Given the considerable emotional, physical and financial investment associated with IVF treatment, it is essential that factors known to affect success be identified and regulated in advance of initiating treatment and to always thoroughly and meticulously “plan the trip before taking the ride.” This article addresses the most important considerations in this regard:
- Factors influencing embryo quality
- Age and, diminishing ovarian reserve (DOR): Women are born with all of the eggs they will ever have. After the menarche (the time of the onset of menstruation), a monthly process of using up numerous eggs continues until the number of eggs remaining in the woman’s ovaries falls below a certain critical threshold, at which time ovarian function starts to decline and the woman becomes relatively resistant to ovarian stimulation with fertility drugs. This phase of the woman’s reproductive life is referred to as the climacteric. The onset of the climacteric is heralded by a decline in the woman’s overall egg population, i.e. diminishing ovarian reserve-DOR as evidenced by increasing basal blood concentrations of FSH (progressively rising above >9.0MIU/ml) and AMH levels falling below 2ng/ml or 15picomols/L ) The climacteric ultimately ends (after 4-6 years ) with depletion of the functional egg supply, cessation of ovulation and of menstruation (the menopause).The timing of the onset of the climacteric varies from person to person. Genetic factors, exposure to environmental toxins and radiation, disease, drugs and pelvic disease associated reduced ovarian blood flow can all influence the timing of the onset of both the climacteric and the menopause. Most American women will enter the climacteric in their late thirties to mid-forties and go into menopause around 45-52yrs of age.
As a woman advances beyond 30 years of age, her eggs are progressively more likely to be numerically chromosomally “abnormal” (aneuploid). By way of example, a women < 35 years, is likely to have about 50% of her “mature eggs” being aneuploid at the time of ovulation or egg extraction. By the time she reaches 40 years of age about 90% are likely to be aneuploid and by 45Y of age about 95% of her mature eggs will likely be aneuploid. Bear in mind that upon fertilization, aneuploid eggs will either not fertilize or if they do, will propagate aneuploid and “incompetent” embryos which will either arrest during development, fail to attach to the uterine will be lost early in gestation as chemical pregnancies or as miscarriages. Infrequently, an aneuploid embryo will attach and develop further, to produce a chromosomally defective babies (e.g. Down syndrome).This inevitable increase in the rapid rise in the prevalence of infertility, miscarriage, and birth defects that occur as the age of conception increases beyond the age of 35 years. The progressive decline in the woman’s egg population or DOR also serves to explain the accompanied decline in response to fertility drugs and in large part, why treatment of infertility becomes progressively less successful with advancing maternal age. Simply stated, egg (rather than sperm) aneuploidy is a primary rate-limiting factor in human reproduction. While DOR and declining egg quality both commonly occur as the woman approaches her 40’s, there is often a tendency to believe that DOR and a decline in egg quality go hand in hand. This is a misconception because often times the climacteric and DOR will set in at a younger age, before age related egg aneuploidy becomes a factor. Thus, a 38-40 year old woman who has a normal basal FSH/AMH (i.e. has not yet entered the climacteric) would probably respond relatively well to modest ovarian stimulation with fertility drugs, produce numerous growing follicles/eggs and embryos, while woman B, also 40 years of age has entered the climacteric (with elevated basal FSH and low AMH) and thus has DOR would produce significantly fewer follicles/eggs in response to aggressive ovarian stimulation with fertility drugs. However, provided the protocol used for ovarian stimulation used were optimal selected, both women, would likely have the same incidence of egg/embryo aneuploidy. However, since Woman A would probably yield more eggs/embryos than woman B, the former likely have more available from which to select the best quality embryos for transfer, she would have more embryos available for transfer and thus the chance of her having a chromosomally normal embryos transferred would be greater than for Woman B and her chance of success would likewise be improved. Since egg aneuploidy precipitates embryo aneuploidy and the latter is at the root of the age-related decline in IVF success, many IVF programs recommend preimplantation genetic sampling (PGS) of embryos for chromosomal integrity. PGS methods such as next generation gene sequencing (NGS) and comparative genomic hybridization (CGH) allow for evaluation of all 23 pairs of chromosomes. Embryos that have all 46 chromosomes intact (euploid) have a much higher chance of propagating a viable pregnancy and are far less likely to result in early pregnancy loss and chromosomal birth defects. For those women whose age and/or degree of ovarian resistance make having a baby with their own eggs unappealing or unlikely, ovum donation (using donated eggs from a young donor (usually compatible and anonymous) is an excellent option. In fact IVF-ovum donation is one of the most successful methods of achieving pregnancy, regardless of the woman’s age.
- The protocol selected for controlled ovarian stimulation (COS): Aside from the woman’s age, the single most important factor influencing IVF success is the protocol selected for ovarian stimulation. This is most important when conducting IVF in older women and those with DOR.
In order for any organism to attain an optimal state of maturation (ripening) it must first undergo full growth and development. A fruit plucked from a tree before having developed fully ,or a poorly developed fruit, might still ripen (mature) on the shelf and might even appear as enticing as one that had previously undergone proper development, but it will lack the same quality. The same principle applies to the optimal maturation of human eggs. Proper development as well as precise timing of the initiation of egg maturation through meiosis (a process that in nature, is triggered by the LH surge, and in IVF, by the administration of hCG), is a vital requirement for optimal maturation, fertilization and ultimately to embryo quality. In fact, in cases where follicle/egg development is compromised or hCG (which initiates maturational division of egg chromosomes-meiosis), is administered prematurely or too late, the incidence of aneuploidy can rise well above the age-related threshold level, compromising IVF outcome. During the normal, ovulation cycle, ovarian hormonal changes are regulated to avoid irregularities in production and interaction that could adversely influence follicle development and egg quality. As an example, while small amounts of ovarian male hormones (androgens) such as testosterone, enhance egg and follicle development, over-exposure to excessive concentrations of the same hormones can seriously compromise egg ( and subsequently, also) embryo quality . It is important to recognize that the pituitary gonadotropins, LH and FSH, while both playing a pivotal role in follicle development, have different primary sites of action in the ovary. The action of FSH is mainly directed toward granulosa cells (which line the inside of the follicles) proliferation and estrogen production. LH, on the other hand, acts primarily on the ovarian stroma (the connective tissue that surrounds the follicles) to produce male hormones (predominantly testosterone). Only a small amount of testosterone is necessary for optimal estrogen production while over–production has a deleterious effect on granulosa cell activity, follicle growth/development, egg maturation, fertilization potential and subsequent embryo quality. Furthermore, excessive ovarian androgens can also compromise estrogen-induced endometrial growth and development. It follows that protocols for controlled ovarian stimulation (COS) should be geared toward optimizing follicle and egg development while avoiding over exposure to testosterone The fulfillment of these objectives requires an individualized approach to COS and that the administration of human chorionic gonadotropin (hCG) to “trigger” ovulation, be timed precisely. In conditions such as polycystic ovarian syndrome (PCOS), which is characterized by increased blood LH biological availability there is also an increased ovarian male hormone (androgen) production. It is therefore not surprising that “poor egg/embryo quality” is often a feature of this condition. The use of an LH/hCG -containing preparation such as Menopur can in my opinion, further aggravate this effect. Thus I recommend against the exclusive use of Menopur (and similar menotropins), in PCOS patients, preferring FSH-dominant products such as Follistim, Puregon and Gonal F. While it would seem prudent to limit LH/hCG exposure in all cases of COS, this appears to be most needed in older women, women with DOR and PCOS women who tend all to have increased LH biological activity. Use of GnRH-Agonists (e.g. Lupron/ Buserelin) and GnRH-Antagonists (e.g. Cetrotide, Ganirelix, Orgalutron): It is common practice to administer gonadotropin releasing hormone (GnRH) agonists and more recently, GnRH-antagonists to prevent the “premature” release of LH late in the cycle of ovarian stimulation with gonadotropins. GnRH agonists exert their LH-lowering effect over a number of days. They act by causing an initial outpouring and then depletion of pituitary gonadotropins. This results in the LH level falling to within negligible concentrations, within 4-7 days, thereby establishing a relatively “LH-free environment”. GnRH Antagonists, on the other hand, act by rapidly (within a few hours) blocking pituitary LH release, so as to achieve the same effect. The most commonly used protocols for ovarian stimulation:
- Long Gonadotropin releasing hormone agonist (Lupron/Superfact)-GnRHa Protocols: A protocol I commonly prescribe is the so-called “long protocol”. I usually start the patient on a birth control pill given for at least 10 days and then overlap the pill with an agonist (Lupron) for 3 days and then stop the BCP while continuing with daily injections of Lupron until a period ensues in 3-5 days. This protocol can also be used without prior use of a BCP. Here Lupron is given, starting a week or so prior to menstruation. The agonist Agonist/antagonist conversion protocol (A/ACP) here precipitates an initial rise in FSH and LH level, which is rapidly followed by a precipitous fall to near zero. This is followed by uterine withdrawal bleeding (menstruation), whereupon gonadotropin treatment is initiated while daily Lupron injections continue, to ensure a relatively “LH-free”
- Agonist/antagonist conversion protocol: This is a modified long protocol, where the agonist is supplanted by an antagonist (Ganirelix/Orgalutron/Cetrotide) given daily from the onset of the ”Lupron-only long protocol in cases of DOR because it avoids some of the resistance sometimes caused by the agonist during COS.
- Late antagonist protocol: Here an antagonist is started about 76-8 days into the COS process. While I never prescribe this protocol because in my opinion it is less effective in women with DOR, it is important that if it is used, it NOT be launched coming off a BCP as this will often delay antral follicle development, prolong the stimulation and lead to discordant egg development. It should in my opinion only be used in cases where no BCP is used to launch COS and only in younger women (<35Y who have normal ovarian reserve.
- Flare GnRHa protocols: Another approach to controlled ovarian stimulation is by way of so-called “flare protocols”. This involves initiating COS gonadotropin therapy simultaneous with the administration of GnRH agonist. The intent is to deliberately allow the agonist (Lupron/Superfact) to effect an initial surge (“flare”) in pituitary FSH release, so as to augment ovarian response to the gonadotropin medication. Unfortunately, this approach represents “a double edged sword” as the resulting increased release of FSH is likely to be accompanied by a similar rise in blood LH levels that could evoke excessive ovarian stromal androgen production. As mentioned, the latter might potentially compromise egg/embryo quality, especially in older women, and women with PCOS, whose ovaries have increased sensitivity to LH. I believe that in this way, “microflare protocols” can potentially; hinder endometrial development; compromise egg/embryo quality and reduce IVF success rates. Accordingly, I do not prescribe “flare protocols”.
- Agonist/antagonist conversion protocol + estrogen priming (A/ACP+ E2V): When it comes to very poor responders who have severe DOR, I tend to prescribe an A/ACP in combination with estrogen priming using estradiol valerate. The estrogen augments follicle response to gonadotropins by priming the granulosa cells in the follicles to FSH. However, it is important to be aware of the fact that when using the A/ACP+E2V protocol the woman often stimulates several days longer than usual before reaching adequate follicle development. His is not detrimental in any way. Use of the A/ACP protocol in women with severely diminished ovarian reserve markedly reducing the cycle cancelation rate to under 10% (i.e. much lower than expected). Many patients who had previously been told that they should give up on using their own eggs, and switch to ovum donation because of severe DOR, have subsequently achieved viable pregnancies using this approach.
- Augmentation with supplementary human growth hormone: In women who have very severely diminished ovarian reserve, I often supplement ovarian stimulation with the administration of human growth hormone. The latter is believed to fuel mitochondrial activity and so augment follicular response to COS
Selecting embryos for transfer to the uterus:
- Blastocysts versus cleaved embryos: Today it is well recognized that embryos that fail to survive to the blastocyst stage of development are almost always “incompetent” and unworthy of being transferred to the uterus. Had such embryos been transferred earlier they would not have propagated a pregnancy anyway. Because of this, I, urge most patients to confine the selection of embryos for embryo transfer to those that make it to the blastocyst stage by day 5-6 post-fertilization, resulting in increased success rates.
- PGS, Staggered IVF and Embryo Banking: When Levent Keskintepe PhD and I first introduced PGS with full chromosomal karyotyping into the clinical IVF arena, we had no idea that it would so rapidly help fashion the field. Now PGS has become part of the IVF main stream. Using NGS and CGH technology (among other methods) embryos are fully karyotyped in one cycle when following a biopsy where one or more cells are removed for PGS testing they are vitrified (cryostored) while awaiting the results whereupon they are subsequently transferred (in a subsequent cycle) to the (i.e. Staggered-IVF). And when numerically chromosomally normal (euploid) eggs are transferred into a hospitable uterine environment the chance of a successful pregnancy increases significantly, the risk of miscarriage rate declines and the risk of a chromosomal defect such as Down Syndrome is kept to a minimum. This has led to the approach gaining widespread use. Unfortunately, in my opinion, this method is now being “over-used”. In fact, some programs virtually recommend PGS embryo testing to all of their IVF patients. In my opinion this is wrong and fraught with often unforeseen circumstances. Bear in mind that PGS does NOT improve an embryo’s quality, it simply selects those embryos that are more likely to be “competent”. Egg/embryo “competency” remains primarily a function of age and the protocol used for controlled ovarian stimulation (COS). Consider the following:
- PGS does not improve IVF success in younger women: In younger women (<35Y) with normal ovarian reserve, the selective transfer of 2 PGS-untested blastocysts (advanced embryos) will yield about the same chance of success (50-60%) as would the transfer of 1-2 euploid embryos. This is because 1:2 untested blastocysts are likely to be euploid anyway and each euploid blastocyst has about a 60% chance of resulting in a live birth. On the other hand in older women (36-45y) where depending on age , at best between 1:3 and 1:20 blastocysts are likely to be euploid, PGS –based selection of embryos definitively improves the chance of selecting the most ”competent” embryos for transfer. Thus, with the exception of younger women who have DOR those with “unexplained IVF failure” and women with RPL where embryo selection would improve success, PGS should in my opinion be largely confined to embryos derived from the eggs of women in their later 30’s and beyond. Staggered IVF with or without Embryo banking (stockpiling) is however an ideal method for blunting the effect of the biological clock and has particular merit in cases where the woman is older or has DOR and wants to avoid resorting to the use of donor eggs. In such cases, by selectively stockpiling and banking of PGS-euploid embryos over several cycles and then selectively transferring only them at a later date (Staggered IVF with Embryo Banking), it is now possible to improve IVF success rate per embryo transferred, reduce the miscarriage and chromosomal birth defect rate and provide women with an opportunity to have >1 baby over time.
- Embryo mosaicism: Not all aneuploid embryos are doomed. There is a condition where a chromosomally normal embryo might subsequently become aneuploid during regular (mitotic) cell replication. Here, some of the embryos cells are chromosomally intact (euploid) while others are aneuploid. The condition is referred to as “mosaicism”. Mosaic embryos can autocorrect during subsequent development and produce normal offspring. This means that many aneuploid embryos should be banked and stored for possible future dispensation after full disclosure to the potential parents.
- Sperm Quality and ICSI:
While in the vast majority of cases embryo aneuploidy results from egg rather than sperm problems, few would argue that sperm quality is also a very important factor in insuring embryo quality. Nevertheless, the advent of intracytoplasmic sperm injection (ICSI) has all but removed male infertility as an impediment to IVF success. The procedure of ICSI, involves the direct injection of a single sperm into each egg under direct microscopic vision. Initially ICSI was only used to achieve fertilization in cases of male infertility. However, today it is often used routinely to optimize the fertilization rate in all cases of IVF. When ICSI is conducted in cases of male infertility, there is a slight increase in the risk of chromosomal deletions leading to miscarriage. However, when ICSI is done in the absence of male infertility the miscarriage and IVF birth rate appears to be unaffected. It is relevant however that when ICSI is performed for male infertility there might well be a subsequent increased risk of male factor infertility in the offspring.
- Receptivity of the uterine lining (endometrium):
While there are many factors that affect implantation, the most important boil down to the following four (4)
- Contour of the uterine cavity: It has long been suspected that anatomical defects of the uterus might result in infertility.
While the presence of myomas (fibroids) in the uterine wall, are unlikely to cause infertility, an association between their presence and infertility has been observed in cases where they distort the uterine cavity, or protrude as submucous polyps through the endometrial lining. It would appear that even small submucous myomas have the potential to prejudice implantation. It is likely that any surface lesion in the uterine cavity, whether an endometrial, placental or fibroid polyp (no matter how small), or intrauterine adhesions, has the potential to interfere with implantation by producing a local inflammatory response. Unfortunately, a hysterosalpingogram (HSG) will miss the diagnosis in approximately 20% of cases. The only reliable methods for diagnosing even the smallest of such lesions, is through the performance of a sonohysterogram (fluid ultrasound) or by hysteroscopy. Either such procedure should ideally be performed within a year of doing IVF.
- Endometrial thickness. In 1989, I first reported that preovulatory endometrial thickness, will help predict which women will succeed after embryo transfer and who will not. With conventional IVF (where the woman receives fertility drugs and has her own fresh embryos transferred to her uterus), there needs to be at least 8mm thickness (preferably >9mm). I in fact, do not perform embryo transfers if the lining is <8mm in thickness by the day of hCG trigger (in fresh cycles) or at the start of progesterone administration in embryo recipient cycles (FET, egg donation, Gestational surrogacy and donated embryo transfers).Hitherto, attempts to augment endometrial growth in women with poor endometrial linings by bolstering circulating estrogen blood levels (through the administration of increased doses of fertility drugs, aspirin administration and by supplementary estrogen therapy all yielded disappointing results. Some time ago, I reported on the ability of vaginally administered Sildenafil (Viagra) to significantly enhance uterine blood flow and estrogen delivery to the endometrium and thereby improve endometrial development. In the process many women with intractably poor endometrial development have achieved healthy pregnancies.
- Immunologic factors:The implantation process begins six or seven days after fertilization of the egg. At this time, specialized embryonic cells (i.e., trophoblast), which later becomes the placenta; begin growing into the uterine lining. When the trophoblast and the uterine lining meet, they, along with Immune cells in the lining, become involved in a “cross talk” through mutual exchange of hormone-like substances called cytokines. Because of this complex immunologic interplay, the uterus is able to foster the embryo’s successful growth. Thus, from the earliest stage, the trophoblast establishes the very foundation for the nutritional, hormonal and respiratory interchange between mother and baby. In this manner, the interactive process of implantation is not only central to survival in early pregnancy but also to the quality of life after birth.
Considering its importance to successful procreation, it is not surprising that failure of proper function of this immunologic interaction during implantation has been implicated as a cause of implantation dysfunction leading to IVF failure, unexplained infertility, and recurrent pregnancy loss (RPL). A partial list of immunologic factors that may be involved in these situations includes anti-phospholipid antibodies (APA), antithyroid antibodies (ATA), and, perhaps most importantly, activated natural killer cells (NKa). Presently, these immunologic markers in the blood, can be adequately measured by only a few (less than a half dozen) highly specialized reproductive immunology laboratories in the United States:
- Antiphospholipid Antibodies (APA). A large body of literature has confirmed that patients who experience repeat IVF failures often have increased levels of circulating APAs. Compelling evidence has also demonstrated that up to 50% of women with pelvic endometriosis and unexplained infertility harbor APAs in their blood
- Natural Killer (NK) Cells: NK cells comprise more than 70% of the immune cells in the uterine lining where they play an important role in facilitating and regulating implantation of the embryo’s root system (trophoblast) and the formation of the placenta. NK cells produce a variety of local hormones known as TH-1 and TH-2 cytokines. Uncontrolled, excessive release of TH-1 cytokines is highly toxic to the trophoblast and endometrial cells, leading to their programmed death (apoptosis) and, subsequently to immunologic implantation dysfunction (IID). Activated NK cells (NKa) spill over from the uterine lining into the peripheral blood where their toxicity can best be measured using the K-562 target cell test. When there is excessive NK cell activation, Intralipid + corticosteroid therapy, initiated 10-14 days prior to embryo transfer, can down-regulate activated NK cells, thereby reducing the risk of implantation failure.
- Antithyroid Antibodies (ATA) A relationship has been established between ATA and reproductive failure (RPL, infertility and IVF failure). I previously reported on the fact that 50% of women who have ATA also have activated uterine natural killer cells. In such cases intralipid/corticosteroid therapy can double IVF success rates. The risk of IIID in ATA positive women appears to be confined to cases where ATAs coexist with Nk cell activation.
- Embryo Transfer:
In the final analysis, the transfer of embryos is the “holy grail”. It would not matter whether the embryos are ”competent” or whether the uterus is anatomically and immunologically “receptive” or not, if they are not transferred using optimal technique and by someone with dexterity, experience and dedicated commitment. First, as stated above, it is my opinion that with few exceptions only day 5 or 6 blastocysts should be transferred. Second, the transfer should be conducted under ultrasound guidance with the woman having a full bladder and finally the assistant performing the ultrasound examination for the transfer needs to be experienced or at the very least should be supervised by someone who has seasoning and expertise. Reporting of Clinic-Specific IVF Success Rates: Currently IVF success rates are reported by the society for Assisted Reproductive Technology (SART) and the CDC annually, on their respective websites. Unfortunately the reporting process currently in use is seriously lacks proper of accountability and oversight. Moreover, by presenting IVF outcome as a function of pregnancy or birth rate per completed cycle of IVF, per egg retrieval or per embryo transfer procedure performed does not serve to compare IVF programs on a level playing. This is because it leaves the door open for even the most honest and well intentioned IVF practitioners to attempt to try and look better by simply altering the mix of the patients they treat and report on or by increasing the number of embryos transferred per procedure. One way to remove such tendencies while still being able to readily compare and evaluate IVF success rates would in my opinion be to express IVF outcome in terms of pregnancy and birth rates (in various age and demographic categories) per embryo that is transferred. This, would immediately allow for reported success rates to be assessed using a level playing field. At the same time, it would discourage the transfer of multiple embryos at a time, and would lead to a further and much needed drop in the still unacceptably high national NF multiple birth rate. In my opinion, by using this approach and expressing IVF success as Viable Conception Rate (VCR) and/or Live Birth Rate (LBR) per embryo being transferred would immediately transfer responsibility for reporting, from the clinical team to the embryology laboratory, where such data is already accessible. This would make it simple for governing bodies to cross-check reported outcomes using existing embryology laboratory oversight mechanisms already in place through the College of American Pathologists (CAP) and in the process, remove one of SART’s biggest impediments to reliable IVF outcome data collection. Our practice has had some of the highest rates of IVF success in the world. I have been working in this field for over thirty years and have helped bring more than 18,000 babies into the arms of loving families. If you have any questions about IVF, fertility, or any other specific concerns, I would be happy to answer them on my open forum, or in a private Skype consultation.