In January, Dr. Carolyn Givens and I attended a meeting in Hawaii organized by the American Board of Bioanalysts (ABB). This organization board certifies and licenses embryologists, andrologists, and a number of other laboratory specialists in the United States. Our meeting was under the direction of the College of Reproductive Biology, a special interest group within the ABB and for which I am the immediate past Chair.

The meeting was small and intimate, a situation always welcomed among reproductive biology professionals. The location allowed for good interaction with embryologists from Japan who have always been a great source of ideas and innovation within our specialty.

In fact, the highlight of the meeting was a series of videos shown by Dr. Yasuyuki Mio from the Mio Fertility Clinic in Yonago, Japan. He was able to take time-lapse cinematography of human embryos in culture, and as a result reported some novel observations on how oocytes fertilize and how embryos develop. The actual moment of sperm entry into the oocyte was recorded and it was possible to see that human oocytes form a fertilization cone (a membrane that helps bring the sperm into the oocyte), shortly after sperm entry. The events that follow (2nd polar body extrusion, which is the egg extruding a set of chromosomes, and pronuclear formation, alignment of the nuclei from the egg and sperm) occurred as expected, but for the first time the male and the female nuclei could be distinguished from each other.

After fertilization, the embryos were seen to change dramatically as they developed. In particular, they appeared more disorganized and untidy immediately after a cell division event and more symmetrical and organized several hours later. This discovery has implications for those embryos that sometimes may appear poorly. It suggests that they may look better later in the day when they are clear of the cell division process. Another important observation regarding blastocysts, is that those that develop 2 inner cell masses (ICM: the precursor cells of the fetus) do so in a predictable way. At PFC, we avoid using embryos with two ICMs whenever possible, as they are likely to lead to the formation of identical twins. A normal embryo should have only a single ICM. Currently, it is possible that one of the ICMs may be small enough to avoid detection. The observation was made that the fine cellular bridges within the embryo cavity appear to correlate to the presence of an extra ICM.

Another notable presentation was that of Dr. Tetsunori Mukaida, of Hiroshima HART Clinic, on sperm morphology. He demonstrated that observing sperm under ultra-high magnification can show structural defects that are not always visible when using standard microscopes. While magnifying sperm thousands of times has its difficulties, Dr. Mukaida reported that sperm with subtle physical defects have a much lower chance of making an embryo that can become a baby. Sperm that are close to perfect in size, shape and structure are difficult to find in any sperm sample and it can take hours just to find a few ideal sperm. However, the extra effort may be worthwhile, especially in patients that have had a previous IVF cycle where the embryos did not develop well or implant after transfer. PFC is currently looking into this technology and we will report more details in a future issue of Fertility Flash.

Attending meetings like this and keeping up with the latest developments in our field is an important part of the culture at PFC. We share the load of traveling to educational events and are always excited to bring home ideas and thoughts to share with our colleagues. PFC is committed to implementing the latest technology and innovations to maximize pregnancy rates for our patients. We will continue to stay updated with all of the research and development in our specialty.

	Joe Conaghan, Ph.D., HCLD is PFC’s laboratory director. Dr. Conaghan is internationally recognized for his work on improving embryo culture conditions. His interests include developing programs for the treatment of severe male factor infertility; diagnosis of genetic disease in embryos; and improved embryo culture.
	Carolyn Givens, M.D. was the first in San Francisco to successfully initiate a pregnancy using intracytoplasmic sperm injection (ICSI). She currently co-directs the Bay Area Pre-Implantation Genetic Diagnosis Program (PGD) and is director of PFC’s PGD program.

At Pacific Fertility Center we aim to help our patients build a healthy family. To build healthy families, maximum pregnancy rates are a goal, but maximum pregnancy rates must be balanced by consideration of risk, the chance of an adverse outcome. High pregnancy rates with minimal risk is PFC’s goal.

The risk of multiple pregnancy has increased as fertility therapy has improved. The wider use of gonadotropins in the 1990s to induce ovulation of multiple follicles, as well as the use of more effective laboratory and clinical IVF methods, resulted in production of more and healthier oocytes and more embryos, and increased the chances of multiple pregnancy. The very dramatic improvement in success rates over this time period resulted in many more children being delivered after fertility therapies, but also more twins, triplets, and higher order multiples.

Over the last twenty years, the incidence of multiple birth has increased nationally. According to the National Vital Statistics Report and the March of Dimes, the incidence of twins has increased by two-thirds, and the number of triplets and quadruplets has increased four-fold since 1980.

It is thought that about one-third of multiple pregnancies arise because women are waiting until later in life to conceive; age is a well-known risk factor for multiples. Another third arise from use of ovulation induction with gonadotropins (Pergonal, Follistim, Gonal-F, Repronex) alone. Less than one fifth of multiples are from assisted reproduction techniques (IVF and related procedures). Assisted reproduction in 2003 accounted for 18% of multiple pregnancies, 16% of twins and 44% of triplets ¹.

The risks to the children of multiple pregnancy are numerous. Low birth weight and very low birth weight are increased in children born as multiples. The chance of low birth weight (<2500g) is increased 8 times in twins. Cerebral palsy is increased 4 times, neonatal death risk by 7 times ^{2, 3}.

The risk to the mother from multiple pregnancy is also increased. Pre-eclampsia, high blood pressure, preterm labor, and premature rupture of membranes are all more common with multiple pregnancy ⁴ .

Multiple pregnancy is also expensive. It is estimated that twins alone cost the healthcare system some $600,000,000. There is clear evidence of an increase in parenting stress and divorce in families of multiples ^{5, 6} .

The need to assure our patients of the highest quality care requires that we bear this in mind – the healthiest pregnancy is a singleton pregnancy.

Pregnancy requires the cooperation of sperm and egg, accurate transcription of the early genetic code in the developing embryo, a fertile spot for attachment to the mother in the uterus, and a route for getting there. All other factors being equal, pregnancy rates almost double when two embryos are transferred instead of one, and increase again when a third and fourth embryo are added. The desire for high pregnancy rates has driven a desire for more embryos to be transferred ⁷ .

Improvements in insemination technique, embryo culture methods, and transfer efficiency have added substantially to pregnancy rates. Each embryo transferred today has a considerably higher chance of producing a pregnancy than an embryo transferred twenty years ago. Such improvements have enabled us to think about ways to reduce the risk of multiple pregnancy by transferring fewer embryos.

The development of blastocyst (day 5 embryo) culture techniques allows the selection of high quality embryos for transfer. The blastocyst stage requires advanced incubation techniques with low oxygen incubators and specialized culture media. A tight quality control system is also required. The blastocyst stage is a more advanced stage in which the genetic code of the embryo is fully activated and working. Only the healthiest of embryos can move to the more advanced stages, allowing selection of the best embryos for transfer.

In 2006 the ASRM published guidelines for number of embryos to transfer:

These guidelines encourage all of us to transfer ‘just enough’ embryos to achieve pregnancy.

Pacific Fertility Center has pioneered techniques of transferring fewer embryos. Last year, in 2007, our program of single embryo transfer in oocyte donation recipients produced a 66% pregnancy rate. The multiple pregnancy rate in this group was minimal. Utilizing a single embryo, two-thirds of patients were able to conceive a singleton pregnancy. This pregnancy rate was very similar to the overall pregnancy rates regardless of the number of embryos transferred.

Today half of our patients using oocyte donation elect to transfer a single embryo. Single embryo transfer is not always possible. Our criteria include age and embryo quality. A young woman (under age 35) with high quality blastocyst stage embryos and a healthy uterus can reliably transfer a single embryo and achieve high pregnancy rates. An older woman (over 40) may need to transfer 3 or more embryos to achieve a good pregnancy rate. Because of the higher number of embryos transferred, the risk of multiple pregnancy remains higher in these older age groups⁹ .

Pacific Fertility Center is very pleased to offer these techniques of single embryo transfer as some of the best and most advanced fertility treatment technology available. We are moving closer to our goal of growing families, one healthy baby at a time.   Philip Chenette, MD

1. Martin, Births: Final Data for 2003. National Vital Statistics Reports, volume 54, number 2, 2005
2. Scher, Ped Res, Vol. 52:671-81, 2002
3. Rutter, J Child Psychol Psych, Vol. 44:326-41, 2003
4. Pinborg, Human Reproduction, Vol. 18:1234-43, 2003
5. Griesinger, Hum Reproduction, Vol. 19:1239-1241, 2004
6. Glazebrook, Fertil Steril, Vol. 81:505-11, 2004
7. Paulson RJ, Fertil Steril., Vol. 53:870-874 , 1990
8. Fertil Steril, Vol. 85, Suppl. 4, 2006
9. Pacific Fertility Center 2007 IVF Statistics

Philip Chenette, M.D. has spent over a decade specializing in the treatment of patients with complex infertility diagnoses, especially in women with decreased ovarian reserve and women over 40.

For patients having their embryos transferred at the blastocyst stage, the grading procedure used to assess the embryos can seem complicated. However, we simply look to see that the embryos are developing normally, are not slowing down, and are preparing for implantation in the uterus.

In the 2 days following fertilization, embryos go through 3 rounds of cell division. The fertilized oocyte divides in 2, these cells each divide again to give 4, and then these divide to give 8. In the resulting 8-cell embryo, each cell should be 1/8 the size of the original oocyte since there is no growth in size, and each cell should be intact and symmetrical. When we assess embryos at this stage, we first count the number of cells and we then assign a grade based on how good the embryo looks. Embryos that have disintegrating or asymmetrical cells are assigned a lower grade.

At this early stage, the individual cells stay together because they are contained within a shell called the zona pellucida. However, as the embryo progresses past the 8-cell stage, dividing to 16 and then 32, the cells attach to each other and cooperate to form a tight ball called a morula. At the morula stage, the cells are pressed so tightly together that individual cells cannot easily be identified or counted. Once the attachments between cells are formed, the cells begin to pump fluid into the center of the ball, giving rise to a tiny fluid filled cavity or cyst. As long as the junctions between cells hold, no fluid can escape from the cyst, and the cyst grows larger as more fluid is pumped in.

These are critical days for the embryo. In addition to forming the central cyst, the embryo is also busy organizing its cells into two distinct populations. As the embryo moves beyond the 8-cell stage, some cells stay on the outside of the ball and some are pushed to the inside. In the typical 16-cell embryo, there are 12 outer and 4 inner cells. At the 32-cell stage, 22 of the cells are outer cells and 10 are inner cells. Creating more outer cells is deliberate, because these cells are needed to maintain the integrity of the cavity as it becomes larger. More importantly however, these cells will become the placenta, and having enough cells to establish the placenta is critical to successful implantation in the uterus. Once the placenta is established, it can feed the inner cells which become the developing fetus.

The appearance of the cyst at the center of the morula marks the next embryo stage, the blastocyst. In assessing the blastocyst, we look at the size of the cyst and the integrity of the outer and inner cells. Depending on the size of the cyst, the blastocyst is referred to as early, expanding or fully expanded. If the cyst has become large enough to cause the embryo to burst through its shell, we call it a hatching blastocyst. Occasionally, we even see fully hatched blastocysts. Hatching is a natural process that frees the embryo from its shell to allow implantation to occur. The more expanded the cyst has become, the more we favor the embryo for transfer.

In addition to looking at cyst expansion, the grade of the blastocyst is further determined by the integrity of the inner and outer cells. Embryos with more cells are better, and the best blastocysts are well expanded with distinct inner and outer cell populations. In poor quality blastocysts, there can be few cells in one or both populations, and/or the cavity can be small. And sometimes, even in embryos with beautiful outer cells, we cannot see any inner cells at all. These embryos are destined to fail since a full blastocyst with 32 cells is incapable of making inner cells if they do not already exist.

The embryos that are most difficult to assess are those where the cavity has just begun to open up, but has not expanded sufficiently to allow us to see inside. These early blastocysts are usually assigned lesser grades as we are unable to determine whether any inner cells are present. We often look at these embryos again several hours later to see if further expansion has revealed the presence of those critical inner cells. We would then re-grade the embryo, if appropriate.

All of this development, from fertilization to blastocyst expansion and hatching, normally follows a tight timeline that is independent of cell number. The embryo attempts to hatch from its shell approximately 5 or 6 days post fertilization, regardless of the number of cells it contains. If development is slow, and cell number is consequently low, the outer cells stretch to enclose the cyst and expansion continues. This is important, as the uterus waits only a few days for the embryo to implant. If the embryo takes too long to make the “right” number of cells for expansion and hatching, it may miss the implantation window. The practical result of this is that we still get high implantation rates even if only early blastocysts are available for transfer.

The above phenomenon is relevant to frozen embryo transfer cycles too, because many embryos lose one or more cells as a result of freezing and thawing. Such embryos still try to form blastocysts according to their original timeline, even though they may have less than the ideal number of cells. The consequences of arriving with plenty of cells but too late for the uterus are worse than having a chance to implant even with fewer cells. As a result, frozen-thawed embryos that have lost a cell or two are not assigned a lower grade since we still consider them to have high implantation potential.  Joe Conaghan, Ph.D., HCLD

In vitro fertilization (IVF) is perhaps one of the most effective options available for the treatment of infertility. This procedure has been available in the developed world for approximately 30 years, and has been responsible for 1-4% of all conceptions. While IVF was originally developed for the treatment of female tubal factor, it has evolved to include treatment of male factor infertility via intra-cytoplasmic sperm injection (ICSI), as well as oocyte quality factor (Decreased Ovarian Reserve, or DOR). With the development of embryo biopsy techniques, IVF has also grown to incorporate pre-implantation genetic screening of embryos (PGD) to avoid genetic diseases in embryos and to screen for normal chromosomes. In the history of mankind, IVF will undoubtedly remain the greatest development for the treatment of human infertility for the foreseeable future.

Since the introduction of IVF, there has been a directly proportional increase in multiple gestation births. Traditionally IVF centers have measured success as the number of live births, irrespective of outcomes. This increase in multiple births is driven by the clinical incentives for live births, but some may also be driven by patient request. Two studies have shown that 20% of European and US infertile couples wanted a multiple birth^{(1, 2)}. Even after counseling regarding the risks of a multiple gestation, many patients still wanted to transfer 2 embryos. As IVF success rates have increased, and as the embryo freezing technologies have improved, a shift in the philosophy of IVF providers is occurring. Success rates are more likely to be measured as “live birth of a singleton (single baby) pregnancy”—in other words, “one healthy baby at a time”.

As the number of babies born after IVF has grown, there has been increased interest in looking at the pregnancy and birth outcomes in the successful IVF population. While potential complications for mother and babies are increased with any multiple gestation, there may also be an increased risk for complications even with IVF singleton babies. However, it may not be the IVF treatment itself that results in this increased risk for complications. The questions that reproductive endocrinologists and high-risk pregnancy specialists are trying to answer are primarily: 1) Is there a higher risk for a baby of any adverse birth outcome if that baby is conceived in an IVF laboratory? and 2) Is there something inherent about a past diagnosis of infertility which places even a singleton gestation at greater risk of pregnancy and birth complications?

IVF Singletons:

A number of large studies have addressed the question of increased risk to IVF babies ^(3-7). They echo a similar theme concerning birth outcomes, most importantly preterm birth <37 weeks, and low birth weight. One study compares the differences in degree of risk of poor outcomes with IVF babies vs. naturally-conceived babies. There appears to be a 93% increased risk for IVF singletons as compared to naturally conceived singletons, and a 57% increased risk for IVF twins versus naturally conceived twins⁽⁶⁾. Certainly the overall chance of a preterm delivery is much smaller for singletons than twins, and a twin pregnancy carries much greater risks overall.

A review of the US birth registry indicates that the proportion of IVF singleton babies born at full term with low-birth-weight is decreasing, but the proportion of IVF singleton babies born prior to full term with low-birth-weight is stable. In either case, the incidence of low-birth-weight is higher in babies born after IVF when compared with the general population. While outcomes of low-birth-weight babies may be getting better, there are still elevated risks for singleton low-birth-weight babies conceived via IVF.

For most of these studies looking at risks for IVF babies, factors known to influence pregnancy and birth outcomes are taken into consideration in the analysis. These important factors include maternal age and prior birth history. However, other factors may also be important but are not as well accounted for: factors such as previous poor obstetrical outcome, smoking status, socio-economic status, performance of fetal reduction procedure (especially for the analysis of the singleton data), types of ovarian stimulation protocols, media used in the IVF laboratory, and/or use of laboratory techniques (ICSI, etc.).

Infertility per se may itself be a risk factor for poorer pregnancy and birth outcomes. In an attempt to answer this important question, IVF outcomes have been compared with either non-IVF fertility treatments such as ovulation induction (OI) or to spontaneous conception outcomes. Numerous studies ^(8-15) have evaluated this question, and shown a higher risk of preterm birth for both IVF and OI babies as compared to spontaneously conceived singleton pregnancies. When evaluating outcomes for sub-fertile women (infertility for greater than 1 year) who spontaneously conceive, again we see a greater risk of preterm deliveries, obstetrical complications and adverse birth outcomes ^(16-18). These studies strongly suggest that there is an inherent characteristic of infertile patients which place them at greater risk of poorer pregnancy and birth outcomes. Whether this is due to uterine or embryo issues is not yet known.

IVF Twins:

Many studies have compared the outcomes for twins conceived via IVF versus spontaneous conception. These outcomes were summarized and reviewed in a meta-analysis of birth outcomes of IVF twins in studies up to 2003 ⁽¹⁹⁾. The specific findings showed an increase in the chances of a preterm birth (57% increase), admission to the neo-natal intensive care unit (two-fold increase), and Cesarean section delivery (33% increase). No other parameters were significantly different from spontaneously-conceived twins.

These differences between twin gestations conceived via IVF versus spontaneously-conceived twins were similar for cycles of twin gestation conceived via ovulation induction (OI). The rate of prematurity seemed to be higher for the IVF than OI group ⁽²⁰⁾.

In conclusion, when comparing singleton or twin gestations conceived via IVF or spontaneously, the degree of difference in the overall risk is greater for the singleton-baby births than twins. This is especially true with regards to preterm delivery which is increased two-fold in IVF singletons and by 40% (adjusted for age) in twins. While most studies have made adjustment for factors which can affect birth outcomes, such as maternal age, some other potential factors are difficult to measure, such as history of infertility or direct effects of IVF technology itself. It appears as though infertility prior to conception may play a larger role in IVF outcomes, for both singleton and twin gestations.  Isabelle Ryan, M.D.

1. Thurin A et al. Elective single-embryo transfer versus double-embryo transfer in in vitro fertilization. N Engl J Med 2004; 351:2392-2402.
2. Ryan GL et al. The desire of infertile patients for multiple births. Fertil Steril 2004; 81; 500-504.
3. Jackson RA et al. Perinatal outcomes in singletons following in vitro fertilization: a meta-analysis. Obstet Gynecol 2004; 103; 551-563.
4. Helmerhorst FM et al. Perinatal outcomes of singletons and twins after assisted conceptions; a systematic review of controlled studies. BMJ 2004; 328; 261.
5. McGovern PG et al. Increased risk of preterm birth in singleton pregnancies resulting from in vitro fertilization-embryo transfer or gamete intrafallopian transfer: a meta-analysis. Fertil Steril 2004; 82; 1514-1520.
6. McDonald SD et al. Perinatal outcomes of singleton pregnancies achieved by in vitro fertilization: a systematic review and meta-analysis. J Ostet Gynaecol Can 2005; 27; 449-459.
7. Bower C et al. Assisted reproductive technologies and birth outcomes: overview of recent systematic reviews. Reprod Fertil Dev 2005; 17; 329-333.
8. French National IVF Registry. Analysis of 1986 to 1990 data. Fertil Steril 1993; 59; 587-95.
9. Frydman R et al. An obstetric assessment of the first 100 births from the in vitro program of Clamart, France. Am J Obstet Gynecol 1986; 154; 550.
10. McFaul P et al. An audit of obstetric outcome of 148 consecutive pregnancies from assisted conception: implication for neonatal services. Br J Obstet Gynecol 1993; 100; 820-5.
11. Tan S et al. Obstetric outcome of In vitro fertilization pregnancies compared with normally conceived pregnancies. Am J Obstet Gynecol 1992; 167; 778-84.
12. Wang JX et al. The obstetric outcome of singleton pregnancies following IVF/GIFT. Hum Reprod 1994; 9; 141-6.
13. Tanbo T et al. Obstetric outcome in singleton pregnancies after assisted reproduction. Obstet Gyncol 1995; 86; 188-92.
14. Rufat P et al. Task force report on the outcome of pregnancies and children conceived by in vitro fertilization (France 1987-1989). Fertil Steril 1994; 154; 550-5.
15. Friedler S et al. Births in Israel resulting from in vitro fertilization/embryo transfer. 1982-1989: National registry of the Israeli association for fertility research. Hum Reprod 1992; 7; 1159-63.
16. Basso O et al. Subfecundity and neonatal mortality: longitudinal study within the Danish national birth cohort. BMJ 2005; 330; 393-394.
17. Basso O et al. Infertility and preterm delivery, birthweight, and Caesarean section: a study within the Danish National Birth Cohort. Hum Reprod 2003; 18; 2478-2484.
18. Pandian Z et al. A review of unexplained infertility and obstetric outcome: a 10 year review. Hum Reprod 2001; 16; 2593-2597.
19. McDonald S et al. Perinatal outcomes of in vitro fertilization twins: a systematic review and meta-analysis. AM J Obstet Gynecol 2005; 193; 141-152.
20. Adler-Levy Y et al. Obstetric outcome of twin pregnancies conceived by in vitro fertilization and ovulation induction compared with those conceived spontaneously. Europ J Obstet Gynecol and Reprod Biol 2007; 133; 173-178.

Isabelle Ryan, M.D. is recognized by prestigious medical associations for her pioneering research leading to new insight into the important clinical problem of endometriosis related infertility. Dr. Ryan is medical director of PFC’s Third Party Parenting Program and Egg Donor Agency.