Fertility and Infertility Research News Portal

“Immature, Mature and Post-Mature Eggs” – Confusing and Misleading Terminology

About 38-42 hours following the onset of the spontaneous LH surge in normally ovulating women as well as after the administration of human chorionic gonadotropin (hCG) to women undergoing ovarian stimulation with fertility drugs, the 46 chromosomes in the human egg begin to segregate…to divide in two. This process, known as meiosis or maturational division is designed to leave the mature egg (MII) with precisely 23 chromosomes in its egg nucleus. The remaining 23 are expelled from the egg nucleus (enveloped on a thin membrane) and come to lie in the viteline space (located between the outer membrane of the egg (the oolema) and the egg’s outer shell (envelopment) or zona pellucida. The purpose of the expulsion of this so called first polar body (PB-1) is to ensure that following fertilization by a mature spematazoon (whose chromosome number has also been reduced from 46 to 23), the resulting embryo so propagated would have regain the full quota of 46 chromosomes that makes up the human genome.

Thus the detection by microscopy, of a PB-1 situated immediately under the zona pellucida, indicates that maturational division (meiosis) has been completed. However, it does NOT confirm that chromosome segregation has take place evenly, i.e. that precisely 23 chromosomes remain in the egg nucleus (i.e. that the egg is “euploid”). The presence in the MII egg of one or more chromosomes above or below 23 in number, is referred to as “aneuploidy”, a condition that almost always inevitably in failed embryo development, failed implantation, miscarriage or a chromosomal birth defect such as Down’s syndrome.

As it turns out, even in younger women a half to one two thirds of MII eggs are aneuploid and this incidence increases rapidly with advancement in age beyond 35 years.

It follows therefore that the absence of a PB-1 on microscopic evaluation (i.e. an MI egg) clearly indicates that the egg had not completed meiosis, and thus “is aneuploid” and “incompetent” (i.e. is incapable of propagating a viable, healthy embryo. On the other hand, the detection of a PB-1 under the zona pellucida (an MII egg) while confirming that meiosis has been completed, offers no assurance that the egg is in fact “euploid” and thus is potentially “competent”. Quite to the contrary…most MII eggs are in fact “aneuploid” and thus totally ‘incompetent”.
It is by and large the chromosomal integrity of the egg, rather than the sperm that determines embryo “competency”. Thus egg “competency” is an essential prerequisite for the propagation of a viable embryo and a healthy baby.

Another interesting fact is that in more than 90% of cases, an embryo that fails to reach the blastocyst stage (>100 cells), will be aneuploid, “incompetent” and thus are doomed from the get go. On the other hand, although not invariably the case, embryos that do make it to the blastocyst stage are much more likely to be euploid and thus “competent.” In fact, even in young women, at least 50% of blastocysts are aneuploid and thus “incompetent.” This percentage increases progressively with advancing age. While age is the main determinant of what percentage of eggs are aneuploid, the protocol used for ovarian stimulation as well as the timing of the hCG “trigger” can also influence the incidence of chromosomal abnormalities. When the hCG trigger is administered too early or too late, the egg might not be developmentally positioned to undergo orderly meiosis and either 1) be unable to expel half its chromosomes as a PB-1 and thus remain an M-I egg, or 2) the PB-1 may be expelled, but could contain an irregular number of chromosomes. In the latter case, the egg would have a visible PB-1 and would thus be labeled as a “mature” (MII) egg, though it would be aneuploid and “incompetent.” The terms “immature” and “post-mature” as applied to eggs , are often interpreted as meaning that the eggs were either harvested (retrieved) too early or too late, and that performing the egg retrieval a day or two earlier or later would have prevented this from happening. This infers that an MI egg was harvested before it was developmentally ready to enter meiosis and that egg post-maturity results from waiting too long before the hCG trigger. This inference is completely erroneous. In fact, an M1 egg could just as easily have resulted from delaying the hCG trigger shot, as it could from administering it too early. Likewise a “postmature” egg can result just as readily from administering hCG too early as too late. For these reasons, the terms “immature” and “post-mature,” as applied to eggs, should be supplanted by the term “dysmature” which simply means that the M-1 or M-2 egg in question is maldeveloped, aneuploid and “incompetent”.

Posted on March 10, 2010 | Filed under IVF Treatment | Permalink

“Immature Eggs” and “Post-Mature Eggs” – Confusing and Misleading Terminology

An M1 or “immature” egg has 23 pairs of chromosomes (i.e. 46-total). Within 38-42 hours of the natural preovulatory surge in Luteinizing Hormone (LH) or following the hCG “trigger,” each chromosome pair divides in two. The first polar body (PB-1) comes to lie outside of the egg membrane, immediately beneath the egg’s outer envelopment (zona pellucida). The PB-1 contains the 23 chromosomes that are expelled from the egg in a membranous envelopment during this maturational or “ripening” process known as meiosis.

The detection of PB-1 under the zona pellucida via microscope, provides evidence that meiosis has taken place. However, it does not provide any indication as to whether more or less than 23 chromosomes reside in either the egg nucleus or in the PB-1.

Likewise, the term M-II egg refers to the fact that meiosis took place but in no way indicates that the matured egg is euploid (i.e., has exactly 23 chromosomes). An egg that has more than or less than 23 chromosomes is referred to as aneuploid and is “incompetent” (i.e., incapable upon fertilization of developing into a chromosomally normal, “competent”) embryo.

This so-called “competency” is an essential prerequisite for an embryo to be able to propagate a viable, healthy baby. It is a fact that most such aneuploid eggs fail to fertilize, or upon fertilization will arrest (stop dividing). Those that continue to divide will often fail to reach the most advanced preimplantation stage of development (blastocyst). In more than 90% of cases, an embryo that fails to reach the blastocyst stage (>100 cells), will be aneuploid and thus “incompetent.” Such aneuploid embryos are in fact doomed from the get go.

On the other hand, although not invariably the case, embryos that do make it to the blastocyst stage are much more likely to be euploid and thus “competent.” In fact, even in young women, at least 50% of blastocysts are aneuploid and thus “incompetent.” This percentage increases progressively with advancing age.

While age is the main determinant of what percentage of eggs are aneuploid, the protocol used for ovarian stimulation as well as the timing of the hCG “trigger” can also influence the incidence of chromosomal abnormalities. When the hCG trigger is administered too early or too late, the egg might not be developmentally positioned to undergo orderly meiosis and either 1) be unable to expel half its chromosomes as a PB-1 and thus remain an M-I egg, or 2) the PB-1 may be expelled, but could contain an irregular number of chromosomes. In the latter case, the egg would have a visible PB-1 and would thus be labeled as a “mature” (MII) egg, though it would be aneuploid and “incompetent.”

The terms “immature” and “post-mature” as applied to eggs , are often interpreted as meaning that the eggs were either harvested (retrieved) too early or too late, and that performing the egg retrieval a day or two earlier or later would have prevented this from happening. This infers that an MI egg was harvested before it was developmentally ready to enter meiosis and that egg post-maturity results from waiting too long before the hCG trigger. This inference is completely erroneous. In fact, an M1 egg could just as easily have resulted from delaying the hCG trigger shot, as it could from administering it too early. Likewise a “postmature” egg can result just as readily from administering hCG too early as too late.

For these reasons, the terms “immature” and “post-mature,” as applied to eggs, should be supplanted by the term “dysmature” which simply means that the M-1 or M-2 egg in question is maldeveloped, aneuploid and “incompetent”.

Posted on March 8, 2010 | Filed under IVF Treatment | Permalink

Upcoming Infertility Seminars Where I’ll Be Speaking

SIRM will be hosting a series of free infertility seminars in March and April where I’ll be speaking on fertility issues and breakthroughs as well as answering questions with other SIRM physicians. The dates are as follows:

March 25th: Easton, PA
March 27th: Clinton, NJ
March 28th: Dallas, TX
April 14th: Peoria, IL

The link below has details, maps and registration info:

SIRM Infertility Seminars

I look forward to meeting you!

- Geoff Sher

Posted on March 5, 2010 | Filed under IVF Treatment | Permalink

ICSI associated with increase in Stillbirth Rate? What a Recent Danish Study Means

A recent Danish study reported a 4-fold increase in still births following IVF with intracytoplasmic sperm injection (ICSI).

In the past, intracytoplasmic sperm injection (ICSI) , an in vitro-fertilization method, has been used predominantly in cases of moderate or severe male factor infertility. More recently many IVF centers have applied ICSI as a preferred method of fertilization in non-male infertility cases, as well. It is well recognized that when ICSI is performed for male infertility there is a definite increase in embryo defects and related miscarriages.

A few years ago, a large study in Sweden (2003) followed by one reported from Egypt (2004) clearly showed that when ICSI is performed for non male factor infertility, IVF outcome is not prejudiced, and the rate of pregnancy loss and birth defects are unaffected.

Since the Danish study does not differentiate between cases where ICSI was done for male factor versus non male factor infertility, it is likely that their finding of an increase in still births might be due to the effect of abnormal sperm on the embryo’s health rather than being due to the ICSI process itself. And… since male factor infertility requires ICSI, there is no avoiding this procedure in such cases anyway.

Posted on February 26, 2010 | Filed under IVF Treatment | Permalink

Embryo Splitting To Increase the Availability of “Competent” Embryos For IVF

I recently received several inquiries from visitors to our SIRM discussion boards (http://forums.haveababy.com/index.php?showforum=1 ) on the subject of “embryo splitting” to try and improve the opportunities to conceive through IVF. Here is one example of such an inquiry that recently appeared on the SIRM-Las Vegas regional discussion board:

“I’ve been reading on embryo splitting (for readers: manually splitting embryos in the lab …..in order to increase the number of embryos to transfer/freeze). The AMA seems to not have an issue with it and understands that it could help infertile couples increase their chances: (www.amassn.org/ama/pub/physician-resources/medical-ethics/code-medical-ethics/opinion2145.shtml)”

Against this background, I wish to share the article below with those of you that may be interested in this topic.

Posted on February 23, 2010 | Filed under IVF Treatment | Permalink

Egg Quality and Ovarian Reserve: The Effect of FSH and Inhibin Blood Levels

In most cases, when basal blood inhibin levels fall, so too will the woman’s Follicle Stimulating Hormone (FSH) level. It is likely that inhibin produced by the granulosa cells lining the cavity of early follicles signal an area in the brain known as the hypothalamus which produces small “releasing hormones” that direct the woman’s pituitary gland to regulate the production and release of FSH. This also explains why basal inhibin and FSH as well as the number of early follicles (antral follicles) represent reasonable measures of ovarian reserve, which in turn correlates with the woman’s potential to produce follicles on her own and/or in response to ovarian stimulation with fertility drugs.

Low FSH/inhibin and antral follicle count (AFC) in the first few days of a woman’s cycle suggests diminished ovarian reserve and serves as a warning that the woman will not produce an optimal number of mature follicles and eggs in response to ovarian stimulation. It also serves to prompt the use of a more aggressive, yet individualized approach to ovarian stimulation.

Most women develop diminished ovarian reserve about 6-8 years prior to the onset of menopause (in the period termed the “climacteric”) but in some cases this happens at a much younger age (i.e. “a premature climacteric”). Thus, the basal FSH, inhibin level, and AFC are good indicators of ovarian reserve and the number of follicles that are likely to develop, given an optimal protocol for ovarian stimulation. However, these parameters alone are not good predictors of subsequent egg and/or embryo quality. It is the woman’s age and the protocol designed to effect ovarian stimulation that plays a major role here, and these measurements can aid in designing the ideal stimulation protocol.

Let me explain! Human eggs undergo degradation in quality over time, such that by age 39, an egg (ovulated or harvested at egg retrieval) will on average have about a 20% chance of being genetically/chromosomally normal. This is about one half the chance at age 35 and under. By the time she reaches her mid forties, that number will decrease by half again (i.e. reaching less than 10%). This “wear and tear” effect on egg quality is an inevitable consequence of the advancing “biological clock“. So, when it comes to egg quality, it is the woman’s age and the protocol of ovarian stimulation that are the most important determinants. You simply cannot stimulate a woman in her 40’s or for that matter a woman with diminished ovarian reserve using the same “recipe” (i.e. the stimulation protocol) as you would prescribe for a younger woman who has normal ovarian reserve. If you do not individualize the protocol of stimulation, you are highly likely to propagate the development of poor quality eggs that have a disproportionately increased likelihood of having chromosomal abnormalities.

Again, the most important factors affecting a woman’s egg quality are 1) the woman’s age, and 2) her ovarian reserve. While these two variables may be linked (women are more likely to develop diminished ovarian reserve as they get older), women sometimes do experience a “premature climacteric” and egg quality deteriorates with advancing age regardless of ovarian reserve. Thus, these two contributing factors should be seen as related, but independent variables.

So how then does age and/or diminishing ovarian reserve affect egg quality? First, as stated above, it is inevitable that with advancing age, egg quality will decline. Second, for follicles to grow and for eggs to develop normally (an essential prerequisite for proper genetic maturation), the tissue surrounding the follicle (ovarian stroma or theca) must produce testosterone. Stromal testosterone production requires luteinizing hormone (LH), which is produced by the woman’s pituitary gland and is also acquired through some varieties of injectable fertility medications (Menopur, Repronex and Luveris) given in the course of ovarian stimulation. The testosterone then gets carried in “bucket brigade” fashion to the granulosa cells lining the inside of the adjacent follicle(s). Here, under the influence of FSH, it gets converted to estrogen (mainly estradiol/E2). In the process, the follicle grows and the egg it harbors within undergoes vital developmental changes in preparation for final genetic maturation (”ripening”) that occurs with the spontaneous surge of LH that triggers ovulation (or following the administration of the hCG during ovarian stimulation).

Thus, a small amount of testosterone is needed for optimal egg quality (though too much testosterone can be harmful to the egg as I will discuss below). Eggs that have the genetic potential to transform into genetically “competent,” mature eggs will do so within 36 hours of the spontaneous pre-ovulatory LH surge, or following hCG-induced ovulation.

The important consideration here is that there should not be preovulatory over-exposure of the developing egg to testosterone, something that is most likely to happen when older women and/or those with premature diminution in ovarian reserve are prescribed a suboptimal protocol for ovarian stimulation. If there is overexposure to testosterone, egg development and subsequent egg/embryo quality can be severely compromised along with the chance of a healthy pregnancy. Since older women (>39 years) and women with diminished ovarian reserve tend to produce an excess of LH and have a tendency to over-produce testosterone, this is where the problem lies.

What does this all mean in the context of preparing a woman for a cycle of ovarian stimulation? First, it means that a young woman who has diminished ovarian reserve should still be capable of producing produce good quality eggs, albeit in a smaller number, provided that she gets prescribed an individualized and customized protocol that is designed to prevent over-production of ovarian testosterone. Conversely, an older woman with diminished ovarian reserve will, because of the inevitable effect of age on egg quality, produce a higher percentage of poor quality, genetically “incompetent” eggs.

Finally, when it comes to natural cycle IVF, it should be recognized that some women will produce up to 2 or 3 follicles. Some will be smaller than others, but even the smaller ones can yield eggs. However, as with regular IVF (with ovarian stimulation), the quality of her eggs will be inevitably be influenced by her age. Thus the success rate following natural cycle IVF in such cases will be very much lower than for younger women, especially if they have diminished ovarian reserve. The success rate with natural cycle IVF, even in young women, is only about 10% per cycle. In older women, it is under 5%. Furthermore, older women are more likely to have increased LH production. In addition, the LH they produce becomes more potent with advancing age and thus is much more likely to evoke a greater ovarian testosterone response with a negative effects on egg/embryo quality (and correspondingly on the likelihood of IVF success).

In summary, natural cycle IVF is a much less effective and successful form of IVF across the age spectrum. If it is used, it should be confined to younger, ovulating women who have a normal ovarian reserve.

Posted on February 17, 2010 | Filed under IVF Treatment | Permalink

The “Vanishing Twin”: What Does It Mean?

Today, in first world environments where there is ready access to advanced medical technology, many women undergo ultrasound diagnosis of pregnancy as early as 5-6 weeks after their last menstrual period. As a result, multiple pregnancies are usually recognized very early on. Serial ultrasound follow-up examinations in such cases have shown that often times one of the developing babies “mysteriously” vanishes, with the remaining conceptus (baby) proceeding to a healthy birth. Since most multiples comprise twin pregnancies, the term “vanishing twin” is used to describe this situation. While in most cases the loss of a vanishing twin is associated with painless, innocuous bleeding, this is not always the case. In fact bleeding might not occur at all.

The incidence of spontaneous pregnancies resulting in twin births is about 1:80. In women under 35 treated with clomiphene citrate it is about 1:20. After ovarian stimulation using injectible gonadotropin fertility medications, it increases to 1:5 and after IVF (given the current tendency to replace multiple embryos), the incidence of twins is about 1:3.

What many fail to appreciate is that about 1 in 10 spontaneous pregnancies start off as twins, but as the pregnancy advances into the 1st trimester and beyond, one twin will “vanish” (absorb) while the other will continue as a healthy, unaffected singleton. When this happens, the painless mild bleeding (or spotting) raises understandable concerns by the affected woman who often asks:

Q. – Am I about to miscarry?

Answ: The bleeding results from the absorption of one of the pregnancies, and since the vast majority of twin pregnancies have separate and independent placentas, the loss of one will accordingly usually not affect the remaining twin. As long as the bleeding remains mild and she does not experience an increase in cramping and pain over a period of a few days, the pregnancy will probably not be lost. In fact, in the majority of such cases this is precisely what happens.

Q. How long will I continue to bleed?

Answ: In most cases – unless the pregnancy is destined to miscarry completely – the bleeding will remain painless, mild, and will stop within a week or so. However, this will depend on when the fetus was lost. If it occurred late in the first trimester, the bleeding will usually last longer (even a few weeks) than when the pregnancy is lost earlier. It should be borne in mind however, that some women don’t bleed at all, and their body reabsorbs the one twin with no outward indication of the loss.

Q. How will the loss of one twin affect the surviving one?

Answ: In the majority of cases, the other baby will usually progress unaffected to a healthy birth.

Q. – Will there be any remaining evidence of the vanished twin at birth?

Answ: Usually not! Sometimes a small area of scarring or “thickening” of part of the placenta will be seen. However, this will usually only occur in cases where the first twin succumbed late in the first trimester (10-12 weeks) or in the 2nd trimester.

Q. – Could the vanishing of one twin have been prevented? Did I do something wrong?

Answ: The vanishing twin is not the result of something the mother/father did – or failed to do. In most cases, the vanishing twin is lost because it is chromosomally or genetically abnormal. In a small number of cases it could result from alloimmune implantation dysfunction. Since many vanishing twins are lost very early in pregnancy, before most women will have undergone an ultrasound to confirm pregnancy, most cases go undetected. The woman would have no knowledge that she had been carrying more than one fetus. In fact, as stated above, many more of us begin life as twins than was previously thought.

Vanishing concepti can also occur within higher order multiple pregnancies. A triplet pregnancy can reduce spontaneously to a twin or singleton and so can a quadruplet pregnancy.
In the final analysis, individuals and families who experience the vanishing of a conceptus will experience anxiety and even panic when bleeding starts, and a sense of relief when it finally stops, and they learn that the remaining fetus and the pregnancy have survived. Thereafter there will inevitably be a sense of loss, sadness and even grief, especially if they had anticipated and looked forward to a multiple birth.

Posted on February 11, 2010 | Filed under IVF Treatment | Permalink

Share Your Stories on the SIRM Facebook Page

Many of our patients have shared their inspirational stories on our Facebook page. We would love to hear yours! Just follow this link:

http://www.facebook.com/pages/Sher-Institutes/213018641760

Posted on February 5, 2010 | Filed under IVF Treatment | Permalink

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.

Posted on February 3, 2010 | Filed under IVF Treatment | Permalink

Staggered IVF Using CGH Egg/Embryo Selection To Optimize 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 all evolve to the betterment of all.

Posted on February 2, 2010 | Filed under IVF Treatment | Permalink

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.

Posted on January 29, 2010 | Filed under IVF Treatment | Permalink

DQ-alpha Matching in IVF: The Controversy, How Does it Affect 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 aloimmune 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. Aloimmune 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 aloimmune 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 can now manage both aloimmune 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 is value in the clinical management of cases o immunologic implantation failure. Presently the K-562 target cell test remains the gold standard for measuring uterine NK cell activity.

Posted on January 23, 2010 | Filed under IVF Treatment | Permalink

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:

Posted on January 21, 2010 | Filed under IVF Treatment | Permalink

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 inflamation, pelvic adhesions, fibroids etc):
   A hysterosalpingogram (HSG) should be performed within a week of the cessation of menstruation. This out-patient 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 (eg; endometriosis, chronic inflamation, 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 acurately. 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) tha 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 te 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 Assesment

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%.

Posted on January 17, 2010 | Filed under IVF Treatment | Permalink

The General 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 inflamation, pelvic adhesions, fibroids etc):
   A hysterosalpingogram (HSG) should be performed within a week of the cessation of menstruation. This out-patient 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 (eg; endometriosis, chronic inflamation, 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 acurately. 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) tha 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 te 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 over the counter, 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 Assesment

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 a 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, and 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%.

Posted on January 15, 2010 | Filed under IVF Treatment | Permalink

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.

Posted on January 9, 2010 | Filed under IVF Treatment | Permalink

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.

Posted on January 6, 2010 | Filed under IVF Treatment | Permalink

Will Lowering An Elevated FSH Level Improve Response To Fertility Medications?

The short answer is NO. A woman with diminished ovarian reserve, as indicated by an elevated day-3 blood FSH level, does not have the time to waste on relatively low-efficacy treatments. Regardless of age, she should be looking preferentially at IVF rather than alternatives such as Controlled Ovarian Hyperstimulation (COH) with or without intrauterine insemination.

The basal blood FSH value is a reflection of ovarian reserve (how many eggs are still left in the ovaries) and is not the CAUSE of a poor follicle/egg production. It is also NOT the cause of poor egg quality.

What is true is that women with diminished ovarian reserve are “poor responders” to COH (produce fewer eggs). Their stimulation protocols must be individualized so as to down-regulate their own pituitary production of LH, in advance of initiating COH. This means that agonist “flare” protocols and the administration of LH antagonists (e.g. Ganirelix, Cetrotide) starting 5-7 days after COH with gonadotropins has commenced, should be avoided. Also, they should not receive much LH/hCG-containing fertility drugs such as Repronex or Menopur. Failure to recognize this can compromise egg/embryo quality and decrease the likelihood of a pregnancy (see my previous post on IVF Stimulation Protocols)

Day-3 FSH levels will often fluctuate from month to month. A higher or lower FSH level on day 3 of a particular cycle does NOT mean that she will respond better if stimulated with fertility drugs in that cycle.

Simply stated, there is no point in postponing ovarian stimulation for a subsequent cycle to wait for the Day-3 FSH level to come down. It only puts an extra burden on an already taxed biological clock and the bottom line is that it won’t help. You cannot suddenly grow more eggs for recruitment by waiting for the FSH to fall to a lower level. Likewise, it is useless to go on to a birth control pill or take an estrogen/progesterone preparation to lower the blood FSH level in the hope of improving subsequent ovarian response to gonadotropin stimulation.

Posted on December 30, 2009 | Filed under IVF Treatment | Permalink

Egg Banking of Genetically (CGH)-Tested Eggs Could Change The Face Of IVF-Ovum Donation

For more than a quarter century, medical scientists have attempted to defy the biological clock by freezing a woman’s eggs to preserve her fertility. Most of these efforts have failed. Consider the fact that since the birth of the world’s 1st “frozen egg baby” in the mid 1980’s, fewer than 1,000 such births have been reported worldwide. Compare this to more than 3 million IVF babies born in the same time period and approximately 25,000 IVF births per year from frozen embryos.
Until very recently, the reported statistical chance of a frozen and thawed egg ultimately resulting in a baby has been under 4%.

There are two major reasons why egg freezing has been such a dismal failure. The first is the fact that even in young women; more than 60% of eggs are chromosomally defective and thus non-viable, making egg freezing a hit and miss gamble to begin with. The second reason is that prior methods used to slowly freeze human eggs, caused ice crystals to form within the egg, damaging it to the point that a high percentage of frozen eggs did not survive the freezing process (let alone make it to the viable embryo stage.

In October 2008, we reported in the Journal “RBM Online” on a process that dramatically improves the frozen egg birthrate 7-8 fold. It involves identifying and selecting chromosomally normal eggs using a genetic test known as Comparative Genomic Hybridization (CGH), and then applying a new ultra-rapid (600 times faster) freezing method called vitrification to preserve these chromosomally normal eggs safely and indefinitely in a state of suspended animation.

The process of combining CGH testing with selective egg vitrification, and the banking of only chromosomally normal eggs has several potential applications, the most obvious of which are:

• Fertility Preservation, for women who want to defer childbearing till later

• Fertility Rescue, for women who want to store and preserve their eggs pr before undergoing fertility-threatening cancer treatment

• Donor Egg Banking, where selected viable eggs would be stored and subsequently made commercially available for IVF and embryo transfer to women for whom egg donor-IVF provides the only means by which they can go from infertility to family.

Rather than waiting, often on a long waiting list, to access a preferred fresh egg donor, patients worldwide would be able to choose one or more eggs from a web-based catalogue. The web-based catalogue would give detailed information on the background of the egg donor(s) that produced the eggs the patient is potentially interested in purchasing. Upon purchase, the frozen eggs would be shipped to the patient’s Physician. The entire process from beginning to end should not take longer than 4-6 weeks to complete.

So, what advantages would a CGH- Donor Egg Banks have over the banking of non-genetically tested eggs?

1. Each CGH-normal egg, upon being thawed is about 7 times more likely to result in a live birth.

2. Given this improved viability, pregnancies resulting from such embryos are 3-4 times less likely to miscarry than embryos derived from non-CGH tested eggs.

3. Because the baby rate per CGH-normal embryo transferred is about 60% (more than 80% of embryos derived from CGH-normal eggs would likely also be chromosomally normal), there is no need to ever transfer more than 2 such embryos at one time. As a result, the risk of triplets or greater is negligible.

4. Given that such embryos are derived from eggs that have been chromosomally tested as normal, there would be a minimal risk of chromosomal birth defects such as Down’s syndrome.

The cost in the United States of donor egg IVF is the highest in the world (all told, about $25,000-$30,000 per cycle). As a consequence, many women/ couples currently travel abroad for lower cost treatment. In fact, such “medical tourism” is fast becoming a significant industry in. European countries such as Spain and the Czech Republic, India, South Africa and Australia.

In the United States, much of the cost of donor egg IVF relates to the amount paid as donor stipends. As currently conducted, IVF using donor eggs is both complex and cumbersome. It requires donor selection which is often a tedious process that takes months to complete, independent consultation and medical evaluations of both the recipient and the egg donor, thorough microbiologic and genetic testing of all parties involved, followed by synchronization of the recipient’s menstrual cycle with that of the chosen egg donor. Thereupon, the donor is stimulated with fertility drugs and monitored. At precisely the right time, the donor receives a trigger shot with human chorionic gonadotropin (hCG) and the recipient’s hormone treatment regime is altered to coincide. About 36 hours later, the recipient’s eggs are harvested under anesthesia and fertilized with designated sperm. The embryos are cultured and when, 3 to 5 days later they reach a certain stage of development, are transferred to the recipient’s uterus.

By allowing patients to procure one or more banked CGH-normal eggs (derived from fully pre-tested donors) for fertilization and subsequent transfer of the resulting embryo(s) to a hormonally prepared uterus markedly improves efficiency without compromising success rates.

A few IVF programs and egg donor agencies already offer their clients access to Donor Egg Bank services. However, the dismal reported average baby rate per non-CGH tested frozen egg (i.e. <4%) means that a recipient would need to purchase numerous eggs to have a realistic chance of a successful pregnancy. By offering patients fully karyotyped, CGH-normal banked eggs; they would not need to purchase more than 1or 2 eggs for about $3,000 per egg at a time. Since each CGH-normal egg would likely yield about a 27%-28% baby rate (i.e. a 7 to 8-fold improvement over other options), the birth rate following the transfer of 2 resulting embryos would be about 65%. This success rate is as high as any comparable reported results with conventional IVF-egg donation and it would come at 1/2-1/3 the current cost for such services in the United States.

Posted on December 25, 2009 | Filed under IVF Treatment | Permalink

Human Cloning: What Does It Involve And Would It Be Going Too Far?

Following the successful cloning of an adult sheep announced in Scotland a few years ago, various commentators – from scientists and theologians to physicians, legal experts, talk-radio hosts and editorial writers – raised concerns about the prospect of cloning a human being. At the request of the President, the National Bioethics Advisory Commission (NBAC) held hearings and prepared a report on the religious, ethical, and legal issues surrounding human cloning, recommending a moratorium on efforts to clone human beings.

As background, the term “cloning” refers to three procedures, each with very different objectives. The three different types of cloning are:

1. Reproductive Cloning: The objective is to replicate an existing animal by removing the DNA of one of its cells and swapping this with the DNA in an egg from the same species. Following this process of “artificial fertilization”, the resulting “pre-embryo” is either transferred directly to the uterus or is allowed to divide several times with the resulting embryo being transferred. Reproductive cloning has been used to clone sheep and other mammals. In the process however, serious genetic and developmental defects have been noted in more than 30% of offspring. The likelihood that the same attrition rate would occur in humans, coupled with the belief that cloning disregards the sanctity of human life, has caused many medical ethicists to brand this procedure as morally repugnant. There have been a few claims of successful cloning in humans, but to date, none of these have been substantiated.
2. Embryo Cloning: This is an experimental medical technique referred to as “embryo splitting”. If successful it would produce monozygotic (identical) twins or triplets by replicating the process that nature uses to produce identical twins or triplets. By this process, the embryo is virtually sliced in half or into thirds, allowed to develop further and the separate sections are transferred to the uterus. This procedure would be unlikely to produce an increased risk of birth defects, and while it could have the potential to enhance the likelihood of pregnancy in infertility patients, who have been classified as “poor responders”, its introduction would almost certainly evoke an ethical firestorm. This having been said, possible advantages could be:
   a. The potential to achieve improved IVF success following the acquisition of a single good quality embryo.
   b. By increasing the number of transferable embryos derived from fertilization of each egg, there could be the potential to obtain acceptable IVF pregnancy/birth rates in women who, because of age and or “poor response to fertility drugs,” are otherwise hard pressed to produce even a single “viable” embryo with IVF.
3. Bio-therapeutic Cloning: The purpose of this method is to generate embryos for research. This would involve mainly the harvesting of early embryonic “stem cells” that have the potential to develop in different tissue types, dependent upon the environment into which they are delivered and the stimulus evoked. The production of a healthy replica of a diseased tissue or organ could be vastly superior to relying on organ transplants from other people. What is more, the supply would be unlimited. Theoretically at least, there would be no risk of post transplant organ rejection and therefore no need to use immunosuppressive drugs.

A poll conducted a few years ago revealed that: 90% of Americans thought that cloning was bad; 67% felt that cloning animals was also a bad idea; 45% believe that humans would be cloned within a decade; 90% believed that human cloning is “against G-d’s will”; while 23% thought it was not.

Posted on December 23, 2009 | Filed under IVF Treatment | Permalink

Follow Heidi through her IVF Journey on Oprah.com

One of my current IVF patients is journaling her IVF experiences for Oprah Winfrey’s website. You can read her journal here. She gives an inspiring and very human perspective on what so many women go through!

Posted on December 19, 2009 | Filed under IVF Treatment | Permalink

How Many Times Should You Try IVF Before Giving Up?

Because of the emotional, physical, and financial toll exacted by IVF, it is preferable that a couple undertake the process with the mindset that they will be in it for more than one attempt. If a couple can only afford one treatment cycle, IVF may not be the right course of action. Recall that on average, with conventional IVF, there is only about one chance in three that it will result in a live birth, and there is a tremendous letdown if it fails. It is thus unreasonable to undergo IVF with the attitude that “if it doesn’t work the first time, we’re giving up.” In vitro fertilization is a gamble even in the best of circumstances.

Statistically speaking, a woman under 40 years of age, using her own eggs, having selected a good IVF program is likely to have a better than 70% chance of having a baby within three completed attempts – provided that she has adequate ovarian reserve, (the ability to producing several follicles/eggs in response to gonadotropin stimulation), has a fertile male partner (or sperm donor sperm) with access to motile sperm, and has a normal and receptive uterus capable of developing an “adequate” uterine lining. Women of 39-43 years of age who meet the same criteria, will likely have about half that chance (35%- 40%).

When the most “competent” embryos are selected for transfer using a new genetic process (introduced into the clinical arena by SIRM in 2005), known as comparative genomic hybridization (CGH), the birth rate per single, completed IVF cycle is likely to exceed 60% (regardless of the age of the egg provider) and, more than 85% within three such attempts.

Unfortunately, there will inevitably always be some women/couples who in spite of best effort at conventional IVF will unfortunately remain childless. In my considered opinion, it rarely advisable to undergo more than three IVF attempts using the same approach each time. There is of course one important caveat: in women where the reason for repeated IVF failure is finally uncovered, it would indeed be justifiable (assuming there are sufficient emotional, physical and financial resources) to continue trying, using a defined and new approach that addresses the reason for prior failures. Simply stated, “the time to stop trying is when there is no remediable explanation for repeated failure to achieve a viable pregnancy”.

One very interesting case comes to mind. It happened a few years back when I consulted with a 42 year old Australian patient (she happened to also be a physician) who had undergone 22 prior failed attempts at IVF elsewhere. After determining that the reason for prior failures (at least in part) was due to a hitherto unrecognized immunologic implantation dysfunction (IID), I took her through yet another IVF attempt using selective immunotherapy. She conceived (using her own eggs) and went on to have a healthy baby boy. This case serves to point out that the time to stop doing IVF should not always be based on the number of prior failed attempts alone.

When conventional IVF (with or without egg donation and/or CGH embryo selection) fails to yield a successful outcome, other options such as ovum donation, IVF surrogacy, or adoption should be considered.

Although it is the right of any healthy women who has a uterus and is capable of producing even one follicle/egg to have the right to decide on doing IVF using her own eggs, given the very low success rate after 43 years of age (less than 10% per attempt and under 25% within 3 tries) it is my opinion that women over 43 years should be advised to rather do egg donor IVF. Here, regardless of the age of the embryo recipient, the IVF birth rate after a single attempt is about 60% – and better than 80% within three IVF attempts.

Couples who choose to undergo IVF should be encouraged to view the entire procedure with guarded optimism, but nevertheless must be emotionally prepared to deal with the ever?present possibility of failure. It is important for IVF patients to be made to realize from the outset that an inability to become pregnant should never be considered a reflection on them as individuals.

Posted on December 19, 2009 | Filed under IVF Treatment | Permalink

IVF and Immunology: The Role of Immunologic Factors and Selective Immunotherapy

Few innovations in IVF have evoked the degree of controversy and bad press as has the thesis that reproductive immunologic factors play a role in unexplained IVF failure and recurrent pregnancy loss, and that immunotherapy presents a valid treatment of these problems unexplained IVF failure and recurrent pregnancy loss.
This having been said, the fact remains that there are thousands of women with repeated and unexplained IVF failure, and/or who have suffered recurrent miscarriages, that following diagnosis and slectively immunotherapy, have subsequently gone on to have healthy babies.

The question then is, why this militant obsession to denounce the validity of an immunologic component in reproductive failure?

Bear in mind that there is very little we currently do in the clinical realm of assisted reproductive technology (ART) that could stand the test of being supported by the results derived from randomized controlled statistical analyses. This does not in any way mean that their use is invalidated. Such gold standard statistical testing requires that the influence of all variables be controlled, other than the one(s) being evaluated (kept constant). This would allow the one in question to be subjected to randomized testing. Only in this way can the results be truly and objectively validated.

Such stringent criteria are simply impossible to apply in the ART setting. That is why virtually every clinical treatment/process currently used in the field gained acceptance through a process of longitudinal efficacy testing …..by trial and error, not through gold standard statistical testing.

Let me illustrate with an example: No studies have ever definitively established a real benefit in choosing IVF over alternatives such as gamete intrafalopian tube transfer (GIFT) or Zygote intrafallopian tube transfer (ZIFT) . Yet no one would argue that IVF is far more effective than either alternative. In fact, both GIFT and ZIFT have fallen into disrepute, are hardly ever used anymore, and have been relegated to the pages of history.

Similarly, there are no gold standard randomized statistical studies that have demonstrated a benefit in using adjunct procedures such as assisted hatching (AH), embryo co-culturing, nuclear cytoplasmic transfer, intracytoplasmic sperm injection (ICSI), or the preferential use of one IVF protocol over another. Yet good and honorable physicians who practice in this arena tout such approaches as being efficacious and even selectively superior, often with “dubious conviction.” Why then have many such treatments become entrenched in the IVF therapeutic armamentarium, and why the double standard when it comes to Reproductive Immunotherapy in IVF?

Consider the following: Embryo abnormalities account for 75% of IVF failure, while implantation problems (including immunologic factors) only account for 25%. It follows that it would be impossible to statistically assess the role of immunologic factors or the effect of selective immunotherapy on IVF outcome unless we can be sure that the embryo(s) being transferred is/are “competent” (i.e. are chromosomally normal and thus upon reaching a receptive uterus, would propagate a viable pregnancy). ..

Microscopic embryo grading falls far short of the mark in reliably differentiating between “competent” and “incompetent” embryos. The suggestion that preimplantation genetic diagnosis (PGD) with commercially available fluorescence in-situ hybridization (FISH) can assess embryo “competence”, is erroneous. In fact, PGD/FISH does not help much, because at best, it can only examine half of the embryo’s chromosomes).

The recent introduction of a new genetic test known as comparative genomic hybridization (CGH) performed on embryos could change all this. Unlike FISH, CGH reliably identifies all the chromosomes in the embryo, thereby allowing for reliable differentiation between chromosomally normal (“competent”) and abnormal (“incompetent”) embryos, thus permitting the selective transfer of competent embryos to the uterus.

Using CGH embryo selection, we will finally be able to control for the important variable of embryo “competence”, and thereby much more reliably evaluate the role of immunologic factors in the genesis of implantation dysfunction. This in turn will help us better assess the benefit of selective immunotherapy in rationally addressing this issue.

Stated otherwise: If by transferring only “competent” embryos to the uteri of women who have been diagnosed with immunologic implantation dysfunction, we can demonstrate a significantly improved birth rate with immunotherapy than without such treatment, then the controversy can finally be put to rest. Such a study is currently underway, and while complete interpretation must await its completion and full statistical analysis, preliminary findings suggest a benefit through selectively treating such cases with Intralipid (IL) steroids (e.g. dexamethasone and prednisone) and/or heparin therapy. Hopefully this matter can finally be put to rest, allowing fact to prevail over emotion.

Posted on December 16, 2009 | Filed under IVF Treatment | Permalink

Ectopic Pregnancy: Causes, Diagnosis and Treatment

An ectopic pregnancy is defined as a gestation that implants outside of the uterus. The most common site is in the fallopian tube, but it can also occur in the ovary, the cervix, outer surface of the uterus or elsewhere with the abdomen. An exrauterine, intraabdominal ectopic pregnancy can even develop into an advanced and even full term gestati. However, such fetuses are usually severely developmentally compromized and rarely, if ever survive.

About 1:200 naturally conceived pregnancies and 1:30 IVF gestations are ectopic. On very rare occasions (1:2,000), a tubal ectopic pregnancy occurs in combination with another pregnancy (usually in the uterus. Timely, early ssurgical removal of the tubal component often is followed by the intrauterine pregnancy progressing normally to delivery at term.

Ectopic pregnancy is one of the most dangerous complications of gestation. If undetected, the pregnancy will continue to grow and will typically rupture; resulting in calamitous intra-abdominal bleeding. If not treated quickly, such an event could be fatal.

Monitoring pregnancies both hormonally and with ultrasound technology now makes it possible to completely prevent catastrophic events associated with ectopic pregnancies. Within the last two decades, treatment of ectopic pregnancies has evolved from emergency surgery with tubal removal and blood transfusion, to out-patient surgery with tubal repair or even treatment with medication. The key with ectopic pregnancy is to diagnose early and manage the outcome instead of waiting for events to unfold.

The fertilization of the human egg normally takes place within the fallopian tube. The embryo then takes about 5 to 6 days to complete its journey to the uterus, where it implants into the endometrium. Anything that delays the passage of the embryo down the fallopian tube can result in the embryo hatching and sending its “root system” into the wall of the fallopian tube and initiating growth within the tube. One of the most common predisposing factors is pelvic inflammatory disease (PID) in which microorganisms, such as Chlamydia and Gonococcus, damage the inner lining (endosalpinx) and eventually also the muscular walls of the tube(s) by creating scar tissue.

The endosalpinx has a very complex and delicate internal architecture, with small hairs and secretions that help to propel the embryo toward the uterine cavity. Once damaged, this lining does not regenerate. This is one of the reasons why women who manage to conceive following surgery to unblock fallopian tubes damaged by PID, have about a 1 in 4 chance of a subsequent pregnancy developing within the fallopian tube. Another cause of ectopic pregnancies are congenital malformations of the fallopian tube associated with shortening of, or small pockets and side channels within, the tube. These can interrupt the smooth passage of the embryo down the fallopian tube. There has even been some suggestion that premature appearance of hormones like progesterone, which relax muscle contractions within the fallopian tube, may also create an increased risk of ectopic pregnancy.

A woman who has had one ectopic pregnancy has an almost four-fold higher risk of another ectopic implantation in a future pregnancy. With every subsequent ectopic, this risk increases dramatically. Since the lining of the fallopian tube does not represent an optimal site for healthy implantation, a large percentage of pregnancies that gain early attachment to its inner lining will be absorbed before the woman even knows that she is pregnant. This is often referred to as a tubal abortion.

When an ectopic pregnancy occurs after ART, it is most likely the result of a uterine contraction causing a carefully placed embryo to be ejected into the fallopian tube. Various strategies to reduce the risk of this occurring are typically employed. The use of ultrasound guidance to place embryos and the use of minimal fluid to transfer them helps. There is some evidence that transferring blastocysts that are ready to implant instead of earlier embryos may also reduce the incidence. Sometimes however, despite the best laid plans, ectopic pregnancies do occur.

Diagnosis of an Ectopic Pregnancy
The easiest and most common method of diagnosing an ectopic pregnancy is by tracking the rate of rise in the blood levels of the “hormone of pregnancy,” human chorionic gonadotropin (hCG). With a normal intrauterine pregnancy, blood levels of hCG will usually double every two days throughout the first nine to ten weeks. However, an increase of at least 60% is still reassuring. A slower rate of increase in hCG more commonly suggests an impending miscarriage of one or more of the embryos that have implanted. However, it might be a sign of an ectopic pregnancy. Thus, the hCG levels should be followed serially until a clear pattern emerges.

The diagnosis of an ectopic is most often determined by a vaginal ultrasound examination. Performed by someone with sufficient expertise using a modern ultrasound machine, this test should reveal an ectopic pregnancy before it ruptures and becomes a surgical emergency. If the tube has already ruptured or internal bleeding has occurred, ultrasound examination will detect the presence of free fluid in the abdominal cavity, which is a more ominous sign.

If there has been a significant amount of intra-abdominal bleeding, irritation of the peritoneal membrane will cause the abdominal wall to become tense and, depending on the amount of blood in the abdomen, to distend. In such cases, any pressure on the abdominal wall will evoke significant pain and when a vaginal examination is done, movement of the cervix can be excruciatingly painful – especially on the side of the affected fallopian tube.

The most common conditions that must be ruled out when an ectopic pregnancy is suspected are:

• A hemorrhagic cyst of the ovary
• Appendicitis
• Acute pelvic inflammatory disease (PID)
• An inevitable miscarriage

Solutions: Surgical and Medical Management

Surgical: In some situations, laparoscopy is performed for diagnostic purposes. This may be necessary if a woman has a heterotopic pregnancy; one embryo implanted in the uterus and one in the fallopian tube. If an ectopic pregnancy is in fact detected, a small longitudinal incision over the tubal pregnancy will allow for its removal, without necessitating removal of the tube. In such situations, it may be possible to save the normally implanted embryo. Bleeding points on the fallopian tube can usually be accessed directly and bleeding can often be stopped through the laparoscope. Sometimes the damage to the fallopian tube has been so extensive that the entire tube will require removal. On occasions where very severe intra-abdominal bleeding heralds a potential catastrophe, a laparotomy is performed to stop the bleeding more rapidly. In such cases, a blood transfusion is usually required and may be life saving.

Medical: The introduction of Methotrexate (MTX) therapy for the treatment of ectopic pregnancy has profoundly reduced the need for surgery in most patients. MTX is a chemotherapeutic that kills rapidly dividing cells, such as those present in the “root system” of a developing fetus. Low doses of MTX are used to treat ectopic pregnancy since the fetal tissue is very sensitive. Accordingly, the side effects for the treatment are minimal. It is important to confirm that the ectopic pregnancy has not yet ruptured prior to administering MTX and is not too far along to be treated safely in this fashion.

The administration of MTX is by intramuscular injection. Prior to its administration, blood is drawn to get a baseline blood hCG level. After the injection of MTX the patient is allowed to return home with strict instructions that she should always have someone with her and never be alone in the ensuing week. The concern is that if she was to be on her own and internal bleeding occurred, she might not be able to get to the hospital quickly enough. In reality, this situation rarely occurs, but it is wise to be cautious. Instructions are also given to look for early signs that might point towards a worsening situation such as the sudden onset of severe pain, light-headedness or fainting. The patient returns to the doctor’s office four days later to check the blood HCG level, noting that it may have risen a bit. Three days later (7 days after MTX), the level is checked again. By this time, the HCG level should have dropped at least 15% from the value on day 4. If not, a second MTX injection is given and the blood levels are tested twice weekly until HCG level is undetectable. Once this occurs, vaginal bleeding will usually begin within a week or two.

Recent advances in the field of ultrasound diagnosis along with the introduction of MTX therapy have revolutionized the treatment of ectopic pregnancy and have significantly reduced both the high morbidity and mortality rates previously associated with this condition. When an ectopic pregnancy occurs following infertility treatment, there is the added advantage that the physician will be on the lookout for the earliest possible signs of trouble. The performance of a vaginal ultrasound within two weeks of a positive blood pregnancy (hCG) test following IVF allows for early detection of the unruptured pregnancy and timely intervention with MTX and/or laparoscopy.

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Posted on December 13, 2009 | Filed under IVF Treatment | Permalink

Single Embryo Transfer: One Embryo, One Baby

With the evolution of new and advanced techniques of in vitro fertilization, resulting in improved embryo implantation and birth rates, there has come about a substantial increase in the rate of multiple births. Largely as a result of this, obstetrical outcomes are poorer following IVF than following spontaneous pregnancy. In fact, multiple births are responsible for a markedly higher risk of prematurity with low or very low birth weight, as well as perinatal death and more frequent lingering neurologic complications as well as an increased risk of malformations.

Consider the fact that when comparing singleton with triplet pregnancies:
• Twins have 3-times, and triplets, a 6-times greater perinatal mortality rate
• Twins have 6-times, and triplets, an 11-times greater likelihood of developing cerebral palsy
• 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.

The anguish of losing one or more of your children at birth or watching them endure a life-long disability is a situation no parent would wish to face, yet it is a frequent consequence of multiple births. So why then do so many IVF practitioners still insist on transferring multiple embryos at a time? The following are the main reasons for this:

• 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.
• The relative inability hitherto, to reliably differentiate between embryos that will propagate a healthy pregnancy (i.e. “competent” embryos) and those that will not (“incompetent” embryos): Most IVF patients erroneously believe that a “pretty”, embryo (one given a high grade because it fulfils the microscopic criteria of “good quality”) should invariably propagate a baby. This is simply not the case. Consider the fact that such a microscopically “good quality” embryo from a 30-year-old has about an 8-times greater chance of resulting in a normal birth than would an identical looking embryo of a 45 year old! This confronts IVF practitioners with a “damned if you do, damned if you don’t” situation; driven by patient pressure to achieve a pregnancy and by competing market forces, they still too often choose to transfer multiple embryos, often with disastrous results. It is generally true that declining egg / embryo “competency” with advancing age justifies transferring more embryos in older women – especially in those over 40 years of age – but this still needs to be carefully measured against the risk of multiple gestations.

Not only is multiple gestation the most common complication of infertility treatment, it has also become the most costly in terms of its social impact. If all of the factors associated with multiple gestations are considered, including the costs of antenatal maternal hospitalization, neonatal intensive care for premature infants, as well as the costs of chronic medical care, rehabilitation and special education, the projected annual cost of IVF-associated multiple gestations in the United States is approximately $1.5 billion as compared to about $550 million for all the other IVF cycles performed.

On the positive side is the fact that the last decade has seen a slight but significant decline in the IVF twin pregnancy rate from about 25% to about 22%, as well as a decline in the incidence of triplets from 5% to about 3%. Still, IVF multiple birth rates are about ten times higher than those associated with natural conception. Clearly, multiple pregnancy (especially high-order multiples) represents a complex problem that can no longer be justified as an acceptable outcome following IVF treatment.

Numerous studies have demonstrated that the cumulative birth rate after single embryo transfer (SET), followed by subsequent transfers of individually 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 <40>

When we use the term “competent” embryo, we mean one which, upon being transferred to a “receptive” or “hospitable” uterus, will in most cases propagate a viable gestation. By far the single most important determinant of embryo “competency” is its karyotype (the number of chromosomes present). Aneuploid embryos (those with too many or too few chromosomes) are uniformly “incompetent” while “euploid” embryos (those with the correct number) are by and large “competent”. An incompetent embryo will not result in a normal pregnancy. In most cases it will either fail to implant or will miscarry early on. It follows that the ability to efficiently identify competent embryos for selective individual transfer would likely represent a “game changer” in the IVF arena.

In the past, the ability to select competent embryos for transfer has been thwarted by:

a. Lack of reliability of microscopic morphologic (appearance) embryo grading.

b. The inability of traditional pre-implantation genetic diagnosis/sampling (PGD/s) and chromosomal evaluation (karyotyping) by conventional Fluorescence In-Situ Hybridization (FISH) to be able to access all of the embryo’s chromosomes

Let’s take a look at the merit and reliability of several current methods used to select the best embryo(s) for transfer:

• Microscopic Embryo Grading: Currently, most IVF centers culture embryos in groups and then perform a single microscopic evaluation (at 2, 3 or 5-6 days) prior to transferring one or more to the uterus. This approach is limited in scope and in its ability to reliably discriminate between “competent” and “incompetent” embryos, since chromosomally abnormal embryos are often identical in appearance to those that are normal. Embryos should be at 2 to 4 cells at 48 hours after egg retrieval and about 6-9 cells by 72 hours. The cells in an embryo are also referred to as “blastomeres”. Ideally the blastomeres should be of even size, and there should be less than 20% fragmentation, or blebbing. This is where portions of the embryo’s cells have broken off and are found lying free as debris inside its substance. Most IVF clinics “grade” each embryo using one of many scoring systems. Unfortunately, there is no agreement at all as to which system to use. But regardless of the microscopic grading system used, one thing is certain…they all lack reliability because they cannot evaluate the chromosomal integrity of the embryo.

• Blastocyst Embryo Transfer: A blastocyst is an embryo which has developed to the point of having 2 different cell components and a fluid cavity. Human embryos, in culture in an IVF lab, or developing naturally in the female body, usually reach the blastocyst stage by day 5 or 6 after fertilization. Many “incompetent” embryos are culled out as the embryo progresses to the blastocyst stage. Thus, those embryos that make it to blastocyst are much more likely than their day-3 counterparts to be competent. Embryos that do not reach blastocyst are in >95% chromosomally abnormal (aneuploid) and would not have been worthy of transfer earlier on anyway. Routinely taking embryos to blastocyst is thus a good idea since if they do not make it they are incompetent anyway.
  Following fertilization, the cells of the embryo divide progressively over several days until after 4 days the embryo reaches an advanced (100 cell or more) stage known as a morula. One or two days later the morula will have differentiated further, developing a defined fluid filled cavity within its structure. Only about 40% of a younger woman’s embryos make it to this highly advanced or blastocyst stage of development. With few exceptions, embryos that fail to progress to the blastocyst stage are in fact aneuploid and therefore incompetent. By waiting five or six days post fertilization to select and transfer only blastocysts to the uterus, we can improve the likelihood that those being transferred are more likely to be the ‘competent” ones.

• Embryo Soluble Human Leukocyte Antigen-G (sHLA-G) Expression: sHLA-G is a compound released by early embryos into the media in which they are cultured. Recent studies have shown that its presence in sufficient concentration is an indication that the embryo is more likely to implant and develop into a healthy baby. About 8 years ago we published several articles on the use of this method in more than 1,000 women undergoing IVF. The findings revealed that women under 39 had a 45%-50% viable pregnancy rate when 2-3 embryos that expressed sHLA-G concentrations above a defined threshold level were transferred at a time. Although not a “silver bullet”, sHLA-G testing is definitely a helpful tool.

• Conventional Fluorescence In-Situ Hybridization (FISH): FISH is a method used to identify up to 12 of the 23 chromosome pairs in the embryo for abnormalities. The process requires removal of one of the 3-day old embryo’s cells (blastomeres) by a process referred to as Pre-Implantation Genetic Diagnosis/Sampling (PGD/s). Unfortunately, the remaining chromosome pairs cannot be accessed by conventional FISH and attempts to perform 23 chromosome FISH still lack sensitivity and specificity. In fact there remains about a 45% likelihood of an aneuploidy involving one or more of the remaining chromosomes – even when conventional FISH results are reported as “normal”.

• Comparative Genomic Hybridization (CGH): This very promising method for egg/ embryo selection was introduced into the clinical arena by SIRM in 2005. It represents a real break through in the IVF arena. Unlike conventional FISH testing which can only reliably recognize 12 of the embryo’s 23 chromosome pairs, CGH allows for identification of ALL the chromosomes and in the process, overcomes the inadequacies associated with most other methods of embryo selection. A study we published in Fertility & Sterility in May, 2005 demonstrated a birth rate of more than 70% in women who received just one CGH-selected embryo. Our follow-up study reported in Fertility and Sterility (December 2009) confirms that through CGH embryo selection we finally have a highly reliable method for differentiating between “competent” and “incompetent” embryos. Even without CGH embryo selection, “one embryo/one healthy baby” is now even more attainable. However, the introduction of CGH has made embryo selection much more scientific, virtually removing the incentive to transfer multiple embryos at a time.
  One of the perceived disadvantages of CGH embryo selection is the associated increase in cost of such testing. However, while the performance of egg/embryo CGH does increase the cost per cycle of IVF, it actually lowers the “cost per IVF baby”.
  Since CGH testing requires several days or weeks to complete, the use of this technology usually requires that advanced embryos (blastocysts) be frozen and stored (cryostored) in a subsequent cycle . The separation of an IVF cycle is separated into two separate phases to achieve this objective is referred to as Staggered-IVF (St-IVF). Cryostoring blastocysts allow sufficient time for the CGH testing to be completed.
• Vitrification (Ultrarapid Freezing): Until recently cryopreservation of human embryos has been problematic because it often caused ice crystals to form inside the embryo, damaging or destroying it. The recent introduction of ultra-rapid freezing or vitrification (see the article on vitrification elsewhere on this blog) has changed all that. With vitrification, embryos are so rapidly frozen that no ice forms, yielding a post-thaw embryo survival rate of more than 95%. Impressively, birth rates following the transfer of thawed, previtrified embryos hardly differ from those using fresh embryos.

The Hippocratic Oath, decrees that the cardinal rule of medicine is “primum non nocera (”foremost do no harm”). Since multiple pregnancy is the most serious complication of Assisted Reproductive (AR) Medicine, and IVF has been responsible for a virtual explosion in the incidence of twins and higher order multiples, those of us that practice medicine in this arena have a solemn responsibility to educate our patients and then to restrict the number of embryos we transfer at one time. Central to achieving this goal is to optimize the ability to select the most “competent” embryos for transfer.

The transfer of a single, good quality embryo selected using objective methods will be a central part of IVF treatment in the years to come. In contrast to many areas of AR Medicine and infertility where additional evidence is often sought and further trials are warranted, we believe there now is sufficient information to start moving to SET, especially good-prognosis women who are undergoing IVF treatment.

Posted on December 13, 2009 | Filed under IVF Treatment | Permalink