My son is starting his second year residency in obstetrics and gynecology. He, like I was 30 years ago, is turned on by reproductive medicine and enjoys performing gynecologic surgery. When I decided then to specialize in reproductive endocrinology and infertility (REI) I was looking forward to being on the frontier of fertility medicine. The details of Reproductive physiology were being unraveled in real time and IVF had just reported its first successful pregnancies. In those days, microsurgery of the fallopian tubes was commonly performed by REIs as well as endometriosis and fibroid surgery.

During my fellowship, surgery was a huge part of my training. I travelled to Nashville to train with one of the world’s experts in laser laparoscopy. I practiced my tubal microsurgery skills weekly on anesthetized rats in a plastic surgical lab. I assisted on reproductive surgery several cases every week throughout my fellowship.

Myself and other fellows performed research on basic reproductive physiology questions that had yet to be worked out. Personally, my interest was polycystic ovarian disease and its relationship to weight gain. I studied male hormone production in the ovary and the adrenal gland before and after significant weight loss. I discovered that there was an inverse relationship between weight loss and male hormone production and that this was mediated through insulin. These were exciting times. If only we had metformin back then, I would have proven that in addition to weight loss, we could decrease insulin levels and therefore male hormone levels with metformin.

Today, discoveries in reproductive physiology are much more esoteric than it was when I was a fellow. Reproductive surgery, in particular tubal microsurgery and laser laparoscopy for endometriosis and adhesions is usually replaced with in vitro fertilization (IVF) which has become so much more successful, less invasive and therefore a preferable option. Most causes of infertility, if they are not successfully treated with ovulation induction and intrauterine insemination (IUI) can be overcome with IVF.

In the 1980’s when I was a fellow, IVF was grossly inefficient and we had to transfer multiple embryos to achieve a pregnancy. Consequently, triplets and quadruplets were not rare occurrences. In many programs, they constituted over 10% of all pregnancies. Today, we can often transfer one embryo at a time minimizing the risk of multiple pregnancies. We can freeze excess embryos so many patients need go through only one stimulation and retrieval and still have multiple transfers providing them with an excellent chance of conceiving a baby from their efforts.

Today, we get excited about advances in preembryo genetic screening and diagnosis and contemplate the current and future potential of eliminating hereditary medical disorders. This involves highly trained laboratory personnel who perform the latest technologic advances. In 2010, the REI, in general is removed from a hands on involvement with the frontiers of Reproductive Medicine and instead works like a film producer gathering his team including these lab personnel, nurses, etc and directing them as to how to approach his patients’ fertility problems. It used to be that he used the microscope and laser laparoscope to perform the tubal and endometriosis surgery. The IVF retrieval and transfer were new procedures that were still being perfected.

Today, they are the routine cases performed daily by the REI.

My son looks at the REI of today as a doctor who starts his day with 1-2 hours of ultrasound that is part of the daily ovulation monitoring for IUI and IVF. Many REIs no longer perform more surgery than hysteroscopy and occasional laparoscopy or myomectomy in addition to their retrievals. These are all considered routine procedures now. The current frontier in infertility is limited pretty much to the laboratory. Though many of us consider ourselves expert in stimulations, retrievals and transfers and while we know we make a significant difference in our patients’ outcomes our work does not appear or feel as glamorous as it once did. Perhaps, he will decide, as I did, that the pleasure in helping women build their families is sufficient reward. Or perhaps, this Nintendo generation, will seek a more apparently exciting lifestyle. How about that Robotic surgery?

Successful IVF is dependent on many factors.  The quality of the egg and embryo, the placement of the embryo into the uterus and the environment surrounding implantation are all paramount to the ultimate goal of creating a pregnancy that leads to a live baby.

Typically, patients present with their own gametes so the genetics and pregnancy potential of the eggs and sperm is usually predetermined when patients first present to an IVF program.  As a specialist in REI and IVF, I have dedicated my career to optimizing those other factors that we may influence.

In the late 1990’s I recorded data on all my embryo transfers including distance the catheter tip was placed into the uterine cavity, number of cells and grade of the embryos, difficulty of the transfer, use of tenaculum etc.  I presented my results at the ASRM in 2000 that highlighted the two step transfer to the middle of the uterine cavity and replaced the tenaculum with a cervical suture when needed and this radically improved pregnancy rates.

The uterine environment has been optimized through screening for anatomic issues in the uterine cavity with a hydrosonogram to identify polyps, fibroids and scar tissue that may impede implantation.  Hormonally, we have supplemented patient’s cycles with progesterone through both vaginal and parenteral (intramuscular) administration as well as estrogen that we monitor closely after embryo transfer and make adjustments when deemed helpful.

The greatest improvement in pregnancy rates for the past several years however has been due to a Culture Revolution in IVF that is the media environment bathing and feeding the embryos.  All these advances have had a great impact on IVF success rates to the point that 50% of retrievals will result in a pregnancy.  Unfortunately, older patients and some younger ones have yet to share in this success.

Many IVF programs have reintroduced the concept of utilizing a co-culture medium to improve the quality and implantation of embryos. Co-culture is a procedure whereby “helper” cells are grown along with the developing embryo. Today, the most popular cell lines include endometrial cells (from the endometrium, or uterine lining) and cumulus cells from women’s ovaries.  Both cell lines are derived from the patient, thereby eliminating any concerns regarding transmission of viruses. Endometrial cells are much more difficult to obtain and process, while cumulus cells are routinely removed along with the oocytes during IVF retrieval.

Cumulus cells play an important role in the maturation and development of oocytes.  After ovulation cumulus cells normally produce a chemical called Hyaluronan.   Hyaluronan is secreted by many cells of the body and is involved in regulating cell adhesion, growth and development. Recent evidence has shown that Hyaluronan is found normally in the uterus at the time of implantation.

Co-culture of cumulus cells provides an opportunity to detoxify the embryo’s culture medium that the embryos are growing in and produce growth factors important for cell development.  This may explain why some human embryos can experience improved development with the use of co-culture.

Preparation of co-culture cells starts with separation of the cumulus cells from the oocytes after aspiration of the follicles. These sheets of cells are washed thoroughly and then placed in a solution that permits the sheets to separate into individual cells.  The cells are then washed again and transferred to a culture dish with medium and incubated overnight. During this time individual cells will attach to the culture dish and create junctions between adjoining cells. This communication is important for normal development. The following morning, cells are washed again and all normally fertilized oocytes (embryos) are added to the dish. Embryos are grown with the cumulus cells for a period of three days to achieve maximum benefit.

Performing co-culture of embryos has improved implantation and pregnancy rates above and beyond those seen with the IVF advances previously described. More importantly, it promises to offer advantages for those patients whose previous IVF cycles were unsuccessful.

IVF

After eggs are retrieved, they are transferred to the embryology laboratory where they are kept in conditions that support their needs and growth. The embryos are placed in small dishes or tubes containing “culture medium,” which is special fluid developed to support development of the embryos made to resemble that found in the fallopian tube or uterus. The dishes containing the embryos are then placed into incubators, which control the temperature and atmospheric gasses the embryos experience.

A few hours after eggs are retrieved, sperm are placed in the culture medium with the eggs, or individual sperm are injected into each mature egg in a technique called Intracytoplasmic Sperm Injection (ICSI) (see below). The eggs are then returned to the incubator, where they remain to develop. Periodically over the next few days, the dishes are inspected so the development of the embryos can be assessed.

The following day after eggs have been inseminated or injected with a single sperm (ICSI), they are examined for signs that the process of fertilization is underway. At this stage, normal development is evident by the still single cell having 2 nuclei; this stage is called a zygote. Two days after insemination or ICSI, normal embryos have divided into about 4 cells. Three days after insemination or ICSI, normally developing embryos contain about 8 cells. Five days after insemination or ICSI, normally developing embryos have developed to the blastocyst stage, which is typified by an embryo that now has 80 or more cells, an inner fluid-filled cavity, and a small cluster of cells called the inner cell mass.

It is important to note that since many eggs and embryos are abnormal, it is expected that not all eggs will fertilize and not all embryos will divide at a normal rate. The chance that a developing embryo will produce a pregnancy is related to whether its development in the lab is normal, but this correlation is not perfect. This means that not all embryos developing at the normal rate are in fact also genetically normal, and not all poorly developing embryos are genetically abnormal. Nonetheless, their visual appearance is the most common and useful guide in the selection of the best embryo(s) for transfer.

In spite of reasonable precautions, any of the following may occur in the lab that would prevent the establishment of a pregnancy:

- Fertilization of the egg(s) may fail to occur.
- One or more eggs may be fertilized abnormally resulting in an abnormal number of chromosomes in the embryo; these abnormal embryos will not be transferred.
- The fertilized eggs may degenerate before dividing into embryos, or adequate embryonic development may fail to occur.
- Bacterial contamination or a laboratory accident may result in loss or damage to some or all of the eggs or embryos.
- Laboratory equipment may fail, and/or extended power losses can occur which could lead to the destruction of eggs, sperm and embryos.
- Other unforeseen circumstances may prevent any step of the procedure to be performed or prevent the establishment of a pregnancy.
- Hurricanes, floods, or other ‘acts of God’ (including bombings or other terrorist acts) could destroy the laboratory or its contents, including any sperm, eggs, or embryos being stored there.

Quality control in the lab is extremely important. Sometimes immature or unfertilized eggs, sperm or abnormal embryos (abnormally fertilized eggs or embryos whose lack of development indicates they are not of sufficient quality to be transferred) that would normally be discarded can be used for quality control. You are being asked to allow the clinic to use this material for quality control purposes before being discarded in accordance with normal laboratory procedures and applicable laws. None of this material will be utilized to establish a pregnancy or a cell line unless you sign other consent forms to allow the clinic to use your eggs, sperm or embryos for research purposes. Please indicate your choice below:

Success varies with many factors. The age of the woman is the most important factor, when women are using their own eggs. Success rates decline as women age, and success rates drop off even more dramatically after about age 37. Part of this decline is due to a lower chance of getting pregnant from ART, and part is due to a higher risk of miscarriage with increasing age, especially over age 40. There is, however, no evidence that the risk of birth defects or chromosome abnormalities (such as Down’s syndrome) is any different with ART than with natural conception.

Success rates vary with the number of embryos transferred. However, transferring more embryos at one time not only increases the chance of success with that transfer, but will also increase the risk of a multiple pregnancy, which are much more complicated than a singleton pregnancy. The impact of the number of embryos that are transferred on success rates also varies with the age of the woman.

Pregnancy complications, such as premature birth and low birth weight, tend to be higher with ART pregnancies, primarily because of the much higher rate of multiple pregnancies. Nationally, in 2002-2003 about 30% of ART deliveries were twin deliveries, versus 1-2% of spontaneous pregnancies. The risk of pregnancy containing triplets or more was 6% in 2003.

As women get older, the likelihood of a successful response to ovarian stimulation and progression to egg retrieval decreases. These cycles in older women that have progressed to egg retrieval are also slightly less likely to reach transfer.  The percentage of cycles that progress from transfer to pregnancy significantly decreases as women get older.  As women get older, cycles that have progressed to pregnancy are less likely to result in a live birth because the risk for miscarriage is greater.  This age related decrease in success accelerates after age 35 and even more so after age 40.  Overall, 37% of cycles started in 2003 among women younger than 35 resulted in live births. This percentage decreased to 30% among women 35–37 years of age, 20% among women 38–40, 11% among women 41–42, and 4% among women older than 42.  The proportion of cycles that resulted in singleton live births is even lower for each age group.

The success rates vary in different programs in part because of quality, skill and experience but also based on the above factors of age, number of embryos transferred and patient population.  Patients may also differ by diagnosis and intrinsic fertility which may relate to the number of eggs a patient may be able to stimulate reflected by baseline FSH and antral follicle count as well as the genetics of their gametes.  These differences make it impossible to compare programs.

Another factor often overlooked when considering one’s odds of conceiving and having a healthy baby from an IVF procedure is the success with cryopreserved embryos.

Thus, a program which may have a lower success rate with a fresh transfer but much higher success with a frozen embryo transfer will result in a better chance of conceiving with only a single IVF stimulation and retrieval.  Success with frozen embryos transferred in a subsequent cycle also allows the program to transfer fewer embryos in the fresh cycle minimizing the risk of a riskier multiple pregnancy.  It may be more revealing to examine a program’s success with a combination of the fresh embryo transfer and frozen embryo transfers resulting from a single IVF stimulation and transfer.  For example, at East Coast Fertility, the combined number of fresh and frozen embryo transfers that resulted in pregnancies from January 1, 2005 to April 2006 was.  The number of retrieval during that time was.  The success rate combining the fresh and frozen pregnancies divided by the number of retrievals was 77.2%.  The high frozen embryo transfer pregnancy rate allowed us to transfer fewer embryos so that there were 0 triplets from fresh transfers during this time.

What can I do to increase my odds?

Patients often ask if there are any additional procedures we can do in the lab that may improve the odds of conception.  Assisted hatching is the oldest and most commonly added procedure aimed at improving an embryo’s ability to implant.  Embryos must break out or hatch from their shell that has enclosed them since fertilization prior to implanting into the uterine lining.  This can be performed mechanically, chemically and most recently by utilizing a laser microscopically aimed at the zona pellucidum, the shell surrounding the embryo.  Assisted hatching appears to benefit patients who are older than 38 years of age and those with thick zonae.

Recently a protein additive called “Embryo glue” was shown to improve implantation rates in some patients whose embryos were transferred in media containing “Embryo glue”.  Time will tell if the adhesive effect of this supplement is truly increasing success rates and warrants wide scale use in IVF programs.

Embryo co culture is the growth of developing embryos is the same Petri dish as another cell line.  Programs utilize either the woman’s endometrial cells obtained from a previous endometrial biopsy or granulosa cells obtained at the time of the egg retrieval from the same follicles aspirated as the eggs.  Growth factors produced by these endometrial and granulosa cell lines diffuse to the developing embryo and are thought to aid in the growth and development of the embryo.  It appears to help patients who have had previous IVF failures and poor embryo development.

Embryo

Many IVF programs have reintroduced the concept of utilizing a co-culture medium to improve the quality and implantation of embryos. Co-culture is a procedure whereby “helper” cells are grown along with the developing embryo. Today, the most popular cell lines include endometrial cells (from the endometrium, or uterine lining) and cumulus cells from women’s ovaries.  Both cell lines are derived from the patient, thereby eliminating any concerns regarding transmission of viruses. Endometrial cells are much more difficult to obtain and process, while cumulus cells are routinely removed along with the oocytes during IVF retrieval.

Cumulus cells play an important role in the maturation and development of oocytes.  After ovulation cumulus cells normally produce a chemical called Hyaluronan.   Hyaluronan is secreted by many cells of the body and is involved in regulating cell adhesion, growth and development. Recent evidence has shown that Hyaluronan is found normally in the uterus at the time of implantation..

Co-culture of cumulus cells provides an opportunity to detoxify the embryo’s culture medium that the embryos are growing in and produce growth factors important for cell development 1,2.  This may explain why some human embryos can experience improved development with the use of co-culture.

Preparation of co-culture cells starts with separation of the cumulus cells from the oocytes after aspiration of the follicles. These sheets of cells are washed thoroughly and then placed in a solution that permits the sheets to separate into individual cells.  The cells are then washed again and transferred to a culture dish with  medium and incubated overnight. During this time individual cells will attach to the culture dish and create junctions between adjoining cells. This communication is important for normal development. The following morning, cells are washed again and all normally fertilized oocytes (embryos) are added to the dish. Embryos are grown with the cumulus cells for a period of three days to achieve maximum benefit.

Performing co-culture of embryos has improved implantation and pregnancy rates above and beyond those seen with the IVF advances previously described. More importantly, It promises to offer advantages for those patients whose previous IVF cycles were unsuccessful.

References:
1. Barmat LI, Worrilow KC, Paynton BV. Growth factor expression by
human oviduct and buffalo rat liver coculture cells. Fertil Steril 1997;
67:775–9.

2. Fukui Y, McGowan LT, James RW, Pugh PA, Tervit HR. Factors
affecting the in vitro development of blastocysts of bovine oocytes
matured and fertilized in vitro. J Reprod Fertil 1991;92:125–31.