Birds, Bees and Bunnies: The Biology of IVF
This article discusses the basic biology behind the process of In Vitro Fertilization (IVF) and may be helpful to couples who plan to undergo IVF. Technical terms may be found in a glossary at www.fertilitylifelines.com.
In the high school classroom, perhaps we giggled a bit about these slightly embarrassing topics. Now that we are trying to get pregnant we need to pay attention. The story of reproduction is fascinating and complex and the more we know the more we can understand some of the terms used in IVF clinics that affect our outcomes.
In nature, for most women, the production of a single egg each month is automatic. The hormone changes required are treated lightly here. In IVF, we must go to enormous lengths to be able to obtain this precious cell (the egg) in the laboratory. More of the story can be found in articles on how the gonadotropin hormones work and how egg retrieval is done, elsewhere on this web site.
We begin with a consideration of the egg, which measures a tenth of a millimeter, is barely visible, but is the largest and most complex single human cell. It provides the energy and structure to form a healthy embryo which can develop into a baby composed of a few trillion cells. A perfectly competent egg is ready to be fertilized and develop to the 4 cell stage with a little help from the sperm. The sperm will provide 23 chromosomes, half of the genes and the centriole, critical to organize cell division. But (as some would argue, typical of the man), there is no energy provided by the sperm nor is there any follow-up responsibility. The leading contributions for assuring a next-generation must depend on the egg.
The tiny egg which has been resting in the ovary for 20-40 years must suddenly, within weeks, awaken, mature and develop the ability to combine with the sperm and divide. Rapid and complex changes give the egg competence to do these tasks. The more we know about the complexities of the process it seems amazing that it ever works. Indeed, too often it does not. There are many inefficiencies in reproduction, especially in humans. The development of the eggs, the fertilization process, and early development often fail in nature. Even the most fertile young woman has about a 20% chance to conceive in one cycle. Throughout evolution, this has turned out to be just fine for the human species. Other animals are different. Famously the rabbit produces several eggs each month, and to make sure the timing is right the female bunny, the doe, ovulates by reflex after intercourse. Pregnancy follows automatically. Considering our behavior, it is a good thing that this is not the case in humans.
However, the good luck of the bunny is not helpful to infertility patients, who are often frustrated with the low percentages which can be less than 5% per cycle. They have to spend time and money to improve their conception possibilities. In fact, after IVF, ongoing healthy pregnancy rates can be in the range of 40% in one month. There is much technology involved to produce multiple eggs and embryos.
Now let’s look at the details of the process from the perspective of the biology of reproduction. How does a woman conceive? Only 5% of all the eggs harvested in IVF treatments result in a baby. Many of the errors which arise can be traced back to the egg. The most obvious example of this is the well known adverse influence of the advanced age of the woman. In many instances, an egg from a 40-year-old will look great under the microscope, but it is doomed from the start by genetic errors. Please refer to a separate article on IVF and the age of the woman. The resting egg within the primary follicle is surrounded by a single layer of cells, the granulosa cells. Selected follicles grow to become secondary or antral follicles in a phase called initial recruitment. This occurs before the woman even has a period. Then suddenly a new phase of recruitment begins with the onset of the menstrual cycle. Much research has been directed at the signals which initiate the selection of about 20 follicles to suddenly begin to grow at once. As yet this signal is not clearly identified. But in any case, independent of gonadotropins, this cohort of follicles does emerge. As they grow they do become sensitive to the gonadotropin (gonad /growth) – follicle-stimulating hormone (FSH). In IVF, added gonadotropin will be given by injection to promote the growth of these follicles.
The granulosa cells multiply and as the follicle develops, fluid is secreted forming an antrum which can be visualized on ultrasound as it reaches about 2 mm in size. Counting the number of these antral follicles can be predictive of the numbers of eggs retrieved in IVF cycles.
Secondary or antral follicle surrounded by granulosa cells. From www.dkiimages.com
About half of the antral follicles will result in an egg in the laboratory during IVF. The numbers will vary from one month to another, but typically within a narrow range. One does not see the antral follicle count jump from 3 to 15 in one month. There is no amount of drugs which can force more follicles to develop than there are antral follicles. The limit is really set at the beginning of the cycle. Counting the antral follicles is probably more valid than the measurement of blood FSH on day 3, in evaluation of fertility potential.
Mitochondria from www.nsf.org
Another problem which can arise as the cytoplasm begins to mature relates to the tiny energy factories themselves, the mitochondria. Yes, those were in your biology book too. The mitochondrial numbers increase enormously from about 10,000 in the primordial egg, to several hundred thousand in the mature egg. The mitochondria are the tiny engines which burn or oxidize fuel and produce energy for the egg. In older women, eggs may have low numbers of mitochondria. This may be an aspect of the eggs’ incompetence. Attempts in the past to restore mitochondria by injection of healthy cytoplasm from egg donors have not been successful. For complex reasons this research area is not being pursued.
The all important FSH hormone stimulates the development of the cumulus cells which are tightly bound to the egg and provide nutrients and molecular signals. These assist in rapid growth of the egg cell from 35 to 90-120 microns in diameter as it acquires the proteins and RNA which will be utilized in fertilization and development.
In IVF, the immature egg is easily recognized by the presence of the nucleus (germinal vesicle). Once it disappears, we know that maturation is occurring.
Germinal Vesicle Egg
In the meantime, a tiny amount of LH is required to initiate the breakdown of the nucleus, (germinal vesicle breakdown) which marks the beginning of nuclear maturation and the egg evolves to the stage known as Metaphase I.
The development of the nucleus must be coordinated with the simultaneous development of the cytoplasm. Estrogen provided through the cumulus cells helps to activate or suppresses selected genes in the cytoplasm. These lead to the maturation steps required. Just the right amount of estrogen is needed, and this hormone production becomes the marker used for follicular development. Daily blood tests can indicate indirectly that the follicles are producing estrogen well or not so well.
The other ovarian hormone, progesterone is involved in egg membrane and cytoplasmic maturation as well (Chian review). If there is a surge of LH, the process of luteinization can begin in the follicle, causing the production of rising progesterone levels. Too much progesterone from premature luteinization can occur and this causes atresia of the eggs and all is lost. This is the reason in IVF the pituitary gland is usually suppressed by the agonists or antagonists. See further discussion of antagonists at www.fertilitylifelines.com and elsewhere on this website.
Simultaneously, one other element of maturation occurs at the level of the membrane around the egg. Cytoplasmic maturation coincides with one more element. The membrane surrounding the cytoplasm has to mature. Most amazing at the submicroscopic level is the critical cortical granule release system. When one sperm enters the egg, this system instantly solidifies the membrane and prevents the entry of another sperm.
Granulosa cells communicate with the egg by tiny projections which create an intimate relationship. These cells must remain attached to the immature egg so that nourishing factors are transmitted which promotes the development of the cytoplasm. The cytoplasm, the bulk of the egg, contains the submicroscopic mitochondria and other organelles. The maturation of the organelles must occur hand in hand with the maturation of the nucleus. IVF patients will recognize the cytoplasm as the major part of the egg, the place where sperm are inserted with a needle in intracytoplasmic sperm injection, ICSI.
Later, when the egg is mature, the granulosa cells are washed off with an enzymatic solution. Then we can grasp the egg with a pipette and inject a single sperm (ICSI). This will be covered later.
These connections are not a one way street from cumulus cell to the egg. The egg, in turn, is controlling or influencing the functions of the rapidly expanding group of surrounding granulosa cells. It is sending signals which promote the very development of the follicle which expands eventually to approximately 2 cm. Some of the cells are morphed into theca cells, and later will be called on to produce progesterone. This dynamic expansion has no rival in the body. The rapid development of the needed blood supply is another of the diverse and intricate responses required.
During stimulation in IVF, high doses of FSH and sometimes LH are used to recruit multiple follicles and aid in their maturation. For many years there has been a concern that gonadotropin stimulation and the effect on the oocytes could be a factor that limits the success of IVF (Paulson et al 1991). The blood supply is impaired by crowding of follicles when we stimulate for IVF. Low oxygen within the follicle leads to lower oocyte competence in some of the eggs. This is one disadvantage of ovarian stimulation (Von Blerkom 1997)
Van Blerkom ( 1997) and Munne (1997) suggested that drug stimulations might predispose to chromosomal abnormalities in the human embryo. Jackson et al (1998) asserted that accelerated ovulation induction response was linked to abnormal nuclear formation which resulted in embryos with a defect called multinucleation.
On the other hand there have been many studies reassuring us that the outcomes of IVF are not revealing an epidemic of abnormal babies. Furthermore, reassuring results were noted recently in a comparison of stimulated vs natural cycle derived oocytes (Zeibe et al, 2004). Most likely these embryos which become impaired are falling by the wayside and never implant.
But let’s get back to the biology of the cumulus-egg complex. In addition to links to the cytoplasm, there is an active two-way communication from the cumulus involving nutrients and signals to the nucleus to begin its required maturation steps. This begins the process which results in the transition of the nucleus from the packed short chromosomes of the germinal vesicle of the most immature egg, to the metaphase II mature structure which is ready for fertilization.
In the natural cycle, the dominant follicle produces androgens (Anderiesz, and Trounson, 1995) which cause atresia, a type of cell death of the remaining follicle and eggs. Additionally, the rising level of estrogen signaling through the inhibin-based feedback mechanism tells the pituitary gland to reduce the level of FSH which reaches the smaller follicles. These follicles are thus deprived of the stimulation they need to continue to grow. Thus in most cycles, after the largest follicle is about 14 mm, only one egg retains the competence to become fertilized and develop. The concept of acquiring healthy eggs for in vitro maturation (IVM) rests on the appropriate harvesting of oocytes after maturation has begun and before atresia has begun. In any egg retrieval, there are likely to be one or more eggs that are atretic.
Once the follicles have been recruited, the stimulation by FSH is necessary for continued growth. But the timing and amount of FSH can be critical (Thomas and Vanderhayden). Premature stimulation can interfere with egg competence including errors in the chromosomes.
In IVF, ovarian stimulation overrides the influence of the dominant follicle. We promote the continued development of all the follicles by giving FSH in huge doses relative to the amount naturally produced by the pituitary. This is called stimulation or hyperstimulation of the ovaries and is a key to IVF.
An important function of the granulosa cells as they relate to IVF is the fact that these little cells are powerful factories producing estrogen. As mentioned earlier this is a need to promote the development of the egg. The healthy follicles produce one particular type of estrogen called estradiol in science language, but called E-2 in the IVF clinic. Normal follicles produce dramatically increasing amounts. Unhealthy follicles produce less. The total amount of E-2 can be measured in the blood as an indicator of the response and health of the follicle. This indicator ultimately reflects on the health of the eggs. Additionally, too much estrogen being produced can be an indicator of the possibility of ovarian hyperstimulation, a dangerous syndrome in about two percent of IVF cases.
As the egg matures the chromosomes numbers are reduced to half of an adult cell and the egg is ready to receive a contribution of DNA or genetic material from the sperm.
The final trigger for resumption of nuclear maturation and competence in IVF treatment is the result of the surge of LH like activity provided by its chemical cousin, HCG. Suddenly the close bond between the granulosa cells and the follicle are broken. The egg in cumulus is preparing to be cast-off down the tube to meet a sperm and the final nuclear development occurs as the magical metaphase II stage is quickly reached. At this point, the egg has half the copies it will need as it has cast off 23 copies of its DNA into the polar body. The observation of the polar body is the sign of maturity is welcomed in the IVF lab as the signal that the nucleus has reached this stage.
Mature egg with granulosa cells in IVF
Egg with granulosa from www.vetmed.auburn.edu
Sperm from WWW.3dscience.com
M II with nuclear material in the polar body and nucleus (blue) from www.reproduction-online.com
In the IVF lab the ideal egg is the metaphase II which is abbreviated MII using roman numerals, “M two’s” as we call them. The polar body is easily seen once the granulosa cells have been stripped away.
If the first cell division goes awry, the wrong number of chromosomes results. The errors in general are known as aneuploidy. One possibility is that the egg retains one chromosome too many. This is called trisomy, three of one set of chromosomes. Down’s syndrome is caused by triploidy of the number 21 chromosome for example. Many miscarriages are due to trisomies of certain chromosomes. Another derangement of numbers is monosomy (one /chromosome), in which the egg is left with one copy of the pair required for normal reproduction and development. Turner ’s syndrome and some miscarriages are due to monosomy.
Often fertilization can be accomplished by simply allowing the sperm and eggs to mix in vitro. The other prominent structure in the egg is the zona pellucida (clear zone) or jelly coat, which must be traversed by the sperm and usually does so with ease in natural fertilization.
The natural process is depicted in a clip graphically: www.health.howstuffworks.com/human-reproduction10.htm
The Sperm and Embryo
Sperm penetration and fusion, cortical granule reaction, from www.Wikipedia.com
Once one sperm has penetrated, the cortical granule reaction causes a shield to slam shut just inside the egg membrane within microseconds. This prevents the entry of another sperm. If this system fails and two sperm enter, there are two sets of male chromosomes. This is called polyspermy and actually is observed commonly in the IVF lab. Three pronuclei, a sign that an entire extra set of chromosomes is present is a fatal flaw.
In IVF, in cases of low sperm numbers when the sperm function is inadequate for this task, delicate micromanipulation tools are brought to bear. Using methods developed by cell biologists, the embryologist sits before controls like joysticks on a video game. Imagine threading a bit of angel hair into a speck of dust and you will be able to understand how intracytoplasmic sperm injection (ICSI) is done. Now a daily part of the armamentarium of highly skilled IVF embryologists, it is nonetheless an amazing advance.
ICSI, intracytoplasmic sperm injection. From www.cht.nhs.uk
After fertilization, in the incubator, the pronuclei can be seen on day one. On the first day after insemination, the IVF embryologist looks for the two pronuclei as an indicator that fertilization has occurred. Further scrutiny of these under high magnification gives clues as to the future. Lynnette Scott PhD in Boston among others has developed a system of predicting outcomes based on the tiny nucleoli in the pronuclei. If they line up in an orderly manner the embryos may progress. If they are disorganized there is a good chance development will be impaired.
Laser ICSI used to breach the zona, as used for assisted hatching, from www.hamiltonthorne.com
Counting the chromosomes in the pregnancy tissue is recommended if a miscarriage occurs after IVF. This test can be performed in a genetics laboratory on miscarriage tissue in order to determine if one of these errors caused the loss. When aneuploidy is found in the tissue by chromosome testing, we are reassured that the loss was not due to a disease in the mother.
In IVF a common occurrence is the early pregnancy which attaches and produces a positive test, but then dies within a few days. Because we only know there is a pregnancy by looking at a biochemical reaction, the pregnancy test, this event is called a biochemical pregnancy. Most of these unfortunate occurrences are due to aneuploidy also. Implantation occurs, HCG hormone is produced but often the progesterone level is low, the HCG hormone does not rise and over the first week of the pregnancy it is apparent that the pregnancy is in danger, and then the hormone levels go down over the following week. In essence this may be considered a microscopic miscarriage. A tiny defective implanted embryo is lost which contained placental cells and usually had no fetal development at all.
Thomas FH and Vanderhayden . Oocyte growth and developmental competence in In –vitro Maturation Of Human Oocytes Tan, Chian, Bucket eds. pages 1-15 Informa UK 2007
Harris, Sarah E and Picton HM. Metabolism of follicles and oocytes during growth and maturation. IBID page 15-36.
Written by Joe B Massey MD
Additional references provided on request.