The ability to grow a healthy plant requires that a “good” seed be planted in a “fertile soil” and then be properly nurtured until it can thrive on its own….independent of intervention. Similarly, successful IVF requires the same relationship, only here, a “good seed” is a “competent, chromosomally normal (euploid) embryo and “fertile soil” is a “receptive” uterine lining (endometrium). You cannot expect success through planting a “bad seed” in a “fertile soil” any more than you could if you were to plant a good seed in “poor soil”. No…..in either case successful outcome requires that a “competent” embryo (seed) be located in a receptive uterus (“fertile soil”).
It follows that central to achieving a successful IVF outcome is the need:
1. At the very outset to define the variables that affect embryo quality and uterine receptivity
2. To avoid “one size fits all” protocols for controlled ovarian stimulation (COS). Instead it is necessary to individualize the protocol of the COS to fit the profile of each individual so as to establish an optimally nurturing environment for developing follicles, eggs and uterine lining.
3. To precisely time the egg retrieval (ER) and then safely extract, eggs from the woman’s ovary (ies).
4. To expertly transfer the best quality advanced embryo(s) [blastocyst(s)], gently and delicately to the uterus. To support early embryo implantation through optimal hormonal supplementation during most of the 1st trimester.
Controlled Ovarian Stimulation (COS): 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 regulate the timing of ovulation so that her eggs can be surgically retrieved once they reach maturity and before they spontaneously ovulate.
In cases where the woman has previously received fertility drugs, the subsequent COS protocol is in part based upon her prior response to the most recent such treatment regime. If the cycle of COS the woman is to undergo is her first, the dosage and regimen of COS is determined by the biochemical measurement of her ovarian reserve (cycle day 3 basal blood FSH levels and measurement of antimullerian hormone (AMH) concentrations, any time in the cycle). The woman first undergoes a thorough general physical and Gynecologic evaluation before initiating COS. She will usually start by taking a balanced, monophasic birth control pill (BCP) within a few (5-6) days of her period starting. A base-line ultrasound examination is usually performed at the start of spontaneous or hormone-induced menstruation, to count antral follicles and to look for ovarian cysts, The duration of BCP therapy should in my opinion be at least 10 days, but this can be prolonged (cyclically) even for several months, without prejudice, if need be. The goal is to adjust the duration on the BCP so as to “orchestrate” that ovarian stimulation can commence on a set date.
At a predetermined time, while still taking BCP, the daily injection of a gonadotropin releasing hormone agonist (GnRHa); e.g., Lupron or Buserelin/Superfact etc., is initiated. A combination of agonist + BCP is then continued for another three days whereupon the BCP is abruptly stopped while agonist injections continue until menstruation ensues (usually within about 3 to 7 days). In this way we are able to schedule each cycle of IVF to the convenience of both the patient and the medical team. Additionally, the combined use of BCP + agonist reduces virtually eliminates the risk of the BCP suppressing subsequent response ovarian stimulation with gonadotropins, and well as the likelihood of ovarian cyst formation, thereby largely avoiding the need to delay or cancel the cycle of treatment.
As soon as menstruation begins, blood is drawn for measurement of estradiol [E2] concentration. Provided that the [E2] is under 70 pg/ml (or 200pmol/L) , the patient will be ready to initiate ovarian stimulation with gonadotropins. If the [E2] is greater than 70 pg/ml, a repeat ultrasound is done to look for one or more estrogen-producing functional ovarian cysts. If such a cyst is detected, it is my personal preference that (rather than wait for it to resolve on its own), that an immediate transvaginal ovarian cyst aspiration be performed (under local anesthesia) whereupon the [E2] will usually rapidly fall to below 70pg/ml within 24-72 hours. Within a few days of menstruation starting, COS with gonadotropins (e.g., Gonal F, Follistim,) is commenced. As soon as gonadotropin administration commences the agonist injections are either continued (at a reduced daily dosage) throughout the stimulation process, until the hCG trigger (i.e., The Long-Agonist Protocol) or in women with moderately or severely diminished ovarian reserve (DOR) where the AMH is <1.5 ng/ml, the protocol is modified to an agonist/antagonist conversion protocol A/ACP) where, coinciding with the onset of gonadotropin therapy, the agonist is supplanted by low-dosage GnRH antagonist (e.g., Ganirelix, Orgalutron or Cetrotide – the A variation on the A/ACP theme is used in some women who have severely diminished ovarian reserve (DOR) where the basal AMH is <0.5ng/ml . Here, upon supplanting the agonist with an antagonist we add Estrogen Priming. The protocol is referred to as A/ACP with Estrogen Priming. With this protocol variation, the woman receives twice weekly intramuscular injections of estradiol valerate (Delestrogen) and/or vaginal estradiol suppositories beginning with the switch from the agonist to an antagonist at the time of the onset of bleeding ( about 8 days before commencing COS) with gonadotropins. This is continued until at least 50% of the follicles reach 14mm in diameter, whereupon estrogen priming it is stopped. The exact type of gonadotropin medication administered as well as the dosage/stimulation regime will vary from patient to patient. It is however my personal preference to prescribe recombinant DNA- FSH (e.g., Follistim, Puregon or Gonal-F), rather than urinary derived products, (e.g., Bravelle) which I believe to be more subject to batch-to batch variation in potency.
Two days after commencing gonadotropin therapy, I reduce the dosage of Follistim/Gonal F/Puregon and add 75U (1 vial) of Menopur daily. The FSH/LH therapy is maintained until at the point of optimal follicle development as determined by serial ultrasound examinations and blood [E2] measurements. Thereupon, 10,000U hCG (Profasi, Novarel or Pregnyl) is injected. I personally do not advocate supplanting this with 250mg of DNA recombinant hCG (Ovidrel) because, in my opinion, this dosage is too low to achieve optimal biological potency. If Ovidrel is to be used, the dosage should in my opinion be doubled. However, since this is much more expensive than Profasi, Novarel or Pregnyl, and there are no medical advantages, I do not use Ovidrel in my IVF patients. On occasion I do make adjustments to the dosage of gonadotropins, but not to the dosage of hCG.
Some people halve the trigger hCG dosage to 5,000U in very high responders, in the hope that this will reduce the risk of severe ovarian hyperstimulation syndrome (OHSS). In my opinion, it does not do so. Moreover, at such a low dosage, in the very patients that have a multitude of follicles, it does not achieve optimal receptor saturation in the follicle cells, and results in too many “immature” eggs being harvested at egg retrieval. Others favor the use of an agonist (rather than an hCG trigger). The intent is that the agonist, by causing LH to be expunged from the pituitary gland, will achieve a more “natural” maturation of eggs and reduce the risk of OHSS. I’m not convinced of such a benefit. Besides, it is hard to determine how much LH is released following the administration of such an “agonist trigger”.
Commencing 7-8 days after the initiation of gonadotropin therapy, the patient undergoes serial (usually daily) ultrasound and plasma estradiol evaluations to monitor her ovarian response. These assessments are aimed at determining the ideal day for administering the “hCG trigger”. Both the agonist/antagonist and gonadotropin injections are discontinued on the day of the trigger and the patient is scheduled for ER approximately 36-38 hours thereafter. Patients who because of immunologic implantation dysfunction (IID) require selective immunotherapy with a heparinoid (Clexane or Lovenox) plus corticosteroids (dexamethasone, prednisilone, prednisone). These are commenced with the initiation of Gonadotropin stimulation, followed by daily administration. Heparinoid injections are discontinued approximately 12 hours prior to the egg retrieval and re-started after the embryo transfer procedure.
I no longer advocate the use of aspirin for two reasons: First, it has no clear benefit. Second, by prolonging the bleeding time, it can result in excessive bleeding at egg retrieval and concealed intrauterine bleeding at embryo transfer (ET). The latter will often go undetected and could result in failed implantation. Those patients who have a thin endometrial lining receive sildenafil (Viagra) vaginal suppositories (four times daily) from the onset of gonadotropin therapy to the day of the hCG “trigger”, in an attempt to improve the thickness of their uterine linings. In some cases, where a deficient endometrium is detected a few days into the cycle of stimulation, the administration of Viagra can improve the lining within 48 hours.
Patients who have activated natural killer cells (NKa) as measured by a K-562 target cell test and/or cytokine uterine studies receive Intralipid (IL) therapy or IVIG by intravenous infusion 7-14 days prior to expected date of embryo transfer. In the event of a positive (and rising) blood beta hCG level (which suggests that implantation is in progress), the IL/IVIG infusions may be repeated once and then discontinued (in cases of autoimmune implantation dysfunction). In cases of alloimmune implantation dysfunction, Intralipid or IVIG should be administered every 2-4 weeks thereafter, until the 24th week of pregnancy. My IVF patients will almost always receive oral corticosteroids (dexamethasone, prednisilone 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 with rising blood hCG levels (a positive blood pregnancy test) 9-11 days after egg retrieval continue taking corticosteroid and heparin (as applicable) beyond the ultrasound confirmation of pregnancy, which is performed at the 6-7 gestational week until the 10th week of gestation.
In cases where the blood pregnancy test fails to reveal an appropriate increase in the quantitative beta hCG concentration, heparinoid (Clexane, Lovenox) therapy is discontinued, and the corticosteroid dosage is slowly tailed off and stopped over 1-2 weeks. Pregnant women continue on corticosteroid as well as heparinoid treatment until the 10th week of pregnancy, whereupon the heparin is discontinued and the corticosteroid is tapered off over 1-2 weeks and stopped. 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) involves a non-surgical procedure performed under conscious sedation. At SIRM, a qualified anesthesiologist is in attendance with the patient during the procedure. This is performed in a dedicated procedure room, 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 day or so. 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.
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 Percutaneous, 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 (seen after spinal injury, prostatectomy or advanced diabetes), 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.
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-nucleate embryos”.
Intracytoplasmic sperm injection (ICSI): Today, at SIRM, we perform virtually all IVF using ICSI. Studies have shown that there are really only advantages to this policy. The ICSI procedure involves the direct injection of a single sperm into each egg under direct microscopic vision. 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. I personally 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.) The Graduated embryo scoring (GES) system which was developed by us in 2001. It provided a method whereby we score each individual embryo out of a maximum of 100 points. Embryos that on day 3 post fertilization have a GES of >70/100, are the ones that are most likely to develop into blastocysts and propagate healthy babies. However, we now grow almost all embryos to blastocysts so the GES scaring method has become somewhat redundant.
Microscopic Embryo and Blastocyst Grading: While embryos may be transferred 3 days after fertilization when they are divided to the 5-9 cell stage, it is my strong preference to wait until day 5-6 and then only transfer them if they reach the expanded blastocyst (100 cell+) stage of development. This is because embryos that fail to become blastocysts are almost always chromosomally abnormal (aneuploid) and are almost always incapable of propagating a healthy baby and are thus not worth transferring anyway. This does not mean that ALL those embryos that progress to blastocysts are chromosomally normal (euploid). They are not… but they do represent the ones that are most likely to be so. 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) Those blastocysts that contain more cells have a more expanded blastocele, a more prominent inner cell mass and have a well differentiated trophectoderm are the ones that are most likely to be “competent” (i.e. likely to propagate a viable pregnancy).
Preimplantation Genetic Testing (PGT) for the evaluation of eggs and embryos: In 2007, we became the first to report on the clinical value of counting all the embryo’s chromosomes (karyotyping) to identify those that had all 46 chromosomes (were euploid) and thuas the nes that were most likely to propagate viable pregnancies and the least likely to miscarry or result in chromosomal defects such as Down syndrome. We used a method known as metaphase CGH which now has largely been supplanted by next generation gene sequencing (NGS). NGS testing currently represents the most reliable method available to achieve this goal and is fast establishing a “new standard of care” in the field. In fact, the transfer of even a single CGH- normal embryo to a “receptive” uterus can yield a >50% chance of a live birth.
For older women and those with diminished ovarian reserve (DOR), repeated egg retrievals combined with CGH embryo selection will permit stockpiling of “competent embryos” over a number of cycles. This process of “Embryo Banking”, by allowing women to vitrify (freeze) and store their embryos for subsequent dispensation can stall the biological clock and prolong fertility potential.
Undoubtedly, embryo transfer (ET) is one of the rate limiting steps in IVF. It takes confidence, dexterity, skill and a soft touch to perform a good transfer. Of all the procedures in ART, this is arguably the most difficult to teach a training physician to conduct. 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 undoubtedly 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.
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, wherein the patient/couple is/are given instructions on post-transfer care and follow-up.
Post-ET Hormonal Supplementation
This 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 beta hCG pregnancy test is negative or the plasma hCG levels fail to rise appropriately in the ensuing days, all hormonal support is abruptly discontinued. An ultrasound examination is performed approximately 2-3 weeks after the chemical diagnosis of pregnancy is made by blood testing, at which time designated patients with viable pregnancies receive a final administration of Intralipid (in some cases additional monthly doses of Intralipid must be administered).
Embryo Freezing (Cryopreservation)
There have been dramatic advances in the technology of freezing and storing human embryos for future use. We freeze embryos as blastocysts. The recent introduction of an ultra-rapid freezing technique known as “vitrification” has revolutionized embryo freezing. This method, by freezing embryos faster than the blink of an eye, eliminates ice formation in the blastomeres. 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.
I do not intend, and it would indeed be presumptuous to suggest that the above approach should serve as a template for other RE’s to adopt. To the contrary, there are many alternative approaches that could well be equally efficacious, or perhaps even better. However after having been influential in the births of >18000 IVF babies, over the last 3 decades all I can say is that IVF is not a pure science nor a pure art. Rather it is an art-science blend, where experience and seasoning really counts. What is described above has resulted from my own prolonged trial and tribulation. Most importantly, it works.