Category Archives: In Vitro Fertilization

In Vitro Fertilization

Anti-Mullerian Hormone: The Blood-Based Biological Clock?


Many women choose to delay starting a family for various reasons, but how long is too long to wait? Is there some way to determine the time remaining on a woman’s “biological clock” to help guide family planning? A new biomarker measured in blood, anti-Müllerian hormone (AMH), has been proposed to do exactly that but there are some important limitations that must be considered before rushing out to the closest doctor’s office to request an AMH measurement.

First, some background. Women are born with approximately one million primordial ovarian follicles and only about one thousand of these remain when a woman reaches menopause. Over the course of a woman’s reproductive years, these primordial follicles come out of hibernation and develop into immature follicles by accumulating theca cells that produce testosterone and granulosa cells that convert testosterone to estradiol. Each cycle, in response to follicle-stimulating hormone (FSH), one of these immature follicles becomes the dominant, mature follicle that ultimately releases an egg through the process of ovulation. Some immature follicles exit the development pathway and become nonviable while others continue to develop for possible selection as the dominant follicle in a subsequent cycle. The key point is that the granulosa cells of these immature follicles produce AMH, which can be measured in serum or plasma as a direct reflection of the number of immature follicles. If more immature follicles are present, the serum/plasma AMH concentration will be higher. If fewer immature follicles are present, the AMH concentration will be lower. At first glance, measuring AMH would seem to be the ideal way to determine a woman’s reproductive lifespan – if AMH is high, many immature follicles remain and menopause is years away.

Unfortunately, it’s not quite that simple. While elevated AMH concentrations do reflect a large number of immature follicles, this doesn’t necessarily guarantee fertility. Polycystic ovary syndrome (PCOS) is a condition marked by the presence of many immature AMH-secreting follicles and women with PCOS typically have elevated serum/plasma AMH concentrations. AMH has been shown to inhibit the effects of FSH and AMH excess prevents immature follicles from reaching the final stages of development, resulting in impaired fertility for many women with PCOS. While an AMH concentration within the age-appropriate reference interval is a favorable indicator of fertility, higher is not necessarily better as very high AMH concentrations may indicate an underlying anovulatory condition.

At the other extreme, low age-specific serum/plasma AMH concentrations have been associated with impaired fertility in women in their 30s and may predict earlier menopause but low AMH concentrations are substantially harder to interpret in girls and younger women – precisely the population for whom an early estimate of reproductive lifespan would be most valuable. Low AMH concentrations in healthy women in their teens and 20s have not been associated with impaired fertility and survivors of childhood cancers with low AMH concentrations have achieved pregnancy. Furthermore, circulating AMH concentrations are reduced by lifestyle factors like oral contraceptive use and smoking, complicating the connection between AMH concentration and reproductive lifespan.

While studies of large numbers of women show that a low age-specific AMH concentration is associated with earlier menopause, it’s difficult to predict the age at menopause for an individual woman using a serum/plasma AMH concentration. The rate of decline in serum/plasma AMH concentrations varies from woman to woman, meaning that two women with identical AMH concentrations one year may have very different AMH concentrations the following year. Furthermore, the onset of menopause is a complex trait determined by genetic factors, environmental exposures and other influences like smoking, alcohol consumption and previous pregnancies. Ultimately, while AMH does reflect the number of immature follicles, its ability to predict onset of menopause and guide family planning decisions is questionable at the present time.

Currently, the most appropriate clinical use of AMH measurement is to predict response to ovarian stimulation in women undergoing in vitro fertilization (IVF). Women with a high AMH concentration (and a large number of immature follicles) who undergo IVF are at increased risk of ovarian hyperstimulation syndrome (OHSS), a potentially fatal condition marked by abdominal fluid retention, blood clots, altered electrolyte concentrations and kidney failure. Using a moderate ovarian stimulation protocol in women with a high AMH concentration has been shown to reduce the risk of OHSS while increasing the number of pregnancies and live births per IVF cycle started. At the other end of the spectrum, women with a low AMH concentration are enrolled in a more intensive stimulation protocol to maximize egg retrieval while those with undetectable AMH are offered alternate treatment options as the chance of IVF success is low.

It’s possible that one day AMH may be routinely measured to predict the onset of menopause but for now, its most promising uses are limited to PCOS diagnosis (still some kinks to be worked out there too) and customization of ovarian stimulation protocols to improve IVF outcomes while minimizing the occurrence of OHSS.

Can a personalized approach improve IVF success rates?


Test Tube Baby

This post was written by Robert D. Nerenz, PhD, an assistant professor of pathology and laboratory medicine at the University of Kentucky, in Lexington.

In the United States, an estimated one in seven couples experience infertility and for many of these couples, in vitro fertilization (IVF) represents their best chance of achieving pregnancy. However, IVF cycles constitute a significant expense (approximately $12,500 per cycle), disrupt patients’ daily lives and only result in a healthy, live birth 30% of the time! Furthermore, the majority of IVF cycles performed in the United States involve the transfer of multiple embryos. This is of particular concern because multiple embryo transfer carries a finite risk of a multiple gestation pregnancy. Bringing multiple infants to term is associated with an increased risk of poor fetal and maternal outcomes including decreased birth weight, increased rate of fetal death, preeclampsia, gestational diabetes and preterm labor. Clearly, there is a significant need to improve IVF success rates while also minimizing the likelihood of multiple gestation pregnancies.

One strategy that may accomplish both of these goals is to perform “single embryo transfer” by implanting one embryo that has a high likelihood of producing pregnancy and, ultimately, a live birth. This is the focus of an upcoming symposium at the AACC meeting to be held July 29th at 10:30 am in Atlanta, Georgia. Fertility clinics around the world currently attempt to do this by observing embryos under a microscope and choosing the best embryo on the basis of its physical appearance. Unfortunately, this approach does not provide any information about the embryo’s genetic status. This is an important limitation because aneuploidy (the gain or loss of a chromosome) is the most common cause of pregnancy loss. It is also estimated to occur in ≥10% of clinical pregnancies and becomes more frequent with increasing maternal age.

To ensure that aneuploid embryos are not selected for transfer, several research groups have developed methods collectively known as comprehensive chromosome screening (CCS). CCS involves culturing embryos for 5-6 days, removing a few cells from the trophectoderm (the outer cell layer that develops into the placenta), isolating the DNA from those cells and assessing the copy number of each chromosome using techniques such as quantitative PCR, comparative genomic hybridization, or single nucleotide polymorphism arrays. Following determination of the embryos’ genetic status, only embryos with the normal number of chromosomes are chosen for transfer. In multiple prospective, randomized controlled trials described here and here, CCS has been shown to increase the pregnancy rate and decrease the frequency of multiple gestation pregnancies. As a result, CCS is beginning to make the transition from the research setting to use with patients.

The ability to transfer only euploid embryos represents the most promising application of novel technologies to IVF but ongoing research is focused on other ways to improve the IVF success rate. Many different groups are analyzing the culture medium that embryos are grown in prior to implantation. It is hoped that this will provide information about the embryos’ metabolic health and might help identify which embryos are most likely to result in pregnancy and live birth. Other groups are evaluating endometrial gene expression profiles to assess endometrial receptivity and ultimately determine the best time to perform embryo transfer. While both of these approaches have technical limitations and are not quite ready for primetime, they have the potential to greatly improve our current standard of care and may be ready for clinical use in the near future.

Preimplantation Genetic Diagnosis and Screening


BlastocystPreimplantation genetic testing is a way of examining the genetic features of a developing embryo during the process of in vitro fertilization, before pregnancy. After the egg is fertilized with sperm, the embryos develop to the cleavage-stage. On day 3 after fertilization, a single blastomere is removed from the embryo for genetic evaluation using techniques such as PCR, FISH, or comparative genomic hybridization.

Preimplantation genetic diagnosis (PGD) is used to select embryos without certain genetic disorders. This testing including three major groups of disease: sex-linked disorders, single gene defects, and chromosomal disorders.

Preimplantation genetic screening (PGS), is not used to detect disease, but as a screen to select embryos for such things as: matching HLA type in order to be a tissue donor for an affected sibling, selecting gender, selecting embryos with the least predisposition for developing certain cancers, and selecting embryos with a higher chance of implantation and therefore increase the likelihood of achieving pregnancy. Medscape has an excellent overview of PGD and PGS.

For women of advanced maternal age or couples with known genetic mutations, the ability to screen for embryos free of certain genetic mutations is reassuring. However, as with many medical interventions associated with human reproduction, PGS has raised ethical questions. For instance, as stated earlier, PGS can be used to select for a preferred gender. In some cases this is to avoid a sex-specific disease. Other times this is done for so-called "family planning" or "gender balance." In other words, selecting a gender because of personal preference. Some feel this is discriminatory and should not be allowed. In other cases, embryos have been tested so that the resulting child would be compatible to serve as a stem cell donor for a sick sibling (much like the popular fiction book "My Sister's Keeper").   There have also been cases where parents have requested the selection of affected embryos so that the child has the same minor disability, such as deafness or dwarfism, as the parents. Some preimplantation genetics laboratories agree to do this type of testing and some do not.

The New York Times recently ran an article discussing this issue. The article states that:

"In the United States, there are no regulations that limit the method’s use. The Society for Assisted Reproductive Technology, whose members provide preimplantation diagnosis, says it is 'ethically justified' to prevent serious adult diseases for which 'no safe, effective interventions are available.' The method is 'ethically allowed' for conditions 'of lesser severity' or for which the gene increases risk but does not guarantee a disease."

The January issue of Clinical Chemistry published a Question and Answer piece entitled "The Ethical Implications of Preimplantation Genetic Diagnosis." A podcast interview with two of the authors is also available.

The paper summarized the opinions of an ethicist, an attorney, and the director of a preimplantation genetics laboratory. The ethicist indicated that in the past, PGD has focused mainly on reducing the risk of transmitting serious diseases. In the future, he sees a shift away from lifesaving interventions to more ‘eugenically’ inspired interventions. That is, looking for traits that parents do not want in their children and selecting for traits that they do want in an attempt to pass them on. The morality of eugenics is a key moral as this technology moves forward.

Indeed it will be interesting to see where the future of this technology lies. Although it is practiced routinely, the indications, utility, and outcomes of PGD and PGS are still being defined.

Predicting the Success of In Vitro Fertilization


IVFWhat are my chances of getting pregnant? More specifically, if I am having trouble getting pregnant, will I be successful if I undergo the process of in vitro fertilization (IVF)? These are important questions especially since infertility treatments (especially IVF) is time consuming and costly.  

There are tools to assess a woman's fertility status. For instance, Fertistat  is a free on-line questionnaire that was developed at Cardiff University in the UK. Its primary function is as a fertility awareness tool.

A recent publication by Choi et al  reported a personalized model for predicting the success rate of women undergoing their first cycle of IVF. It should be noted that this method is now patented and marketed by Univfy Inc. and this blog post is in not an endorsement for this product.  Their model utilizes patient demographics and reproductive history, age at time of first IVF, BMI, smoking status, gravity, parity, pregnancy losses before 20 weeks, number of ectopic pregnancies, antral follicle count, day 3 serum FSH the year, patient diagnosis, male partner reproductive health including age, total motile sperm count, use of sperm extraction method and use of donor sperm. They developed their model using data from 13,076 first IVF treatment cycles performed at three clinics in Spain, Canada, and the United States. Then they created their PreIVF-D by combining the data from the three clinics and they used a training set of 1,061 independent cases. Finally they tested their model using another set of data from 1,058 patients.  They found that the most important prognostic contributors to their model were patient age, total motile sperm count, BMI, day 3 serum FSH, and antral follicle count.

The authors conclude that age-based estimates of live birth probabilities in IVF treatment are not optimal and that their PreIVF-D model performs better with a 35.7% improvement in the ability to predict live birth. Notably, the area under the curve (AUC) for the age-based prediction was 0.614 and for Pre-IVF-D it was 0.634.  This means that the two models were essentially equivalent.  There are two limitations to this study that should be pointed out. First, the authors discuss that it is not possible for them to prove that PreIVF-D works for a clinic outside this study. In fact, there may be clinic-specific trends (such as referral types, regional BMI, and treatment approaches) that significantly affect their predictive model. Second, the authors do not mention the methods that each clinic used for FSH measurement. According to 2013 CAP surveys, there are up to 2 fold differences in FSH results depending on the method used. It would be interesting to know if the clinics utilized one FSH method or several as this may contribute to significant clinic-to clinic variations.

Personalized prognostic tools are likely the way of the future in reproductive medicine & infertility treatment. Although their utility now may be limited, they will undoubtedly continue to improve and evolve just as predictive models for assessing risk of downs syndrome has evolved over the past 20 years.   

Assessing Ovarian Reserve


OvariesWomen in their mid to late 30s and early 40s with infertility constitute the largest portion of the total infertility population. These women are also at an increased risk for pregnancy loss. This reflects a decline in oocyte quality and a diminished ovarian reserve as a result of follicular depletion. Ovarian reserve is a term that is used to describe the capacity of the ovary to provide eggs that are capable of fertilization resulting in a healthy and successful pregnancy.

While there is no gold standard for assessing the ovarian reserve of individual women, its indirect determination has been used to help direct infertility treatment.

Serum concentrations of follicle-stimulating hormone (FSH) and estradiol on day 3 of the menstrual cycle have been the tests of choice for assessing ovarian reserve. Cycle day 3 is chosen because at this time the estrogen concentration is expected to be low, a critical feature, as FSH concentrations are subject to negative feedback from estradiol. In general, day 3 FSH concentrations >20 to 25 IU/L are considered to be elevated and associated with poor reproductive outcome.   FSH concentrations are expected to be below 10 IU/L in women with reproductive potential.  Concomitant measurement of serum estradiol adds to the predictive power of an isolated FSH determination. Basal estradiol concentrations >75-80 pg/mL are associated with poor outcome. 

Inhibin B is produced by the developing follicles and concentrations peak during the follicular phase. Concentrations of inhibin B can be used in conjunction with serum FSH and estradiol to assess ovarian function. As women age, serum FSH concentrations in the early follicular phase begin to increase. It has been suggested that this is due to a decline in the number of small follicles secreting inhibin B.  Because inhibin is produced by the ovaries, it is thought to be a more direct marker of ovarian activity and ovarian reserve than FSH. In addition, cycle day 3 inhibin B concentrations may demonstrate a decrease before day 3 FSH concentrations. 

Seifer et al reported that women undergoing in vitro fertilization (IVF) with day 3 inhibin B concentration <45 pg/mL had a pregnancy rate of 7% and a spontaneous abortion rate of 33% as compared to pregnancy rate of 26% and abortion rate of 3% in women with day 3 inhibin B concentrations of > 45 pg/mL. 

In recent years, anti-Mullerian Hormone (AMH) has been suggested to be a more useful predictor of ovarian reserve. AMH is expressed by the granulosa cells of the ovary during the reproductive years, and controls the formation of primary follicles by inhibiting excessive follicular recruitment by FSH. In 2005 Tremellen reported that plasma AMH concentrations start to drop rapidly by age 30, and are ~10 pmol/L by the age of 37. David has blogged previously about the use of AMH as a predictor of IVF outcome.   

Using a cut off value of 8.1 pmol/L, plasma AMH could predict poor ovarian reserve on a subsequent IVF cycle with a sensitivity of 80% and a specificity of 85%.     In 2008, Riggs and colleagues confirmed that AMH concentrations correlated the best with the number of retrieved oocytes relative to age, FSH, inhibin B, LH, and estradiol. 

High concentrations of AMH can also be present in women with polycystic ovarian syndrome (PCOS), a cause of female infertility.  Therefore, in PCOS patientsAMH should not be used alone, but should be combined with transvaginal ultrasound to count the number of follicles.

Women who are diagnosed with diminished ovarian reserve should be counseled regarding options such as oocyte donation or adoption.

Anti-müllerian hormone as a predictor of IVF outcome


Human egg Investigators at Brown University have reported that the serum concentration of a hormone made by the ovaries can predict the success of in vitro fertilization (IVF). Specifically, the higher amounts of the hormone were correlated to a greater number of eggs retrieved and the probability of achieving pregnancy.

The study measured blood concentrations of anti-müllerian hormone, or AMH. This is a rather interesting hormone. It plays an important role in both males and females but at different stages of human development.

  • In fetal development, AMH is synthesized by the testes of a male fetus and it functions to suppress the transformation of the müllerian ducts into the uterus and fallopian tubes. In the absence of AMH (i.e. a female fetus), the wolffian ducts fail to develop into ejaculatory ducts of the male reproductive system while the müllerian ducts complete their development.
  • AMH is synthesized by the female ovary but only after birth when it does not affect the development or maturation of the female reproductive tract. It increases slightly at puberty because of it functions to regulate the formation of ovarian follicles (eggs) by opposing the action of another hormone, follicle-stimulating hormone (FSH).

Because of its role in follicle development, AMH can be useful as a marker of ovarian function. In particular it seems to be a good indicator of the growing pool of follicles in the ovary and so reflects the number of eggs in the ovary. That’s exactly what the scientists at Brown University reported.

In 190 women undergoing IVF to achieve pregnancy, the concentration of AMH in a blood sample collected on the first day of ovarian stimulation with FSH was a strong predictor of favorable outcome. Women with the lowest concentrations of AMH had fewer eggs retrieved than those with higher concentrations. Also, the probability of achieving pregnancy was 2.5 times greater in the women with the higher AMH concentrations.

What might these results mean from a practical perspective? The authors of the study comment that IVF patients could be better counseled and IVF management strategies modified by the AMH results. IVF is expensive. Women who seek to achieve a pregnancy by this route are highly motivated to obtain a positive outcome and so they may undergo several IVF cycles in an effort to become pregnant. Those with a low AMH concentration may opt for only a single IVF cycle or seek adoption or other routes of parenthood rather than invest thousands of dollars into what may be an unachievable biological goal.