked to use progesterone suppositories for a specified number of days following the transfer.

Post Transfer Instructions
The patient will be advised to be restful during the first 24 hours after the embryo transfer and to engage in only limited activity during the second 24 hours. She may then carry out her normal level of exercise and activity. It should be noted that physical activity or diet has no impact upon embryo implantation or conception. Once the embryos are transferred, there is really nothing a patient can do to influence the outcome of her cycle. Conception is a natural phenomenon which depends mostly upon the genetic quality of the eggs. Almost without exception "nature" will only allow genetically perfect embryos to survive in order to maximize the chances of the birth of a normal baby.

The outcome of any particular IVF cycle is determined by the quality of the embryos and the post-transfer hormonal support. Good embryos cannot be lost as a result of moving about, and there is no scientific evidence to suggest that any particular activity will cause a woman to lose a pregnancy during the implantation stage. Therefore, we believe that, after the initial 48-hour post-transfer period, the patient should go about her regular daily activities without worrying that she will harm her chances for a successful pregnancy.

During the two weeks after the embryo transfer while a couple is anxiously awaiting the outcome of their IVF cycle, the time will pass more slowly and stressfully if a woman confines herself to bed. Stress can actually be reduced by staying active and being productive. Moderate exercise may also be a useful aid in reducing stress during the two week post-transfer period.

Number of Embryos Transferred
The number of embryos that should be transferred during any single IVF cycle is open to debate. It has been said in the medical literature that transferring no more than four embryos per IVF cycle will yield optimal results. Transferring more than four is believed to result in excess numbers of multiple pregnancies. Experience shows, however, that the chance of a successful IVF outcome may be increased if more embryos are transferred, especially in older patients with unexplained infertility. It is now believed that the risk associated with multiple pregnancies can be safely reduced by eliminating excess embryos via an embryo reduction during the first trimester. This procedure is successful 90% of the time and results in a total miscarriage in only 10% of cases.

At Craft we believe in individualizing the number of embryos transferred. We base our decision on the quality of the embryos as seen under the microscope prior to transfer and on specific patient requests. Embryos that are not transferred may be frozen for use in subsequent cycles.

Results of ICSI
One of the largest series reporting results using IVF/ICSI was from Van Steirteghem et al. at The Brussels Free University in Brussels, Belgium. In their preliminary report on 150 couples who underwent 150 consecutive treatment cycles, 1409 oocytes were injected and 830 were successfully fertilized for a fertilization rate of 59 percent. A total clinical pregnancy rate of 35 percent was achieved. The fertilization rate in this study was not influenced by the standard semen characteristics of concentration, motility, and strict criteria morphology. In another largest case serie on ICSI in the United States, Palermo et al. at Cornell reported successful fertilization in 1,142/1,923 (59 %) metaphase II oocytes injected, and ongoing pregnancies in 84/227 (37%) couples. Neither semen quality nor the source of sperm (ejaculated, surgically retrieved or electroejaculated) affected fertilization rates. They concluded that IVF/ICSI offers fertilization and pregnancy rates comparable to that achieved with normal sperm quality for couples who have failed to achieve fertilization on repeated IVF cycles or have severe impairments in semen quality. In addition, the success of IVF/ICSI was independent of standard semen parameters (density, motility, and morphology).

Factors Affecting Results Of ICSI
1. Spermatozoal factors: Nagy et al. evaluated the effect of spermatozoal factors on results of ICSI in 966 microinjection cycles. Despite no normal forms in a semen preparation, virtual azoospermia or essentially no motile sperm in the ejaculate, pregnancy could still be achieved. Nagy et al. found that the only absolute criterion for successful ICSI is the presence of at least one viable spermatozoon to inject per oocyte in the prepared pellet of the washed semen sample. The only category in which semen parameters had a significantly impaired fertilization and pregnancy rate was when there was no motility of sperm'o. If no motility is present, then viability is often impaired as well.

2. Female factors: Oehninger et al. investigated the role of matemal factors in a total of 92 couples, where 1163 oocytes were injected with an overall fertilization rate of 61 percent. Fertilization rates were unaffected by matemal age, but pregnancy rates were significantly lower with increased matemal age. Pregnancy rates were 49, 23 and 6 percent for couples in whom matemal age was <34, 35-39, and < 40 years. Similar results were found by Sherins et al., with a 30% pregnancy rate for the youngest couples and 13 % pregnancy rate for the couples with the oldest female partners. The rate of aneuploidy increases dramatically for embryos derived from the oocytes of women over 40 compared to those from women less than age 35. Therefore, it is likely that the chance of fertilization is unrelated to female factors, but that the chance of pregnancy occurring after ICSI is related primarily to oocyte factors, if a viable sperm is injected.

3. Oocyte activation: Since oocyte activation normally occurs in association with sperm binding, fusion and penetration of the oocyte, oocyte activation during intracytoplasmic may not necessarily occur. The importance of intentional induction of oocyte activation was demonstrated by Tesarik and Sousa who increased fertilization and pregnancy rates during ICSI with aggressive aspiration and injection of the oocyte cytoplasm. Direct comparison of gentle and vigorous cytoplasm aspiration resulted in an increase in fertilization rates per oocyte from 38% to 80% with increased pregnancy rates up to 52 % with aggressive aspiration/injection. Evaluation of calcium fluxes in oocytes during injections demonstrated an additional peak of intracellular calcium levels for aggressive aspiration, when compared with gentle aspiration. Intracellular calcium changes have long been thought to have a role in oocyte activation. An additional sperm factor may have a role in cytoplasmic activation. Aggressive immobilization of spermatozoa has been used to effect increased sperm membrane permeability. Gerris et al. evaluated the effects of sperm tail breakage on ICSI success by directly comparing fertilization rates achieved using sperm with intact tails compared to sperm with damaged tails. Aggressive immobilization of spermatozoa resulted in an increase in the percentage of normally fertilized oocytes from 36 to 60%. These authors and others" suggested that tail damage induces sperm membrane changes which facilitate biochemical events necessary for sperm nuclear decondensation and pronuclear formation. Palermo et al. have also investigated the effect of aggressive sperm immobilization on fertilization and pregnancy rates. Although there was little improvement in fertilization rates for ejaculated sperm, a dramatic improvement in epididymally-retrieved sperm fertilization rates was seen, from 51 to 84% per oocyte, with an associated increase in pregnancy rates from 5 1 to 82 %. The biochemical basis for the effect of increased sperm membrane permeability to improve fertilization rates is unclear. It is possible that increased permeability of the manipulated sperm resulted in better penetration of ooplasmic factors into the spermatozoon to induce male pronuclear formation. Alternatively, it is possible that increased permeability results in enhanced leakage of toxic factors out of the cytoplasmic droplet of immature epididymal spermatozoa.


4. Cytoplasmic Injection/oocyte injury: Disruption of the oocyte sufficient to cause oocyte demise may occur during ICSI. Results from some of the major centers performing ICSI show rates of oocyte loss after injection of 7 to 14 percent. Although the precise reasons for oocyte injury are not known, it is though to occur as a result of plasma membrane and ultrastructural disturbances associated with injection, damage to the meiotic spindle during injection, and/or extrusion of the oocyte cytoplasm following injection. In addition, other factors such as changes in temperature have been reported to cause irreversible changes in the meiotic spindle of the human oocyte. Clearly, there is a learning curve for embryologists performing the ICSI procedure. As greater expertise is gained, the oocyte injury rate decreases. Palemo et al. have recently described oocyte characteristics that may predispose to oocyte injury. These investigators described an oocyte membrane response of "sudden breakage" during attempted ICSI. The oocytes with this response did not form a normal oocyte membrane funnel around the injection pipette. Instead, the oocyte membrane separated, spilling the oocyte cytoplasm and resulting in a 14 % injury rate, compared to a 4 % injury rate for other oocytes. Oocytes demonstrating sudden breakage were more likely to be retrieved from women who received higher gonadotropin treatment doses, with lower serum estradiol levels at retrieval, yielding immature oocytes, including those requiring maturation in vitro. These observations suggest that ovarian stimulation characteristics may affect the ability of oocytes to successfully undergo ICSI.


Risks Of ICSI
Risks of IVF-ICSI include general risks of IVF as well as the specific risks related to the micromanipulation procedure of ICSI. One of the most significant risks associated with stimulation of the ovaries is the ovarian hyperstimulation syndrome (OHSS). This can manifest as massive ovarian enlargement, peritoneal irritation due to follicular rupture or hemorrhage, ovarian torsion, ascites, pleural effusion, oliguria, electrolyte imbalance, hypercoagulability3l I and sometimes death36 . The syndrome occurs in a moderate form for 3-4% percent of initiated cycles, and in a severe form for 0. 1-0.2 % of the populatioe undergoing controlled ovarian hyperstimulation. Other reported complications of ovarian hyperstimulation are pituitary hemorrhage, endometriotic bloody ascites, and genital cancer. Complications of ovarian retrieval have been reported for transvaginal aspiration as well as laparoscopic aspiration. Complications associated with wmsvaginal aspiration have been reported to occur in 0.3-3 percent of cases and include bleeding, pelvic infections, and abdominal viscera perforation . Laparoscopic complications include hemorrhage, intestinal perforation, infection, and carbon dioxide embolism. The laparoscopic risks are no higher in ovarian retrieval procedures than in other laparoscopic applications.

Finally, pregnancies resulting from ovarian stimulation are at risk for spontaneous abortion, ectopic pregnancy, and multiple gestational. The rate of spontaneous abortion after achieving a biochemical pregnancy with assisted reproduction is approximately 25 percent. These losses are attributed to advanced maternal age and the associated increased prevalence of chromosomal abnormalities, a higher rate of pregnancy loss due to multiple gestations, and early recognition of these pregnancies due to close monitoring. After achieving a clinical pregnancy, the chance of a spontaneous abortion occurring for IVF-ICSI cycles ranges from 10-16%. Fctopic pregnancies occur in up to 3-5.5 % of gestational cycles and can be life threatening. The etiology is usually pelvic adhesions and tubal damage from pelvic inflammatory disease or previous surgery. Multifetal pregnancies occur in 22 percent of cases of IVF with embryo transfer, and 44 to 46 percent of IVF/ICSI cases. Multifetal pregnancies are considered a complication of assisted reproductive techniques because of the associated increased incidence of preeclampsia, placenta previa, placental abruption, premature rupture of membranes, and postpartum hemorrhage. Most importantly, multiple gestations are almost universally associated with prematurity and the associated complications to offspring, including cerebral palsy and intracranial hemorrhage with mental retardation or blindness. To prevent multifetal pregnancies and their attendant complications, it would be preferable to avoid assisted reproduction unless it is specifically indicated, and limit the number of embryos transferred. Where there is government regulation of IVF, including England, Australia and France, transfer of only 3 embryos is allowed and multifetal pregnancies are less commoe. Unfortunately, there is significant pressure to transfer more than three embryos by couples in the United States who are desperate to conceive. In general, for women less than 35 years of age, only 3 embryos should be transferred.

Birth Defects After Assisted Reproduction
The bypass of natural barriers to fertilization, possible genetic defects in men with severe male infertility, and the use of severely abnormal sperm for intracytoplasmic sperm injection has engendered concern over the impact of ICSI on the genetic complement of the offspring . Previous studies have suggested no increase in birth defect rates when IV-F alone was used to induce conception. Van Steirteghem reported no increase in the congenital malformation rate in their center after ICSI when compared with the general population. Of 877 children born after ICSI procedures, 23 (2.6 percent) had major congenital malformations compared to 2.0 to 2.8 percent in the general population and 1.9 to 2.9 percent of children resulting from assisted reproductive techniques.

Sex chromosomal abnormalities have also been reported in ICSI cases. In't Veld et al. reported on 12 patients with ICSI pregnancies who underwent prenatal diagnosis for advanced matemal age. Three of the 12 women had twin pregnancies for a total of 15 diagnostic procedures by amniocentesis or chorionic villus sampling. A total of five chromosomal abnormalities were detected: two cases of XXY, one complex mosaic 45,X/46,X.dic(Y)(q11)/46.X.del(Y)(qll), and two cases of 45 XO. This high rate of sex chromosome abnormalities has not been corroborated by other studies. The Brussels group reported on a total of 585 prenatal diagnoses performed in pregnancies established by ICSI. A total of six sex chromosome abnormalities (1.0 percent) were detected compared to 0.2 percent in the general population. This difference did not achieve statistical significance. Govaerts et al. reported on 55 karyotypes obtained by amniocentesis or chorionic villus sampling in pregnancies from ICSI and found no sex chromosome abnormalities. When sex chromosome abnormalities have been identified it is unclear whether they are related to the ICSI procedure itself or can be ascribed to advanced matemal age. What is reassuring is that the rates of nonsex chromosomal abnormalities in the ICSI population published to date do not exceed the rates seen in the general population. The relationship of ICSI to sex chromosomal abnormalities in offspring may be related to the association between Y chromosomal abnormalities and severe male factor infertility. Several investigators have reported that up to- 13% of men with azoospermia or severe oligospermia may have deletions of 15,000 to 200,000 base pair lengths of Y chromosome. At least one gene (DAZ; deleted in azoospermia) is deleted in 13 percent of patients with non-obstructive azoospermia.

Although chromosomal abnormality rates in offspring after these procedures have not exceeded those in the general population, experience with these techniques is brief. Genetic counseling, preimplantation genetic diagnosis, and state of the art prenatal diagnosis must also be available to couples enrolled in assisted reproductive programs. Genetic counseling should be available to all couples. All couples undergoing micromanipulation procedures are strongly urged.to have prenatal diagnosis with amniocentesis or chorionic villus sampling. The need for prenatal diagnosis is dependent on whether the couple would consider terminating the pregnancy if the results are abnormal. If the couple would carry a pregnancy to term regardless of the results of prenatal diagnosis, then the procedure of prenatal intervention would carry risks to the fetus without benefit and therefore cannot be required.

Associated Procedures
In certain cases, manipulation of the embryo can enhance implantation and subsequent pregnancy. Common practice is to use acidified Tyrode's solution (pH 2.35) to thin the zona pellucida. An increase in implantation from 18 % to 25 % is achievable for oocytes with poor prognosis with this intervention, refeffed to as assisted hatching. Assisted hatching is not applicable only for male factor infertility, in fact, it is a treatment for specific defects in embryo development or oocyte abnormalities. However, it is a micromanipulation technique that is routinely performed during IVF at many centers.

The application of micromanipulation techniques in the IVF laboratory has allowed the development of analysis and selection of embryos with specific genetic, chromosomal or biochemical characteristics prior to transfer of those embryos. An embryo at the four or eight cell stage is isolated and an individual cell is extracted for evaluation. Chromosome-specific sequences can be identified using fluorescent hybridization probes or, alternatively, polymerase chain reaction (PCR) amplification of individual alleles on the chromosomes themselves may be applied to identify the genotype of the "biopsied" embryo. These techniques can allow identification of embryos at high risk of carrying X-linked diseases such as hemophilia A or von Willebrand's disease. In addition, specific genetic defects such as the homozygous delta-F508 mutation of the CFTR gene, associated with the development of a severe form of cystic fibrosis, can also be identified. These techniques have been applied for couples known to be at high risk of having children with specific genetic diseases. "Biopsied" embryos have been successfully transferred resulting in pregnancies and live births'. These micromanipulation techniques are highly labor intensive and carry some potential pitfalls. For example, if both parents in a couple are heterozygous for the delta-F508 CFTR mutation, then an individual embryo has a one-in-four chance of being homozygous for that gene mutation. PCR has the potential for identifying the homozygous condition since the delta-F508 mutation is a deletion of 3 base pairs in the normal allele. The normal CFTR gene is 3 base pairs longer than the delta-F508 mutated allele, and a heterozygous carrier would be expected to have both alleles detectable with PCR. However, PCR involves magnification of the single signal present on both chromosomal alleles; if one allele is preferentially bound, read and amplified with PCR, then the results of PCR analysis are significantly confounded.

For sex chromosomal analysis, this evaluation is more accurately performed (up to 95.5 % efficiency) using different colored fluorescent probes". A test for both X and Y-specific sequences is possible and provides further confirmation of the results of these tests. Given the extensive manpower needed for single-day biopsy and evaluation of the results of embryo biopsy, this technique is limited to those cases where life threatening genetic defects can be reliably detected prior to embryo transfer to prevent the potential termination of a fetus later in development.

Summary
Since the first U.S. report of a successful delivery from in vitro fertilization in 1983 the advances in the field of assisted reproduction and micromanipulation have been truly dramatic. Perhaps the most exciting advances have been in the area of male factor infertility. Couples who previously would have been offered donor insemination or adoption are now achieving . pregnancies despite severe impairments in semen quality, the presence of only single numbers of sperm in the ejaculate or unreconstructable reproductive tract obstruction. Techniques of micromanipulation that were revolutionary less than five years ago are now obsolete, replaced by even more successful methods. Even non-obstructive azoospermia due to maturation arrest or other impairments in germ cell maturation have been added to the list of treatable factors in male infertility since sperm can frequently be extracted directly from testicular parenchyma that is surgically biopsied. For patients without sperm in the testicular parenchyma, round spermatid or secondary spermatocyte injections are possible.

Several important questions remain with regard to IVF-ICSI:
(1) What should be the specific indications for IVF and IVF-ICSI'? Should IVF alone ever be used for male factor infertility?
(2) What are the reasons for failure to achieve pregnancy after IVF-ICSI that still represent over half of our attempts at achieving ongoing pregnancies?
(3) Can we be certain that using severely impaired or less mature sperm will not result in significant birth defects or in genetic abnormalities that could affect the offspring in adolescence or adulthood and
(4) What is the most cost effective approach for the infertile couple with impaired semen parameters? Contemporary application of IVF-ICSI for severe male factor infertility can allow pregnancy rates up to 52% , with ongoing pregnancy and live delivery rates as high as 37 % per IVF cycle attempt. As long as viable sperm are present in the ejaculate or retrievable from the male reproductive tract, then IVF-ICSI procedures can be applied.


Oocyte Donation
Some women are found to be incapable of producing viable eggs. These patients may elect to undergo IVF using eggs donated by another woman. It is our policy not to share a third persons sperm, egg or embryos to the couples, even if they wish. So we never under take cases of donor sperm or donor egg or donor embryo or surrogacy at any cost. We would rather advise the couple to go for adoption in such situations. An egg donor follows the same protocol for medication and oocyte retrieval as all IVF patients. At the same time, the patient receives medication to cause her hormone levels to approximate those of a natural cycle and to mimic those of the donor. After the retrieval, the donor’s eggs will be fertilized with the husband's sperm; the resulting embryos will then be transferred to the patient's uterus.

Variations of the IVF Technique
There are two variations of the standard IVF technique:
Gamete Intra-Fallopian Transfer (GIFT) and Zygote Intra-Fallopian Transfer (ZIFT). In GIFT, medication is used to stimulate the patient's ovaries, and the resulting eggs are retrieved. However, instead of fertilizing the eggs outside of the patient's body, they are placed directly back into the Fallopian tubes (where fertilization normally occurs) along with the husband's washed sperm. With ZIFT, the eggs are fertilized outside of the patient's body as in IVF; however, instead of transferring the fertilized eggs directly into the uterus they are placed into the Fallopian tubes and allowed to migrate to the uterus on their own.