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CGH Egg/Embryo Selection To Optimize IVF Success

Introduction and Background
Successful treatment of reproductive failure demands full prior identification and treatment of those factors that adversely influence both embryo “competence” (the ability of an embryo to propagate a normal pregnancy) and uterine “receptivity” (i.e., thickness of the uterine lining, immunologic modalities, anatomical integrity of the uterus, as well as infective and biochemical factors).

While advances both in methods and drugs used for ovarian stimulation as well as improvements in embryo culture techniques have undoubtedly had a positive influence, IVF success rates have lagged and even stagnated over the last 10 years. This is largely due to an inability to reliably identify and selectively transfer only “competent” embryos (those that are capable of producing a healthy baby) to the uterus. Even in young women, an embryo that “looks good” under a microscope is not necessarily competent. At best, it has a 25% chance of implanting. Furthermore, this statistic shrinks drastically with advancing age beyond 35 years.

Even the use of preimplantation genetic diagnosis (PGD) using fluorescence in-situ hybridization (FISH) to identify chromosomes does not significantly improve this capability. As a result, many IVF specialists still transfer multiple embryos at a time to increase the odds that at least one competent embryo will reach the uterus and produce a pregnancy. The problem is that while this improves the chance of a pregnancy occurring, it also markedly increases the risk of multiple gestations/pregnancies.

A convenient and applicable parallel that we often draw to illustrate the relative importance of embryo competence versus uterine receptivity (in the IVF equation) is that of a “seed/soil relationship.” The ability of an embryo (the “seed”), upon reaching a receptive uterine environment (the “soil”) to successfully implant and develop into a healthy baby (the “plant”), is no different than what takes place in a regular agricultural setting. In simple terms, it is determined by establishing an ideal seed/soil relationship. It follows that it is no more possible to achieve a viable healthy pregnancy when a “competent” embryo is transferred to a “non-receptive” uterus than when an “incompetent” embryo is transferred to a receptive uterus.

In human reproduction, the establishment of an ideal seed/soil relationship is pivotal, since both embryo competence and uterine receptivity are indispensable to the development of a healthy baby. It is, however, an undeniable fact that reproductive failure (i.e. failed implantation, miscarriages and major birth anomalies) are far more likely to be due to embryo incompetence (70-75%) than to a lack of uterine receptivity (25-30%).

It is mostly (but not exclusively) the embryo’s chromosomal configuration that will determine its “competence”. The number of chromosomes in a cell is referred to as its ploidy. A cell with a normal number of chromosomes is referred to as euploid while one with an irregular chromosome number is aneuploid. It appears that it is the ploidy of the mature egg (rather than the sperm) that determines the post-fertilization chromosome configuration of the embryo. The embryo’s ploidy, in turn, determines its competence.

The relatively lax and unregulated IVF setting in the United States has provided a safety net for those wishing to transfer multiple embryos, and this in turn has led to a virtual explosion in the incidence of multiple births in this country. The enormous short and long term financial costs associated with IVF multiple births (many of which are related to prematurity) represents one of the main reasons why health insurance providers in this country are reluctant to cover the procedure.

There is a profound lack of correlation between the microscopic appearance (grading) of embryos and embryo “competence”. Moreover, Preimplantation Genetic Diagnosis/Screening (PGD/S) of human eggs and embryos for their chromosomal integrity, using traditional fluorescence in-situ hybridization (FISH) is only fractionally more reliable. The reason is that conventional FISH cannot fully access all the chromosomes - in fact, only about 12 of them. Thus, even when FISH reveals that all the accessed chromosomes are normal, there still remains more than a 40% chance of chromosomal aneuploidy involving those chromosomes not targeted by the test…and the incidence increases to above 50% by the time the woman reaches 40 years of age. This constitutes a serious drawback when it comes to attempting to select the most “competent” eggs or embryos for dispensation in ART.

We reported on studies involving the performance of CGH on a fragment of nuclear material (the first polar body or PB-1) that is routinely discharged from the egg during the chromosomal rearrangement that takes place following the “hCG trigger”. The PB-1 has a chromosomal makeup which is a mirror image of the chromosomes in the egg’s nucleus. Several hours following fertilization, the PB-1 divides and a second polar body (PB-2) appears alongside it. PB-1 biopsy involves removal of PB-1 from immediately below the outer envelopment (zona pellucida) of the pre-fertilized egg while a pb-2 biopsy refers to removal of the 2nd PB, soon after fertilization has occurred. Both PB-1 & PB-2 biopsy can be achieved without damaging the egg itself.

The study referred to above was performed on eggs extracted from women aged 25-42 years who underwent ovarian stimulation with fertility agents. We first performed PB-1 and PB-2 egg biopsies before and after fertilization. Thereupon, we biopsied the resulting day-3 embryos by removing a single cell (blastomere) from each. The PB-1, PB-2 and blastomere samples were sent for genetic testing using comparative genomic hybridization (CGH) to identify all of the chromosomes in the samples tested.

Subsequently we transferred up to 2 embryos derived from eggs that previously had tested chromosomally normal to the uteri of 35 women. The study revealed the following remarkable information:

• The chromosomal make-up of the egg, rather than the sperm, is the main determinant of an embryo’s chromosomal integrity and its ability to develop into a baby. (i.e., its “competence”).
• Even in young women, >60% of all mature eggs are likely to be aneuploid and thus incapable of producing “competent” embryos.
• The incidence of egg aneuploidy increases progressively with advancing age such that by the mid-forties it is probably above 90%.
• Eggs that have abnormal quotas of chromosomes (i.e. are aneuploid) will, upon fertilization, invariably propagate aneuploid, “incompetent” embryos. Such embryos will either fail to attach to the uterine lining or will attach and then subsequently miscarry early on in pregnancy.
• Approximately 85% of eggs that have a normal number of chromosomes (i.e. euploid) fertilized with normal sperm will subsequently develop into “competent” embryos.
• The transfer of 1-2 euploid (CGH-tested) embryos to a receptive uterine environment (free of immunologic and anatomical irregularities), has better than a 60% chance of resulting in a live birth.
• Embryos that fail to progress to the blastocyst stage will almost always develop into aneuploid, “incompetent” embryos. This finding all but dispels the erroneous contention that embryos might be better off being transferred to the uterus prior to reaching the blastocyst stage.
• Most IVF failures and early miscarriages are almost always attributable to embryo aneuploidy. It follows that by only transferring euploid, “competent” embryos, this risk will be significantly reduced.

Male Factor Contributions
Fertilization of an egg by a dysfunctional spermatozoon significantly increases sperm contribution to the development of aneuploid embryos. This is more likely to occur in cases of moderate to severe male factor infertility. Given that male infertility is responsible for more than 50% of infertility, it follows that it would be preferable to perform CGH analysis on the embryo (rather than the egg). This would improve the accuracy of CGH-diagnosis in diagnosing embryo competence. Accordingly when predicting embryo “competence” we shifted from egg to embryo CGH testing.

Our subsequent study reported in the prestigious medical journal, “Fertility and Sterility” (December, 2009), described the use of embryo (rather than egg-PB) CGH testing which confirmed the accuracy of this approach and showed that regardless of the age of the embryo recipient, the transfer of 1-2, CGH-normal embryos results in better than a 60% pregnancy rate and a marked reduction in miscarriage rate.

Staggered In Vitro Fertilization (St-IVF)

Staggered-IVF involves the use of CGH testing of day 3 embryos in order to identify those that are the most “competent” (i.e. chromosomally normal) embryos. St-IVF involves dividing the IVF cycle into 2 separate stages. The first stage involves ovarian stimulation, egg retrieval fertilization and embryo or blastocyst biopsy and ultra rapid freezing (vitrification) and storage of all blastocysts. The second stage which occurs, in a subsequent cycle, involves hormonal preparation of the recipients uterus followed by the transfer of one or more blastocysts. The reason for dividing the cycle into two parts is to allow for sufficient time to complete CGH analysis of DNA derived from the biopsied material removed in the second stage of St-IVF).
St-IVF improves the efficiency of the IVF process by:

• Markedly improving the birth rate per embryo transferred
• Avoiding the need to transfer several embryos at a time thereby reducing the likelihood of high order multiple pregnancies (triplets or greater)
• Reducing the incidence of chromosomal abnormalities in those embryos that are transferred

Egg/Embryo Competency Testing using CGH, while clearly a major breakthrough in the IVF arena is not a panacea. First, an embryo diagnosed to have all its chromosomes (euploid) through egg and/or embryo CGH testing will, in about 15% of cases, turn out to be “incompetent.” This is due to the fact that even chromosomally normal cells, upon further division at times can generate some aneuploid cells, leading to a condition known as Mosaicism (the presence of both chromosomally normal and abnormal cells) in the blastocyst. Depending on the percentage of aneuploid cells in the advanced embryo (blastocyst), mosaicism might not be lethal. Second, a competent embryo might fail to continue developing because of poor uterine receptivity rather than embryo aneuploidy. Third, Embryo transfer (ET), another rate-limiting factor in IVF, requires a great deal of technical expertise and there is a wide variation in such expertise.

The above serves to explain why the transfer of “competent” (CGH-normal) embryos will result in a live birth rate of 60-70%...not in 100% of cases.

Use of CGH technology represents a major step toward consistently achieving a pregnancy, but it has limitations. By understanding such limitations, we can work toward achieving even better results: First, an embryo, diagnosed to be euploid through single-blastomere CGH, is not always “competent”. Second, a competent embryo might not attach because of poorly understood uterine receptivity issues. Third, there is a wide variation in technical expertise when it comes to the performance of embryo transfer, a rate-limiting factor in IVF.

Using CGH to Select Eggs for Freezing (Banking)
In the past, egg freezing and banking has yielded dismal success (a 1-4% baby rate per frozen egg). After all, freezing a non-viable egg is a futile and fruitless exercise. The introduction of CGH to select only “competent” eggs for freezing has the potential to revolutionize this field. SIRM recently published a study that evaluated birth rates in women who underwent IVF using frozen/thawed eggs pre-selected by CGH. The study revealed that by combining CGH egg selection with a new freezing technique known as vitrification, birth rates from frozen eggs could be increased as much as 6-7 times over current averages!

About Array CGH (aCGH)
There are 2 types of CGH processes used in IVF: the first is metaphase CGH (mCGH) and the second is Array CGH (aCGH) (also called Microarray). To date we have used mCGH, a labor intensive and complex procedure that requires at least 5 days to perform once. Since a single run-through will yield inconclusive results in up to 20% of the DNA samples tested, we often need to repeat the process more than once in order to reduce the percentage of “inconclusive” results. This explains why it requires several weeks to obtain optimal results with mCGH and accordingly why it is necessary to defer embryo transfer to a subsequent cycle (Staggered IVF).

It is true that Array CGH is a much faster and less complex method for performing CGH. While aCGH can even be completed within several days, its accuracy is still in question, when used to evaluate a minute amount of DNA derived from a single PB or single blastomere. In fact, that is why aCGH (as currently applied to IVF) requires access to much more DNA derived from several cells at a time. This means that by and large, aCGH can only be reliably performed on blastocysts, where there are a large number of cells (>100), and several can be removed for testing without damaging the embryo. The problem is that most (if not all) blastocysts have some aneuploid cells (due to mosaicism). As such, when several cells are removed (biopsied) and CGH-tested at a time, there is presently no way of accurately knowing how to interpret the presence of one or more aneuploid cells and at what cut-off it would still be “safe” to transfer such blastocyst(s).

Also, the cost of performing aCGH is significantly higher per sample tested than for mCGH. Finally, as with mCGH done on day 3 embryos, blastocyst aCGH likewise mandates that the blastocyst(s) be frozen and then transferred in a subsequent cycle. But all this could change when/should it become possible to reliably conduct aCGH on single cells (as required for day 3 embryo testing). When this transpires, (provided that aCGH proves to be reliable when so applied and that the cost of aCGH decreases significantly) then it will become possible to perform fresh embryo transfers in the same cycle that the CGH testing was performed. We anticipate this will likely happen in the next few years. Until then it is our opinion that embryo-mCGH with St-IVF will remain the method of choice.

Conclusions

• CGH Embryo selection St-IVF does not improve embryo quality in a given cycle of ovarian stimulation. Rather, it allows for the identification and selection of high quality, “competent” embryos for transfer. As such, while it dramatically improves the live birth rate per embryo transferred and brings us much closer to a time where single embryo transfers will be come standard, it will not improve IVF outcome per stimulation cycle or per-egg retrieval.
• While CGH testing (as is also the case for FISH-PGD) markedly reduces the risk of numerical chromosomal birth defects such as Down’s syndrome, it does not absolutely preclude their occurrence. In fact, the anticipated error rate could be ≤5%. Thus all women undergoing CGH or FISH egg/embryo selection and who seek absolute confirmation that numerical chromosomal birth defects will not occur should still undergo prenatal genetic testing in the 1st or 2nd trimester.
• The potential application(s) of egg/embryo/blastocyst karyotyping through CGH should be regarded as a “work in progress.” Things are still very fluid, and are likely to change over time. Hopefully, with responsibility, honesty, and careful evaluation, this will ultimately evolve to the betterment of all.
  

DQ-alpha Matching in IVF: The Controversy, How It Affects Outcome, & How to Treat!

IVF patients, especially those who find themselves inexplicably repeatedly failing treatment after treatment are no longer willing to blindly accept platitudes from those who would ignore the role of immunologic causes of IVF failure while unable to offer no other alternative plausible explanation. It is largely through the initiative and insistence and persistence of such vocal patients that the veil over the entire issue of immunologic implantation dysfunction has started to lift and has resulted in rapidly growing scientific interest in and attention being paid to the role of selective immunotherapy in IVF.

There are two (2) forms of immunologic implantation dysfunction. The first and by far the most common is autoimmune implantation dysfunction. This variety is usually easily and successfully remedied through treatment with heparinoids (e.g., Lovenox, Clexane), Intralipid (IL), and corticosteroids. The second variety which is often ignored or overlooked is alloimmune implantation dysfunction.

Autoimmune implantation dysfunction is by far the most common variety. It is believed to be implicated in >90% of cases of immunologic implantation dysfunction and occurs when an immunologic reaction is produced by the individual, to his/her body’s own cellular components. Aloimmune implantation dysfunction on the other hand, arises through the reaction of the uterus to an embryo that shares certain genetic (genotypic) similarities (DQa and other HLA genes)with the recipient’s uterus causing immune cells known as natural killer (NK) cells that populate the uterine lining, to start over-producing “ toxins” known as TH-1 cytokines (TNFa and Interferon gamma). Such activated NK cells (NKa+) attack the cells of the embryo’s “root system” (the trophoblast) damaging it and so compromising implantation. Alloimmune implantation dysfunction, while far less common than the autoimmune variety is considerably more complex, much more poorly understood (even by most RE’s) and far more difficult to treat successfully. It involves a reaction by an otherwise normal uterus to the intrusion of one or more embryos that through the contribution of sperm DNA share certain immunogenetic (genotypic) similarities with the recipient.

For some reason, there is a tendency to consider all couples with alloimmune implantation dysfunction (who share DQa similarities) to be incapable of achieving a viable full term pregnancy. Nothing could be further from the truth.

Let me explain: Each individual has two (DQa’s), one is derived from their mother and the other from their father. The fact that many individuals carry identical DQa's (i.e. both are the same), of necessity means their parents must of necessity have had “matching” DQa’s and yet they were born healthy and normal. The reason is that it is not the “matching” DQa that matters. It is whether upon arriving in the uterus, a DQa “matching” embryo encounters activated uterine natural killer cells (NKa+). These NKa+ release large amounts of TH-1 cytokines that attack and damage the cells of the embryo’s “root system” (trophoblast).It is the extent of such trophoblastic damage that will determine whether such an embryo will immediately “die on the vine” (implantation failure) or “limp along” for some time only to be aborted a few weeks later.

It is important to recognize that NK cell activation only occurs after repeated exposures to DQa-"matching" embryos. This explains why a DQa “matching” embryo that reaches the uterus prior to NK cell activation can and often does implant successfully and then go on to propagate a healthy pregnancy. However, with repeated exposures to DQa "matching" embryos, uterine NK cells will ultimately and inevitably become activated. Such NK cell activity will initially often be limited and accordingly TH-cytokine production will wax and wane (in between exposures), allowing .early implantation (albeit with a damaged embryo) to proceed and even proceed for a limited period of time, only to abort in the first trimester. Ultimately, over time following repeated and successive exposures to DQa-“matching” embryos, NK cell activation will become a permanent feature. Once this happens uterine NK cell activation (as measured by the K-562 target cell test) will exacerbate to the point that as soon as the embryo reaches the uterus implantation will be thwarted and the woman will be considered as being "infertile” when in reality she is experiencing a very early, preclinical miscarriage. .

It is important to understand that DQa “matching” refers to a (genotypic) “match” between the male and female partners…rather than a “match” between sperm and egg. An immature sperm contains 23 pairs of chromosomes”…for a total of 46. With maturation division (following meiosis), the immature sperm divides into two mature sperm each of which comprises 23 chromosomes [including only one (1) DQa gene]. Upon fertilization of the egg by such a sperm this single DQa gene is incorporated into the embryo’s genotype. .If that DQa gene “matches” either of the mother’s two (2) DQa’s, then the potential for NK call activation will arise. It follows that if only one (1) of the husband’s two (2) DQa’ genes “matches” either one (1) of the mother’s DQa’ genes, the potential for the resulting embryo to propagate an embryo that containing a DQa gene that “matches” the recipient will be 50%. On the other hand, if both the husband’s DQa’ genes are the same as any one of the mother’s two DQa’s, (or if the mother and father both identical DQa’s genotypes), then 100% of the embryos will “match” and the propensity to activate uterine NK cells will be markedly increased.

What does all this mean when it comes influencing IVF outcome? …….Well, if we are dealing with a 50% chance of embryo DQa “matching” (see above), and we can successfully down-regulate NKa+ through the administration of Intralipid (IL) or immunoglobulin-G (IVIG) in combination with corticosteroids (e.g. prednisone or dexamethasone), then the transfer of a non-“matching” embryo would theoretically provide the same chance of a successful IVF outcome as in the absence of any DQa “matching” between the partners. On the other hand, when the chance of embryo DQa “matching” is 100% (see above) the ability to down-regulate NKa+ with IL or IVIG is diminished as is the likelihood of a successful pregnancy.

What emerges from all this is that not all DQa “matches” are equal. Outcome following IVF treatment (inclusive of IL/IVIG/corticosteroids) is very much influenced by: a) the presence and severity of uterine NK cell activation, b) whether the DQa genotype of both male and female partners “match” absolutely (i.e. both their pairs of their DQa genes “match”), in which case 100% of the embryos will “match” and the prognosis will be poor, c) whether both the male’s DQa genes are identical, in which case, the of a DQa “match” will again be 100% and the chance of a successful IVF outcome will likely be severely diminished.

It is presently not possible to reliably identify the paternal DQa contribution to the embryo. Also, the exposure of DQa “matching” embryos to the uterus will usually activate uterine NK cells. For these reasons, in cases of a 50% risk of a DQa “match”, I usually recommend transferring only one (1) embryo at a time. The reason is my concern that in transferring more than one embryo, uterine exposure to a DQa-“matching” embryo could, by causing local NK cell activation, compromise implantation of a non-“matching” embryo and so, in the process, reduce the likelihood of its successful implantation. In cases of 100% DQa “matching”, this hardly matters since all the embryos would cause NK cell activation anyway.

In truth, when there is a 100% risk of an embryo-DQa “match” between partners (see above) in association with uterine NK cell activation as measured by the K-562 target cell test, the chance of successful pregnancy is very small. In such cases, in my view seeking the help of a gestational surrogate or resorting to the use of donor sperm (ensuring they do not share DQa similarities with the embryo recipient) will in the final analysis become the treatment of choice.

The recent introduction of comparative genomic hybridization (CGH) to identify and select “competent” embryos for transfer can markedly improve the efficiency by which we are able to manage both alloimmune and autoimmune implantation dysfunction.

Lastly; much has been written about the use of endometrial sampling (biopsy) to measure NK cells and cytokine activity. While this is interesting in concept, there is no supportive clinical data to indicate its value in the clinical management of immunologic implantation failure. Presently the K-562 target cell test remains the gold standard for measuring uterine NK cell activity.

Video: A Glimpse Inside Sher Institutes

This is a video that we produced to give insights into our day-to-day events, our staff, our patients and our philosophy:

Infertility Evaluation: A Critical First Step

In cases of infertility, where because of the woman's age (>39 years) or diminished ovarian reserve her biological clock is running out of time, the imperative often exists to move directly to IVF. However, since the majority of infertility (of male and/or female origin) occurs in situations where the woman is younger and has adequate ovarian reserve, there is usually ample opportunity to first consider other less invasive, less expensive and insurance-covered, non IVF options. In such cases, nothing is more fundamental to optimal management than is the initial performance of a detailed and comprehensive basic infertility work-up.

Preparatory Tests

1. On the third day of a spontaneous or progesterone withdrawal menstruation, blood is drawn for the measurement of estradiol (E2), follicle stimulating hormone (FSH), luteinizing hormone (LH) and selectively, for Inhibin-B.


2. Blood should also be drawn (any time) for the measurement of Prolactin, TSH and antisperm antibodies (ASA).


3. Commencing on the second day (2nd) of the menstrual cycle, a basal body temperature chart should be initiated. A thermometer is placed in the mouth for a period of two (2) minutes upon awakening (prior to the ingestion of food/liquid and brushing of your teeth). The temperature should be documented graphically on the basal body temperature chart provided.


4. For women under 35 yrs of age without evidence or symptoms suggesting underlying organic pelvic disease (eg; endometriosis, chronic inflammation, pelvic adhesions, fibroids etc):
   A hysterosalpingogram (HSG) should be performed within a week of the cessation of menstruation. This outpatient procedure involves injection of a radio-opaque dye which outlines the Fallopian tubes allowing the diagnosis of tubal blockage . To a lesser degree, it permits the detection of surface lesions inside the uterine cavity.
   OR
   For all women over 35 yrs of age and for younger women who have evidence or symptoms pointing to underlying organic pelvic disease (e.g., endometriosis, chronic inflammation, pelvic adhesions, fibroids etc): A laparoscopy/hysteroscopy should be performed within a week of the cessation of menstruation. Laparoscopy is a procedure where a telescope-like instrument is introduced through the belly button into the abdominal/pelvic cavity allowing diagnosis and treatment of ovarian cysts/endometriomas/benign tumors, uterine fibroids , tubal blockage, ectopic pregnancy, appendicitis, pelvic adhesions etc. Laparoscopy is usually performed as an out-patient procedure with the patient under general anesthesia. It is one of the only ways to diagnose early pelvic endometriosis accurately. Hysteroscopy is a procedure where a telescope-like instrument is inserted, via the vagina through the cervical canal into the uterine cavity, for the evaluation of the interior of the uterus. It is an important procedure because it allows for diagnosis and treatment of small surface lesions inside the uterine cavity (e.g. polyps, scarring or adhesions) that adversely affect the ability of an embryo to attach to the uterine lining. Such lesions are often missed through the performance of an HSG.


5. Commencing at least 17 days before the expected next menstrual period( ie; usually about 10 days following the initiation of menstruation), urine should be collected twice daily and tested for the onset of the spontaneous LH surge. The initiation of the LH surge usually precedes ovulation by 8 to 36 hours. In order to detect the onset of the LH surge as early as possible, it is important that urine be tested at least twice daily. This is done as follows:
   

A. The bladder is emptied first thing in the morning, upon awakening. One half-hour later urine is collected (only a very small amount is required) and tested using an over-the-counter LH kit (obtainable at a drug store). The earliest sign of any color change should be documented. It need not be a pronounced color change as suggested by the insert in the kit. Any alteration in coloration is significant.

B. The same process of testing is then repeated at night before retiring.

C. At the earliest sign of a color change the couple should:
· Have intercourse, then arrange to have the first in-office physician’s assessment within 6-18 hours following intercourse.
· The woman should RUSH IN to the physician’s office ASAP to have her blood drawn for the measurement of estradiol (E2) l level. Timing is critical, because within approximately 6 hours of detecting LH in the urine, (which roughly coincides with 12 hours after the actual onset of the LH surge), blood estradiol levels start to fall precipitously. If blood is drawn too late, the measurement of estradiol will be of little value.

Note: If the color change is observed in the early morning, the woman should schedule the “first in-office assessment” at the doctor’s office for the afternoon of the same day. If it occurs at night, the doctor’s office should be contacted first thing the next morning and the “first office assessment” should take place within hours.

The First In-Office Assessment

1. A Post-Coital Test (PCT) or Huhner test is performed on the cervical mucus. The purpose of the PCT is to assess sperm survival within the mucus. Since sperm can only survive for six hours in the vagina, a positive PCT is indicative of:
   
   A. Good quality sperm.
   B. Good sperm/cervical mucus interaction, suggesting that there will be safe passage of sperm to the uterine cavity.
   C. Absence of anti-sperm antibodies (ASA) in the sperm or mucus.
   D. That the production of estrogen is adequate.
   E. That the endometrial lining is well primed by estrogen, which is essential for adequate preparation of the uterine lining for implantation.


2. Cervical mucus is cultured for:
   A. Ureaplasma Urealyticum (this requires a specialized medium to transport the specimen to the laboratory).
   B. Chlamydia and Gonococcus (these also require a specialized transport medium).
   C. Aerobic and anaerobic pathogens.


3. A sample of the cervical mucus is allowed to dry on a glass slide and is examined under the microscope for specific features such as “ferning”, which is indicative of an adequate estrogen effect.


4. A vaginal ultrasound examination is performed to detect the presence of at least one dominant follicle that measures 18mm in mean diameter, thus helping confirm that ovulation is imminent. It also allows for the assessment of the thickness and appearance of the endometrial lining. A normal endometrium should measure at least 9 millimeters in sagital diameter at this time.

The Second In-Office Assessment

This visit is scheduled three (3) days after the first office assessment. At this visit, a vaginal ultrasound exam is performed to check whether ovulation has occurred (i.e. whether the egg has been released). The presence of small amount of fluid collecting in the lowermost region of the pelvis or a change in the shape of the follicle is suggestive of ovulation.

The Third In-Office Assessment

The third visit takes place five (5) days after the 2nd visit. At this visit, blood is drawn for the measurement of progesterone (P4) and Estradiol (E2)

The Fourth In-Office Assessment

The fourth and final visit is scheduled for five (5) days after the office assessment. At this visit, an endometrial biopsy is performed. This is a simple in-office procedure, whereby a sliver of uterine lining (endometrium) is removed and sent to the laboratory to evaluate histologic changes in the endometrium.

INTERCURRENT TESTING (i.e. any time in the cycle):

Tests On The Female Partner

1. An immunologic work-up may be required in certain cases of female infertility or where there is a past history of recurrent pregnancy loss. This workup includes measurement of: 1) antiphospholipid antibodies (APA), 2) antithyroid antibodies (ATA) 3) a Natural Killer Cell activity (NKa) test, a.k.a. K-562 Target Cell Test. In select cases, both partners should be tested for alloimmune similarities (DQa and HLA). The blood should be sent to a specialized Reproductive Immunology Reference laboratory, as such tests cannot usually be performed in regular Laboratories because the methods they employ are neither sensitive nor specific enough to be of value in cases of reproductive failure.


2. For patients who anticipate going into an In Vitro Fertilization cycle sometime in the near future, blood should be drawn for the measurement of HIV, Hepatitis B surface antigen, Hepatitis C antibody and RPR (a Syphilis test), blood grouping, RH testing as well as a Rubella antibody test . Such tests will usually not be required in the course of a routine basic infertility work-up. Their performance should be confined to cases where it is anticipated that Assisted Reproductive Technology (ART) procedures such as In Vitro Fertilization or GIFT, will be the primary approach.


3. In select cases, a diagnostic laparoscopy and concomitant hysteroscopy should be performed. The former is the only reliable way to evaluate for endometriosis and to assess tubal patency. A hysteroscopy permits examination of the uterine cavity for surface lesions (polyps, scar tissue, fibroids) and developmental abnormalities (e.g. a uterine septum) all of which can affect reproductive performance.

Tests On The Male Partner

1. A semen analysis is required for accurate measurement of sperm motility and count. Sperm morphology is assessed employing “strict Kruger criteria.” Semen should also be cultured for Ureaplasma Urealyticum, Chlamydia, Gonococcus and for aerobic/anaerobic pathogenic organisms.


2. In addition, the man’s blood should be tested for anti-sperm antibodies (ASA).


3. If In Vitro Fertilization is being considered, the man should also undergo blood testing for Hepatitis B surface antigen, Hepatitis C antibodies, RPR (Syphilis) and HIV.


4. Ideally, the semen should also be sent for a Sperm Chromatin Structure Assay (SCSA) to assess the DNA Fragmentation Index (DFI) which ideally should be <30%.

Unexplained Infertility: True Diagnosis or Cop Out?

For about 10% of all infertile couples, the cause of the infertility cannot be readily determined using conventional diagnostic methods. Such cases are often referred to as "unexplained infertility." The truth, however, is that in most such cases, this diagnosis is in fact “presumptive” because a more in-depth evaluation would have revealed a cause. This having been said, people diagnosed with so called “unexplained infertility” fall into two broad groups:

a. Those couples who don't have any biological problems interfering with pregnancy;
b. Those who do, but the reason cannot be found, due to insufficient medical information or technology.

It is in group b that improved testing techniques have made infertility easier to diagnose and treat. In order to make even a presumptive diagnosis of “unexplained infertility” the answers to the following questions must be in the affirmative.

- Is the woman ovulating normally?
- Is the couple having intercourse regularly in the periovulatory phase of the cycle?
- Are the fallopian tubes normal and open?
- Can endometriosis be excluded?
- Does the male partner have normal semen parameters (most specifically with regard to sperm count and motility)?
- Is the post coital "Huhner" test (a periovulatory examination of cervical mucous, done 6-18 hours after intercourse) normal?

The fewer tests performed, the more likely a presumptive diagnosis. The definitive diagnosis of “unexplained infertility” has a lot to do with the thoroughness of the health care provider in excluding all possible causes.

For Example:
Abnormalities of the fallopian tubes: Adhesions or developmental defects of the finger-like “petals” at their outer ends of the tubes that help sweep eggs inside (fimbriae) can prevent eggs from being collected and transported to the awaiting sperm.

• Chromosomal abnormalities of eggs or embryos: Eggs must be euploid (contain the right number of chromosomes) to be successfully fertilized; embryos must also be euploid in order to implant successfully in the uterine lining. Until recently, there was no reliable method for determining whether eggs and embryos were euploid. The recent introduction of genetic tests such as comparative genomic hybridization (CGH) now allows for identification of all chromosomes in the egg and embryo. As such, CGH represents an important addition to the diagnostic armamentarium.
• Luteinized Unruptured Follicle (LUF) Syndrome: Here, the eggs can become trapped in the follicle and not be released (“trapped ovulation”). In such cases, routine tests done to detect ovulation (temperature charting, urine LH testing, blood progesterone levels) may be normal, resulting in false interpretation that ovulation is actually occurring.
• Ovulation (hormonal) Dysfunction: Abnormalities in ovarian hormone production in the preovulatory phase of the cycle (follicular phase defect) and/or in the postovulatory phase (luteal phase defect) can negatively affect preparation of the uterine lining (endometrium), thus thwarting normal implantation.
• Immunologic implantation dysfunction (IID): Sometimes, the male or female partner’s own immune system can attack sperm cells, killing them or causing them to become immobilized. Also, immunologic dysfunction involving the uterine lining can cause the implanting embryo to be rejected so early that the woman does not even recognize that she had in fact conceived.
• Cervical infection - Ureaplasma urealyticum: Infection of the cervical glands can prevent sperm from migrating through the cervix and uterus to reach the egg in the fallopian tube(s). Such infection will usually not be detectable through routine examination and/or cervical culturing methods.
• Mild or Moderate Endometriosis: Endometriosis is, in 100% of cases, associated with the production of “pelvic toxins” that reduce the fertilization potential of otherwise normal eggs by a factor of 3-5x. In addition, about 1/3 of women with endometriosis (regardless of its severity) have immunologic implantation dysfunction (IID). Furthermore, mild and even moderately severe endometriosis can often only be accurately diagnosed by direct visualization of the lesions through laparoscopy or laparotomy. The detection of IID requires highly sophisticated tests that can only be adequately performed by a handful of Reproductive Immunology Reference Laboratories in the United States. Finally, a condition called nonpigmented endometriosis, in which the endometrium may be growing inside the pelvic cavity with many of the same deleterious effects as overt endometriosis, cannot be detected even by direct vision (at laparoscopy/laparotomy). The fertility of these patients may be every bit as compromised as if they had detectable endometriosis.
• Psychological Factors: The entire reproductive process is governed by the brain. Thus it should come as no surprise that stress and negativity can interfere with hormonal balance and decrease the ability to conceive.

Management of "Unexplained Infertility"
Successful management of “Unexplained Infertility” requires that a very individualized approach be taken. Wherever possible, the underlying cause should first be identified. Problems that involve ovulation dysfunction (hormonal imbalance) require ovulation induction with oral or injectible fertility drugs. Cervical mucous hostility due to infection with ureaplasma (which is transferred back and forth sexually to both partners) requires specific and concurrent antibiotic therapy. In other cases involving younger women (under 39 years) where there is a problem with sperm migration via the cervix and uterus to the fallopian tube(s), intrauterine insemination (IUI) with or without ovulation induction, is indicated.

When these treatments fail, in vitro fertilization (IVF) is needed. This is also generally the case in women over the age of 39 years, women with IID, men or women who harbor antisperm antibodies in significant concentrations, and in cases associated with tubal abnormalities, All cases of intractable, moderate or severe male infertility call for injecting sperm directly into the egg to achieve forced fertilization (intracytoplasmic sperm injection or ICSI).

It is an indisputable fact that most causes of infertility can be diagnosed. In my opinion, it is a great pity that the diagnosis of “unexplained infertility” is often used as an excuse for not having performed a full and detailed evaluation of the problem. Couples should not simply accept a diagnosis of “unexplained infertility” at face value since treatment is most likely to be successful when the specific cause of the problem can be fully identified.

Octuplet Pregnancy: Poor Medical Judgment, Patient Indiscretion, or Both?

The entire debacle surrounding Nadya Suleman and her IVF “Octuplets” raises serious ethical issues. A formal complaint to the California Medical Board was recently filed, resulting in the censuring of the doctor who performed the IVF procedure that led to this travesty. This has sounded an alarm that it is time for all well intended people involved in Reproductive Health Care to take action. The situation represents an example of “medical science gone wild,” but at the same time it evokes concern about the unregulated field of Reproductive Medicine in general and invites the question, “Is this field on the verge of going out of control?” There can be no tiptoeing around the fact that the Hippocratic Oath which binds physicians to “do no harm” was ignored in this unfortunate case.

Notwithstanding the magnitude of understandable outrage surrounding the IVF octuplet debacle, it must be recognized that it merely scratches the surface of a far larger issue, namely, the fact that far too many IVF practitioners in the United States still feel compelled to transfer multiple embryos at a time. Such practice has resulted in a virtual explosion in the rate of IVF multiple births, which are associated with a markedly higher risk of prematurity, low or very low birth weight, perinatal death and more frequent lingering neurological complications, as well as an increased risk of birth defects.

When comparing singleton with twin and triplet pregnancies we find the following:

• Twins have 3-times greater mortality rate and triplets, 6-times greater than singleton pregnancies.
• Twins have a 6-times greater likelihood of developing cerebral palsy and triplets an 11-times greater likelihood.
• Twins are 50%, and triplets 80% more likely to be born prematurely.
• Mothers of twins are 3-times, and mothers of triplets, 7-times more likely to experience serious pregnancy-induced complications.

First, most infertile patients simply do not perceive any great risk associated with multiple gestations, especially when it comes to twins. In fact most, consider multiple pregnancy to be a “bonus”…a favorable outcome. Faced with the high emotional and financial cost associated with IVF treatment, most couples prefer to complete their families in one attempt so as to “maximize the use of their resources.” In fact, when asked, almost 90% of couples undergoing IVF in the United States are desirous of having twins. Some are even interested or covet having high order multiples (triplets or beyond). Education is urgently needed to make IVF candidates fully aware of the risks associated with multiple gestations.

Second is the relative inability to reliably differentiate between embryos that will propagate a healthy pregnancy and those that will not. Most IVF patients erroneously believe that a “pretty”, embryo (one given a high embryo grade because it fulfils the microscopic criteria of “good quality”) should invariably propagate a baby. This is simply not the case.

Numerous studies have demonstrated that the cumulative birth rate after single embryo transfer (SET), followed by subsequent transfers of individual thawed left-over embryos, is as effective in achieving pregnancy as implanting multiple embryos at one time. And by this approach, the risk of multiple births can be virtually eliminated. Moreover, using the SET approach, more than 80% of women under 40 years will have a baby within 4 attempts.

But it was the recent the introduction of genetic tests such as comparative genomic hybridization (CGH) that, by allowing for the identification of those embryos that are most likely to propagate a viable pregnancy, promises to make it even easier to avoid multiple births. Yes indeed, the transfer of a single CGH-selected embryo results in a healthy baby more than 60% of the time. And what is more, such genetic embryo markers can also improve the efficiency of the IVF process by reducing miscarriages and minimizing the risk of chromosomal birth defects such as Down’s Syndrome. With such new technology, the dream of “one embryo, one baby” will hopefully soon become a reality.

No physician seeks to limit the freedom by which he or she practices medicine. On the other hand, when outrages such as IVF-octuplet pregnancies occur, it is time to go back to the drawing board and re-examine/revamp existing practices so as to avoid repetition of such blunders.

Perhaps the time has finally come for mandated regulations that would limit the number of embryos permitted to be transferred to IVF patients in this country.