IVF success rates are dependent upon the number of the mature eggs and “competent” embryos available for transfer. A woman undergoing IVF is given fertility drugs for two reasons: (1) to enhance the growth and development of her ovarian follicles in order to produce as many healthy eggs as possible and (2) to control the timing of ovulation so that the eggs can be surgically retrieved before they are ovulated. In cases where the woman has previously received fertility drugs, the subsequent treatment protocol is largely based upon her response to the most recent such treatment regime. If a woman is receiving gonadotropins for the first time, the dosage and regimen is determined by her cycle day (CD) 3 blood FSH antimullerian hormone (AMH), Inhibin-B concentrations, medical history and body type.

Controlled Ovarian Hyperstimulation (COH)

At SIRM, in most cases, the woman begins her cycle of treatment by taking birth control pills (BCP) and having an ultrasound examination to exclude ovarian cysts. The BCP therapy is continued for 10-30 days before initiating daily injections of a Gonadotropin Releasing Hormone agonist (GnRHa) such as leuprolide acetate (Lupron). Both the BCP and Lupron are administered together for an additional two or three days, whereupon the BCP is stopped. The daily Lupron injections are continued until menstruation begins about 3 to7 days after stopping the BCP. By shortening the duration of time on the BCP it is possible to accurately plan the onset of menstruation. In this way we are able to schedule each cycle of IVF to the convenience of the patient and the medical team. Additionally, the combined use of BCP and Lupron reduces the likelihood of Lupron-induced ovarian cyst formation, thereby largely avoiding the need to delay or cancel the cycle of treatment.

As soon as menstruation begins, blood is drawn and if the plasma estradiol (E2) concentration is less than 70 pg/ml, the patient is ready to initiate ovarian stimulation with gonadotropins. If the E2 level is greater than 70 pg/ml, Lupron therapy is continued at the same (or an increased) dosage for a few more days, whereupon the E2 concentration is re-measured. Subsequent failure of the E2 to fall below 70 pg/ml is an indication for a pelvic ultrasound for the detection of an ovarian cyst, the presence of which usually mandates the performance of an ovarian cyst needle aspiration.
Lupron injections are either continued at a reduced daily dosage or (as is now becoming common practice at SIRM), we stop Lupron and switch to low-dose GnRH antagonist (Ganirelix or Cetrotide). On a designated day (usually within a week and a half of the onset of menstruation), a specified regime of gonadotropins (e.g., Gonal F, Follistim, Bravelle, and Repronex) therapy is initiated. Those patients requiring heparin therapy begin this treatment on the first day of receiving gonadotropins.

In a variation on this theme, patients whom have severely diminished ovarian reserve receive intramuscular Estradiol Valerate (E2V) injections (Delestrogen) and/or vaginal estradiol suppositories for a week or longer, prior to and during gonadotropin therapy.

Those patients who have a history of a poor endometrial lining may be prescribed sildenafil (Viagra) vaginal suppositories and oral terbutaline to attempt to improve the uterine lining.
Patients who have activated natural killer cells (NKa) as measured by a K-562 target cell test receive intralipid (IL) therapy by intravenous infusion 7-14 days prior to embryo transfer.
All IVF patients receive oral corticosteroids (dexamethasone or prednisone) daily, commencing with the start of ovarian stimulation and continuing until the first blood beta-hCG test (i.e., pregnancy test). Women who have rising blood hCG levels (a positive blood pregnancy test) 9-11 days after egg retrieval continue taking corticosteroid and heparin (when applicable) beyond the ultrasound confirmation of pregnancy, which is performed at the 6-7 gestational week.

In cases where the blood pregnancy test fails to reveal an appropriate increase in the quantitative beta hCG concentration, heparin therapy is discontinued, and the corticosteroid dosage is slowly reduced over a few weeks and then stopped. Pregnant women continue corticosteroid as well as heparin treatment until the 8th-10th week of pregnancy, whereupon the heparin is abruptly discontinued and the corticosteroid is tapered off over 1-2 weeks and stopped.

Two days after the initiation of gonadotropin injections, the dosage of gonadotropins is substantially reduced, and is then maintained at this lower level until the administration of HCG. Dosage adjustments are sometimes made during the course of the cycle, based upon the patient’s response to medication.

Commencing seven days after the initiation of gonadotropin therapy, the patient undergoes serial ultrasound and plasma estradiol evaluations to monitor her ovarian response. These assessments are aimed at determining the ideal day for administering 10,000 IU of hCG to trigger the final maturation of the egg(s) and the production of progesterone by the ovaries. Lupron or Ganirelix/Cetrotide and gonadotropin injections are discontinued on this day and the patient is scheduled for egg retrieval approximately 35 hours after receiving the intramuscular injection of hCG. Subcutaneous heparin injections are discontinued approximately 12 hours prior to the egg retrieval and re-started after the embryo transfer procedure.

All patients receive an oral antibiotics beginning about seven days after the initiation of gonadotropin therapy and continuing for a few days after the embryo transfer procedure.

Egg Retrieval (ER)
ER involves a non-surgical procedure where, under direct ultrasound guidance, a needle is passed along the side of a vaginal ultrasound probe through the top of the vagina into follicles (small fluid filled spaces that each contain an egg), within the ovary(ies). The follicular fluid is aspirated and collected in a test tube, which is promptly delivered to the embryologist(s) for analysis and processing. The procedure itself is relatively painless, however patients commonly experience some residual postoperative abdominal discomfort and /or cramping that rarely persists for more than a few hours. Postoperatively, all patients are given detailed instructions and are discharged within an hour or two with a prescription for analgesics (pain killers) and other medications as indicated.

Sperm Processing
A sperm specimen is usually obtained from the male partner through masturbation. On some occasions however, physical, medical and/or religious constraints demand that sperm be obtained through condom collection following intercourse, or by inserting a needle directly into the testicle(s) under local anesthesia and aspirating sperm. The two variations of this procedure are known as Testicular Sperm Extraction (TESE) and Percutaneus Epididymal Sperm Aspiration (PESA). TESE and PESA are procedures of choice in cases where there is blockage of the sperm ducts (as occurs following vasectomy or following severe injury or infection), where the man is born without sperm ducts (congenital absence of the vas deferens), or in cases where as a result of testicular failure, sperm does not reach the ejaculatory ducts. Sometimes, in cases of retrograde ejaculation, sperm can be collected from the man’s bladder. Infrequently, in men with spinal cord injuries, ejaculation is facilitated by electrical stimulation (electro-ejaculation). Donor sperm, obtained from a sperm bank, can be used when indicated.

Sperm must undergo a biochemical and structural changes known as capacitation, before an egg can be fertilized. Capacitation (which under normal circumstances takes place in the woman’s reproductive tract) must be accomplished in the embryology laboratory prior to insemination of the eggs. Motile sperm are processed and activated in specialized culture media and sophisticated techniques are used to enhance poorly mobile sperm.

Fertilization in the Laboratory

Conventional IVF: In vitro fertilization literally means “fertilization in glass”. Fluid aspirated from ovarian follicles is examined in the embryology laboratory. The eggs are identified and extracted, and are placed in a specialized culture medium. Several hours later, approximately 50,000-100,000 processed sperm are placed around each of the eggs. The eggs and sperm are allowed to incubate together in a carefully controlled environment. Approximately 16-24 hours later, the eggs are inspected microscopically for fertilization as evidenced by the presence of two nuclear bodies. These binuclear unicellular bodies are referred to as “pro-nuclear embryos”.

Intracytoplasmic sperm injection (ICSI): The ICSI procedure involves the direct injection of a single sperm into each egg under direct microscopic vision. The successful performance of ICSI requires a high level of technical expertise. ICSI has literally revolutionized IVF. Initially, ICSI was used specifically to achieve fertilization in male infertility. When ICSI is employed in such cases, the IVF birth rate is unaffected by the presence and severity of the male factor. In fact, even when the absence of sperm in the ejaculate requires that ICSI be performed on sperm obtained through Testicular Sperm Extraction (TESE), the birth rate is no different than when IVF is performed for indications other than male infertility. Today, it is commonly used in conventional (non-male factor) cases. At SIRM, we advocate the use of ICSI in virtually all IVF.

Assisted Hatching: In selected cases where it is felt that the zona pellucida (the envelopment of the embryo/blastocyst) is unusually tough or thickened, a process known as assisted hatching (AH) may be employed. The process involves deliberately making a small aperture in the wall of the embryo (usually with a laser) so as to promote hatching (rupturing) and thereby facilitate implantation. It remains controversial as to whether AH actually improves pregnancy rates.

Selecting the Best Embryos for Transfer

Once embryo division (cleavage) has begun, the embryo will continue to divide at regular intervals. (Embryos that divide the fastest are considered the healthiest and the most likely to implant.) Embryos may be transferred 3 days after fertilization when they are divided to the 6-9 cell stage or as expanded blastocysts, 5-6 days post-fertilization when they comprise 100 or more cells with a small collection of fluid inside). Since embryos that fail to become blastocysts are almost always abnormal and incapable of propagating a healthy baby, it is my personal preference to preferentially advocate that blastocysts rather than cleaved embryos be transferred.

1. Graduated embryo scoring (GES) system: The GES system scores each individual embryo out of a maximum of 100 points. Those embryos that are not transferred to the uterus are subsequently re-examined 2-3 days later to determine whether they have developed into blastocysts. Embryos that on day 3 post fertilization have 6-9 cells and have a GES of >70, are the ones that are most likely to develop into blastocysts. Blastocysts are graded on the basis of their cellularity, differentiation of their outer cellular layer (the trophectoderm), the inner core of aggregated cells (the inner cell mass) and the development of a demonstrable collection of fluid inside the blastocyst (an expanded blastocyst) Blastocysts that contain more cells, have a more expanded blastocele, a more prominent inner cell mass and have a well differentiated trophectoderm have the highest grade and are the ones that are most likely to be “competent” (i.e. likely to propagate a viable pregnancy).

2. Early embryo transfer vs. blastocyst transfer: It has long been recognized that the more advanced the embryo’s state and rate of development, the more likely it is to implant successfully into the uterine lining. It is also well established that “poor quality embryos” tend to divide (cleave) and develop more slowly, and are much more likely to arrest before reaching the blastocyst stage. It is therefore not surprising that researchers would focus on trying to grow embryos to the blastocyst stage in order identify “good quality embryos” that are more likely to implant successfully, by their ability to survive to the blastocyst stage of development.

3. CGH analysis of eggs and embryos: This very promising method of selection, currently in use at the Sher Institutes for Reproductive Medicine (SIRM), allows identification of all chromosomes, providing a much more complete analysis than its commonly used alternative – Fluorescence in Situ Hybridization (FISH). CGH performed on the egg/embryo overcomes the inadequacies of previous methods of embryo selection. A recent SIRM study published in Fertility & Sterility (May, 2007) and another soon to appear in the same journal demonstrates a birth rate of more than 60% per chromosomally normal embryo transferred. Now, for the first time, there is a highly reliable method for differentiating between “competent” and “incompetent” embryos.

With the advent of CGH embryo selection, the goal of “one embryo/one baby” is closer than ever to becoming a reality. CGH-based embryo selection also eliminates the current incentive to transfer multiple embryos at a time in order to improve the chance of success. Embryo selection by CGH holds the potential to decrease the cost per IVF baby and lead to a reduction in overall reproductive health care costs.

Embryo Transfer

Undoubtedly, embryo transfer (ET) is a rate limiting step in IVF. It takes confidence, dexterity, skill and a soft touch to do a good transfer. Of all the procedures in ART, this is inarguably the most difficult to teach. It is a true art and we have seen many women fail to conceive simply because this procedure was not performed optimally.

Shortly before the embryo transfer, the embryos/blastocysts are put together in a single laboratory dish containing growth medium. The laboratory staff informs the clinic coordinator that the embryos are ready for transfer, and the coordinator prepares the patient and informs the physician that a transfer is imminent.

Embryos/blastocysts are transferred to the uterus via a thin Teflon catheter. This procedure is conducted under ultrasound guidance with the woman on her back (in the lithotomy position) and with a full bladder.

Today all embryo transfers should be performed under direct ultrasound guidance to ensure proper placement in the uterine cavity. This practice, properly conducted, will significantly enhance embryo implantation and pregnancy rates. We prefer to perform all embryo transfers when the woman has a full bladder. This facilitates the visualization of the uterus by abdominal ultrasound and causes reflex nervous suppression of uterine contractility. The patient is allowed to empty her bladder 10-15 minutes following the embryo transfer.

It is important that the woman be as relaxed as possible during the embryo transfer because many of the hormones that are released during times of stress, such as adrenaline, can cause the uterus to contract. Accordingly we offer our patients 5mg of oral diazepam (Valium) about a half hour prior to the embryo transfer, to relax them and reduce apprehension.

Some IVF programs believe that imagery helps the woman relax and feel positive about the experience, thereby reducing the stress level. In such a program a counselor and/or clinical coordinator may help the woman focus on visual imagery for a few minutes immediately prior to embryo transfer so as to enhance her relaxation.

The embryo transfer procedure: When the woman is sufficiently relaxed, she is helped into the appropriate position and made as comfortable as possible. (In programs that rely on relaxation therapy, a counselor or nurse is usually present at the patient’s bedside, coaching her in relaxation exercises during the procedure.) When the woman is in the proper position, and her bladder is adequately filled, the physician first inserts a speculum into the vagina to expose the cervix and then may clean the cervix with a solution to remove any mucus or other secretions. An abdominal ultrasound transducer is placed suprapubically on the lower abdomen and the uterus is clearly visualized. The physician then informs the embryology laboratory that embryo transfer is imminent and awaits the arrival of the transfer catheter that will be loaded with the embryos.

The physician gently guides the catheter through the woman’s cervix into the uterine cavity. When the catheter is in place, the embryologist carefully injects the embryos into the uterus, and the physician slowly withdraws the catheter. The catheter is immediately returned to the laboratory, where it is examined under the microscope to make sure that all the embryos have been deposited. Any residual embryos would be re-incubated, and the transfer process would usually be repeated to transfer the remaining embryos.

The ET procedure is usually painless and takes less than thirty minutes to complete. Sometimes a prior trial embryo transfer (performed on the 8th day of gonadotropin stimulation) points towards potential difficulty in transferring the embryo(s)/blastocyst(s) to the uterus. In such cases, the procedure may be performed with patient under anesthesia.

Transmyometrial Embryo Transfer (TMET): In rare cases where the shape or partial obstruction of the canal leading in to the uterus (i.e. the cervical canal) severely complicates conventional embryo transfer, this method can be used. The patient is first anesthetized, then, using sterile technique, a needle is passed along the side of a transvaginal ultrasound probe through the wall of the uterus, into the uterine cavity. A very thin catheter is then passed through the needle with its tip protruding into the uterine cavity. The needle is partially withdrawn and the blastocyst(s)/embryo(s) are injected. After the embryo transfer, the woman remains immobile for approximately one hour and is thereupon discharged with specific instructions.

Post Embryo Transfer Management

Immediately prior to being discharged following the embryo transfer procedure, an exit interview is conducted, whereby the patient/couple is/are given directions.
Hormonal supplementation usually involves the administration of intramuscular injections of progesterone and/or vaginal suppositories (comprising estradiol valerate and micronized progesterone) until a blood pregnancy test is performed approximately eight days later (the chemical diagnosis of pregnancy). In selected cases, such progesterone treatment can be replaced with Crinone or Endometrin vaginal applications, once or twice daily. If the pregnancy test is negative or the plasma hCG levels fail to rise appropriately in the ensuing days, all hormonal support is abruptly discontinued.

A positive pregnancy test, followed by an appropriate rise in the plasma HCG concentration, is usually an indication to discontinue daily progesterone injections in favor of three times weekly intramuscular injections of hCG 5000 IU, along with daily vaginal estradiol and progesterone suppositories until the 8th week of pregnancy. In patients who experience an exaggerated response to ovarian stimulation with gonadotropins, hCG administration is withheld (for fear of increasing the risk of severe ovarian hyperstimulation), and intramuscular progesterone injections are continued along with vaginal estradiol and progesterone suppositories until the 8th week of pregnancy. Following an appropriate rise in HCG concentrations, the oral corticosteroids are continued until the 10th week of pregnancy. Thereupon, it is tapered down and stopped within a week or two. Heparin injections are continued until the 10th week of pregnancy and thereupon abruptly discontinued.

An ultrasound examination is performed approximately 2-3 weeks after the chemical diagnosis of pregnancy, at which time, designated patients with viable pregnancies, receive a final administration of intralipid (in some cases additional monthly doses of intralipid must be be administered ).

Embryo Cryopreservation (Freezing)

There have been dramatic advances in the technology of freezing and storing human embryos for future use. We cryopreserve (freeze) embryos as blastocysts. The recent introduction of “ultra-rapid freezing” (vitrification) which so rapidly freezes the embryo that it avoids ice formation in the blastomeres has revolutionized embryo freezing. Now, very few chromosomally normal embryos are lost in the freeze/thaw process and pregnancy rates with thawed pre-vitrified blastocysts are hardly different from that reported following the transfer of fresh blastocysts.