In vitro fertilization (IVF) treatment is rather complex and involves multiple steps, a variety of medications and procedures. All these factors play a significant role in the final outcome of the treatment, which is a healthy live birth. The considerable emotional, physical and financial burden associated with infertility treatment in general and with IVF in specific, demand that factors known to affect outcome be identified and regulated prior to initiating treatment. Some of these factors that impact the outcome of IVF are the following:
- Egg/embryo quality
- Stimulation protocol and timing of ovulation
- Receptivity of the endometrium (implantation)
- Embryo transfer
- Sperm factors
1. Egg/Embryo Quality:
Reproductive age significantly impacts egg and embryo quality, and negatively correlates with IVF outcome. Live birth rates are 50% in women less than age 35 and approximately 25% in women between the ages 40 to 42 per fresh IVF cycle. The decrease in the success rates with IVF or any infertility treatment is due to the decreasing number of eggs within the ovaries. There is an accelerated loss of the follicles/eggs after the age of 32-35 and the fertility potential is significantly decreased thereafter. Several tests can identify patients who may have a suboptimal response to stimulation with IVF. Such tests include day 3 FSH and estradiol levels, Clomid challenge test, inhibin B, Anti-Mullerian Hormone (AMH) levels and antral follicle count in the beginning of the cycle. When there is evidence of decreased ovarian reserve by an abnormal value in one of these tests, a more aggressive approach to infertility treatment is generally taken, such as IVF with aggressive ovarian stimulation.
The method of ovarian stimulation may impact egg/embryo quality. The potential for the eggs to undergo maturation, successful fertilization and subsequent progression to good quality embryos that are capable of producing a healthy offspring is thought to be genetically determined. However, the expression of such potential is susceptible to numerous extrinsic influences, especially to intra-ovarian hormonal changes during the pre-ovulatory phase of the cycle.
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 cell (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 androgens. Only a small amount of testosterone is necessary for optimal estrogen production. 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.
In conditions such as polycystic ovary syndrome (PCOS), which is characterized by increased blood LH levels, there is also an increased ovarian androgen production. It is therefore not surprising that poor egg/embryo quality, immaturity of eggs and inadequate endometrial development are often features of this condition. The use of LH-containing preparations such as Repronex, Menopur or rLH from the beginning of the stimulation may further aggravate this effect. While it would seem prudent to limit LH exposure in all cases of COH, this appears to be more relevant in older women, who tend have higher levels of bioactive circulating LH.
It is common practice to administer gonadotropin releasing hormone (GnRH) agonists (Lupron or Buserelin) and more recently, GnRH-antagonists (Ganirelix or Cetrorelix) to prevent the release of LH with COH. GnRH agonists exert their LH-lowering effect over a number of days by causing an initial outpouring and then depletion of pituitary gonadotropins (FSH and LH). 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 rapidly, within a few hours to block pituitary FSH and LH release, so as to achieve the same effect.
2. Stimulation Protocols and Timing of Ovulation:
Currently, a number of ovarian stimulation protocols are being utilized in IVF. Studies evaluating different types of protocols and medications are limited and not specific to subgroups of patients with different causes of infertility, age or illnesses. Most studies include patients with a variety of different causes of infertility and the medical data generated are not stratified based on multiple factors that might have a significant impact on the final outcome. Some of the protocols that are currently being used and our AACP protocol are discussed below. Ovulation following maturation and development of the eggs should be timed precisely so that the eggs are not too mature or immature. In patients with PCOS or high responders, ovulation may sometimes be triggered early and this may result in immature eggs. In others, eggs may over-mature and that may not yield healthy oocytes if stimulation is prolonged to a point where the follicles are measuring more than 25-30 mm in size. Optimal timing of ovulation should be done based on each individual case.
Long GnRHa Protocols:
The most commonly prescribed protocol for Lupron/gonadotropin administration is the “long protocol”. Lupron is given, starting 1 week prior to menstruation. This precipitates an initial rise in FSH and LH levels, which are 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” environment.
Microflare GnRHa protocols:
Another approach to COH is by way of microflare protocols. This involves initiating gonadotropin therapy simultaneous with the administration of GnRH agonist. The intent is to deliberately allow Lupron to affect an initial surge (“flare”) in pituitary FSH release so as to augment ovarian response to the gonadotropin medication. With this approach FSH is likely to be accompanied by a similar rise in blood LH levels from the beginning of follicular development that could evoke excessive ovarian stromal androgen production. The latter could potentially compromise egg quality, especially in older women, and in women with conditions like polycystic ovarian syndrome (PCOS) whose ovaries have increased sensitivity to LH. It is unknown whether microflare protocols can potentially hinder endometrial development, compromise egg/embryo quality and reduce IVF success rates. Accordingly, we prefer to avoid flare protocols that expose the follicles to high intra-ovarian LH hormone levels in early folliculogenesis.
GnRH agonist/antagonist conversion protocols (AACP) with estrogen priming:
The use of GnRH antagonists as currently prescribed in ovarian stimulation cycles, i.e. the administration of 250 mcg daily from the 6th or 7th day of stimulation with gonadotropins may potentially be problematic, especially in women with high LH and overgrowth (hyperplasia) of ovarian stroma e.g. women over 40 years, women with raised cycle day 3 FSH and/or low Inhibin B, other “poor responders” to gonadotropins, and in some women with PCOS. In such cases the initiation of pituitary suppression with GnRH antagonists so late in the cycle of stimulation fails to suppress high tonic pituitary LH in the most formative (early) stage of folliculogenesis. One of the roles of LH is to promote androgen (male hormone) production which in turn is essential (in small amounts) for optimal follicular growth to take place. In women with high LH and/or ovarian stromal hyperplasia, the failure of conventional GnRH antagonist protocols to address this issue, results in the inevitable excessive exposure of follicles to androgens (mainly testosterone). This may potentially adversely influence egg/embryo quality and endometrial development.
Presumably, the reason for the suggested mid-follicular initiation of high dose GnRH antagonist is to prevent the occurrence premature LH surge. However the term “premature LH surge” is a misnomer and the concept of this being a “terminal event” or an isolated insult may not be accurate. In fact the event results from a culmination (end point) of the progressive escalation in LH (“a staircase effect”) which results in increasing ovarian stromal activation with proportionate growing androgen production within the ovary.
The use of such protocols in younger women or in normal responders will probably not produce such adverse effects, because the tonic endogenous LH levels are low (normal) in such cases and such normally ovulating women rarely have ovarian stromal hyperplasia.
Some form of pituitary blockade, either in the form of a GnRH agonist (Lupron or Buserelin) or a GnRH antagonist (Cetrorelix) is an essential component in ovarian stimulation of poor responders undergoing IVF. If this is not done, a progressive rise in LH–induced ovarian androgens may adversely affect egg development, resulting in compromised embryo quality.
With the long Lupron down-regulation-protocol (where the pituitary gland is largely exhausted of its LH and residual minimal LH is present in the circulation by the time stimulation with gonadotropins begins), the above mentioned adverse testosterone-effect is largely negated. On the down side is the fact that prolonged administration of GnRH agonists such as Lupron (such as with the GnRH agonist down-regulation protocol) could suppress subsequent ovarian response to ovarian stimulation with gonadotropins, by competitively binding with ovarian FSH receptors. Agonist/antagonist conversion protocol (AACP) is introduced in an effort to counter this effect.
With the AACP, low dose antagonist is commenced at the onset of spontaneous menstruation or following bleeding that follows initiation of GnRH agonist (Lupron) therapy using a long-down-regulation protocol arrangement. Preliminary results suggest a significant improvement in egg number, egg/embryo quality as well as in implantation and viable IVF pregnancy rates. The AACP has however, proven to be most advantageous in “poor responders” where additional enhancement of ovarian response to gonadotropins may be achieved through incorporation of “estrogen priming”. Addition of estradiol for a week following the initiation of the AACP, prior to commencing FSH-dominant gonadotropin stimulation appears to further enhance ovarian response, presumably by up-regulating ovarian FSH-receptors.
Serum estradiol levels with the use of GnRH antagonists (Ganirelix/Cetrorelix) tend to be lower in comparison to the GnRH agonist cycles (Lupron). In patients at risk for ovarian hyperstimulation syndrome, where estradiol measurements are closely followed, the AACP is not commonly utilized.
It is remarkable, that while using the AACP + E2V in poor responders whose FSH levels were often well above threshold limits, the cycle cancellation has consistently been maintained below 10% (much lower than expected). Many of these patients who had previously been told that they should give up on using their own eggs and switch to egg donation because of poor ovarian reserve, have subsequently achieved viable pregnancies using the AACP with estrogen priming.
3. Receptivity of the Endometrium:
a) Uterine cavity and the surrounding myometrium
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 (myometrium) away from the endometrial cavity is unlikely to cause infertility, an association between their presence and infertility has been observed. Presence of fibroids or polyps inside the endometrial cavity or fibroids right adjacent to the endometrial cavity may interfere with implantation and also cause recurrent pregnancy loss. Intrauterine adhesions can also negatively impact implantation and also result in recurrent miscarriages. A vaginal pelvic ultrasound provides information about fibroids in the myometrium (muscle layer of the uterus) and also sometimes in the endometrial cavity if the lesion is large enough to be recognized on ultrasound. In order to diagnose intracavitary lesions (inside the endometrium) either a hysterosalpingogram (HSG = dye test with x-ray) or one of the tests underlined below can be performed. HSG not only provides information about inside of the endometrial cavity, but also about the fallopian tubes.
Sonohysterography/Hydrosonography (Fluid ultrasound):
Hydrosonography is a procedure whereby a sterile solution of saline (salt water) is injected via a catheter through the cervix and into the uterine cavity. The fluid distended cavity is examined by vaginal ultrasound for any irregularities that might point to surface lesions such as polyps, fibroid tumors, scarring, or a uterine septum. Small lesions or adhesions may be missed with this diagnostic tool, which is one disadvantage of this test. If a lesion is detected, it requires the subsequent performance of hysteroscopy for removal of the lesion. Additionally, uterine malformations may not be clearly seen or diagnosed with such a diagnostic procedure.
Hysteroscopy:
Diagnostic hysteroscopy is an office procedure performed under anesthesia with minimal discomfort to the patient. This procedure involves the insertion of a thin, telescope‑like instrument known as the hysteroscope, vaginally through the cervix and into the uterus, in order to directly visualize the uterine cavity. The uterus is distended with normal saline fluid, which passes through a sleeve inside the hysteroscope. Diagnostic hysteroscopy facilitates examination of the inside of the uterus under direct vision for defects that might interfere with implantation. The major advantages of hysteroscopy are its 100% sensitivity and 100% specificity for intracavitary lesions. Also, the possibility of treatment during the same procedure eliminates the need for an additional procedure. It is the best imaging technique for all patients and especially those with recurrent pregnancy loss due to developmental abnormalities of the uterus.
b) Hydrosalpinx (fluid filled tube):
This is a relatively common disease of the fallopian tubes and results from distal obstruction (complete closure of the end of the tubes next to the ovaries). Hydrosalpinx can be diagnosed by an HSG or can be suspected only if visualized on ultrasound, although in most cases hydrosalpinx can not be seen on ultrasound. Hydrosalpinx can be secondary to prior pelvic infections that damaged the tubes or from pelvic adhesions or endometriosis related problems and lesions. Whereas IVF bypasses the fallopian tubes, the toxic fluid that accumulates in the tubes due to distal obstruction can drain back into the uterus and significantly decrease the success of pregnancy. It is therefore recommended that the hydrosalpinx be treated prior to IVF cycles. Although there can be different treatment modalities based on patients’ needs and special circumstances, removal of the diseased tubes is the most common and acceptable approach. There is now sound medical evidence that removal of hydrosalpinx prior to IVF improves pregnancy success. In cases of extreme adhesions of the pelvis where removal is not safe, proximal tubal ligation has been shown to be as effective as the complete removal of the tubes in improving pregnancy outcome. Two randomized controlled trials documented improved pregnancy rates with the treatment of hydrosalpinx prior to IVF.
c) Endometrial thickness:
A poor endometrial lining most commonly occurs in women with a history of unexplained recurrent IVF failures or early recurrent miscarriages and is usually attributable to: 1) inflammation of the uterine lining (endometrium), i.e., endometritis, 2) multiple fibroids of the uterine wall, 3) prenatal exposure to the synthetic hormone, diethylstilbestrol (DES), 4) in women who have received clomiphene citrate (Clomid, Serophene). Thin lining (less than 8 mm) has been associated with lower success rates with IVF. When there is such a history of thin endometrium, possible factors in the etiology (causes) should be explored and treated. Additionally, in the next treatment cycle special attention should be paid to the endometrium by accurate and multiple measurements and addition of vaginal or intramuscular estrogen should also be considered. We also utilize vaginal Viagra in such cases and have observed a significant improvement in the endometrial lining and overall pregnancy outcome in most patients.
d) Immunologic factors:
Implantation occurs six to seven days after ovulation. At this time, specialized embryonic cells (trophoblast), which later become the placenta, begin growing into the uterine lining. When the trophoblast and the uterine lining meet, they become involved in a possible 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, it is not surprising that failure of proper function of this immunologic interaction during implantation has been implicated as a cause of recurrent miscarriage, late pregnancy fetal loss, IVF failure, and infertility. Immunologic factors that may be involved in these situations includes anti-phospholipid antibodies (APA) and activated natural killer cells (NKa).
4. The Embryo Transfer:
Embryo transfer (ET) is the last and one of the most important steps of IVF treatment. Correct placement of the embryos in the least traumatic way is of utmost importance. The use of ultrasound guidance to optimally place the embryos in the uterus has become an integral part of the ET process. It has also been shown by multiple studies to improve pregnancy rates. Symptoms such as moderate uterine cramping or bleeding after the procedure are indicators of a difficult transfer. Patients with such a history may benefit from a trial transfer and Dr. Bayrak performs such an intervention at the time of egg retrieval to avoid difficulty at the time of the actual embryo transfer in his patients.
5. Sperm Factor – Sperm DNA Integrity (SDI):
The Sperm DNA Integrity (SDI) like the Sperm Chromatin Structure Assay (SCSA) is a tool for measuring clinically important properties of sperm nuclear chromatin integrity. The results correlate well with the potential of sperm from a given male to produce an embryo that would be competent to produce a live birth. The SDI assay utilizes the metachromatic features of acridine orange (AO), a DNA probe and the principles of flow cytometry (FCM).
SDI assay data are not well correlated with classical sperm quality parameters and have been shown to predict poor reproductive performance. The SDI assay measures DNA damage, which may be present in sperm from both fertile and infertile men. Therefore, this sperm DNA damage analysis may reveal a hidden abnormality of sperm DNA in infertile men classified as unexplained based on apparently normal standard sperm parameters. However, the data suggests that an abnormal SDI assay is more likely to occur in cases of abnormal semen analyses. Thus the assay is ideally suited to fertility clinics to assess male sperm DNA integrity as related to fertility potential and embryo development as well as effects of reproductive toxicants. SDI/SCSA parameters can be independent of conventional semen parameters (normal semen analysis) and a high percentage of DNA damage may identify the cause of infertility in most cases.
