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Birds, Bees and Bunnies: The Biology of IVF – Part 2 (Eggs!)
Posted by: Editor on Feb 09, 2010 in News

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.

CNY Fertility Center has locations in Syracuse, Albany and Rochester, NY. CNY Fertility Center offers couples and individuals affordable fertility treatments including low cost IVF (In Vitro Fertilization) by our experienced and caring staff.

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 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 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 gondadotropin (gonad /growth)follicle stimulating hormone, FSH. In IVF, added gonadotropin will be given by injection to promote the growth of these follicles.

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Primary follicle

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.

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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.

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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.

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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.

 

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MI egg

The development of the nucleus must be coordinated with 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 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 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 promote 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 concern that gonadotropin stimulation and the effect on the oocytes could be a factor which 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 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 which are atretic.

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Atretic egg

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 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 per cent 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.

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Mature egg with granulosa cells in IVF

Egg with granulosa from www.vetmed.auburn.edu

 

 

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Sperm from WWW.3dscience.com

 

 

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MII, as seen in the IVF laboratory with prominent polar body


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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

Related Articles

Birds, Bees and Bunnies: The Biology of IVF – Part 1

Birds, Bees and Bunnies: The Biology of IVF – Part 3 (Sperm & Embryos)

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