Factors Affecting Embryo Development

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Human conception involves the coordination of complex biochemical and molecular intracellular signaling events between human gametes, resulting in the production of viable embryos capable of implantation and the establishment of a viable pregnancy. Successful pre-implantation embryogenesis is critically dependent on the culture environment provided by the IVF laboratory.(1)

Working in the IVF field we are aware and well versed with the phrase “Expectation Versus Reality”. We come across this dilemma several times during the couple’s IVF cycle. The clinician looks at the AFC and AMH and expects a good number of follicles to grow and is baffled with the reality of low response and less number of follicles, on the day of the trigger. In a certain cycle, an embryologist may have a good number of MII and expect good fertilization and blastulation rate, but is met with the reality of low fertilization and less or no blastocysts. This is what makes IVF treatment so mystical and unpredictable. Here 2+2 may not always be 4. Thereby, it’s very important to understand the factors which may affect the embryo development and quality inside the embryology laboratory.

The factors can be broadly classified into three categories: 

  • External – Outside the Laboratory
  • Internal – Within the laboratory
  • Clinical aspects

External Factors:

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While you’re looking around your lab, why not take a quick glance out the window.  Do you see any road resurfacing on the street below?  How about rooftop resurfacing or construction in a nearby building?  What about auto exhaust from idling ambulances or traffic?  Nearby restaurants spreading burnt oil fumes?  Smoke from nearby fires: building fires, tire fires, forest fires.  Seasonal pollutants—the list goes on and on.

But why be concerned about those outside elements?  Because many of the pathogens (viruses, bacteria, fungi), allergens (bacteria, mold), and toxins (endotoxins, mycotoxins) present in those sources are making their way into your lab, and it’s a bad list when it comes to the sensitive environment your patients’ embryos require to maximize their genetic potential, full of toxic, mutagenic and carcinogenic volatile organic compounds (VOCs) including:

  • Ketones, aldehydes, nitriles from disturbed soil in construction.
  • Formaldehyde, acetone, methane, methylene chloride from pressed board and wood in building materials.
  • Toluene and styrene from cement foundations and fresh asphalt, road resurfacing.
  • Sulphur dioxide, formaldehyde, nitrogen, ethylene from cars, ambulances, and medivac exhaust.
  • Polyaromatic hydrocarbons from the fat burning fast food restaurant down the street.
  • Alkenes, benzene, toluene, methane, formaldehyde from your own facility’s generator exhaust.
  • Carbon tetrachloride, chloroform (and much more) from the pesticides being sprayed to kill the bugs in the building next door.
  • Acetone, diethyl ketone, ethanol, and several interesting biologicals and microbial shedding off your lab staff themselves.(2)

seasonal influence of the VOCs over the IVF laboratory’s air, embryo development and implantation rates has been found, in addition to the outside temperature and humidity.

Internal factors

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  1. Now that you are sitting in your IVF lab, take a careful look around the room.  Look at the clean surfaces and equipment, and the lab technicians carefully following protocols to provide an optimal, consistent lab environment, conducive to embryogenesis. Your clinical staff and lab are taking precautions and following strict laboratory practices (GLP) with acceptable pregnancy rates. What could be wrong? As you’re looking around the room, you’re looking past one of the most important variables with the potential to influence your laboratory’s results. In fact, you’re literally looking right through it. Any guesses? Air. That’s right. The ambient air in your lab—clean, though, you think it may be—may actually be adversely affecting your clinical pregnancy rates to a significant degree.(2) This is the same air which is entering from outside, passing through the filters and getting recirculated in the laboratory.

Laboratory personnel alone, constitute one of the greatest sources of bioburden, adding bacteria, viruses and mold spores to the culture environment.

To combat the issue of air, a good laboratory will certainly need an HVAC system that is humming away providing conditioned air and a positive pressure environment for your lab—likely scrubbed with a series of carbon, potassium permanganate and HEPA filters. The design of the IVF laboratory, HVAC and air purification system are therefore critical in determining the future success of human embryogenesis and clinical outcomes.

One of the biggest enemies inside any IVF laboratory is ‘Volatile Organic Compound’, better known as VOC. VOCs are released as gases from solid and liquid materials used in laboratory construction and finishing (flooring, adhesives, paints, sealants, etc.), lab equipment, cleaning fluids, air fresheners, furnishings, cosmetics, floor waxes and cigarette smoke, to name

a few. Opening a new pack of dishes also emits a huge amount of Styrene and Acetone.

Even if you culture under oil, or use table top incubators, or time-lapse imaging, etc.—VOCs can still enter your media and adversely affect your embryos.  If oil soluble, VOCs can easily enter the oil overlay. Once present in the media or oil, the VOCs may have an intimately adverse impact on your culture environment and thus become a permanent threat to your embryos.

We would suggest investing in a VOC meter to monitor the VOC in your laboratory. High VOC is also directly proportional to high number of biochemical pregnancies, as they have an implication on embryo health and development.

Prevention of VOC load in the laboratory:

  • Perfumes and cosmetics should be a complete NO for the personnel working in the laboratory or OT.
  • Open dishes and consumables 2-3 days prior to use, in order to off gas them in the laminar air flow.
  • Personnel to always wear gloves and masks to reduce the load of bacteria and viruses.
  • Install in-line coda filters to the incubators in order to reduce the passage of VOC and dust particles from the cylinder to the incubator.
  • Procure gas cylinders from a reliable vendor.

Use of 70% alcohol and ethanol should be forbidden in the IVF laboratory. Many Labs still use alcohol as their means to clean hoods and instruments. It is not required, and in fact you will be doing more harm than good. There are various alcohol-free disinfectants available in the market, which should be picked in order to maintain the air in the laboratory.

Clinical aspects

There are many clinical aspects that can affect and control how an embryo develops. This includes, but is not limited to, PCOS, thyroid, maternal age, poor ovarian reserve, OAT, diabetes, hypertension, type of stimulation and so on.

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a. PCOS

It is undeniable that women with PCOS undergoing IVF commonly produce a disproportionate number of poor quality eggs, with reduced fertilization potential and which upon fertilization can produce embryos with poor developmental potential. However, there are studies supporting that patients having PCOS and a body mass index (BMI) in the lean rather than the obese range have more favourable assisted reproductive technology (ART) cycle characteristics, but show no clinical outcome differences.(3) PCOS patients are a classic example of ‘more is not always better’. Remember, in IVF we need quality over quantity.

b. Advanced Maternal Age/Poor ovarian reserve

Advanced maternal age (AMA; >35 year) is associated with a decline in both ovarian reserve and oocyte competence. Poor ovarian reserve (POR) indicates a reduction in the quantity of ovarian follicular pool in women of reproductive age group and is an important cause of infertility in many couples.

(i) These women are often diagnosed with low AMH and AFC.

(ii) Ovarian aging leads to a decrease in the quantity and quality of oocytes, and aged oocytes have a reduced number of mitochondria.

Mitochondria are the energy factories of the cells and their lack could lead to lower fertilization rates and poor embryonic development.(4) With depletion in the number of mitochondria, the capability of second polar body extrusion also reduces.

(iii) IVF pregnancies (for which fetal cardiac activity was accurately documented), we have clearly demonstrated a linear increase in miscarriage rates with advancing maternal age. In women younger than 30 years the spontaneous loss rates was less than 6%, whereas, the miscarriage rate in women aged ≥40 years was almost quadrupled (22.2%). For women aged >40 years, the singleton pregnancy loss rate exceeded 27%.(5)

(iv) Women with AMA have a high incidence of Smooth Endoplasmic Reticulum (SER); presence of SER leads to lower fertilization and implantation rate. As an embryologist, one needs to be careful to not deposit the sperm inside the SER, as that would lead to fertilization failure.

(v) Over the years, we have also noticed women with AMA or POR have little or no resistance in the eggs, making them highly susceptible to degeneration.

(vi) Embryo development gives us a lot of information, if you observe and analyse. For instance, fragmentation on Day 2 is different from fragmentation on Day 3 of the embryo. Fragmentation on Day 2 is largely due to incompetence in the quality of the oocyte. Day 3 fragmentation would be because of the sperm.

c. Sperm factor

Sperm plays an important role in embryogenesis and quality of the embryos. This problem may be associated with low sperm production (oligospermia), poor sperm motility (asthenospermia), or abnormal morphology (teratospermia).

(i) Abnormal sperm parameters, particularly teratozoospermia are associated with fertilization disorders in IVF, including failed and delayed fertilization.(6)

(ii) Poor sperm quality is associated with increased aneuploidy and DNA damage (fragmentation, instability and single stranded breaks).(7)

(iii) Other sperm abnormalities have also been associated with failed fertilization and aberrant or arrested embryo development.(8)

(iv) In embryogenesis, the contribution of the sperm begins from Day 3. No fragmentation on Day 2 with an increased fragmentation on Day 3 is a sign of poor sperm. (Teratozoospermia or DNA fragmented sperm)

d. Thyroid and diabetes

Type 1 diabetes compromises female fertility, even before it is diagnosed. Associated hyperthyroidism further reduces fertility.(9) A study done to assess thyroid function and embryo quality found that women with high normal TSH levels or high TSH levels had impaired embryo quality. When diabetes is not well-controlled, it can affect the chance of having a baby. In women, diabetes can result in irregular or absent menstrual cycles, while in men, it can cause problems getting and maintaining erections and lower testosterone levels (the male hormone).(10)

e. Smoking

Smoking has been shown to have a detrimental effect on various parameters of semen analysis. Indeed, men who smoked > 20 cigarettes per day experienced a 19% reduction in sperm concentration, compared with nonsmokers.(11) Some studies have found that the sperm of smokers has increased DNA fragmentation. DNA damaged sperm may lead to problems with fertilization, embryo development, embryo implantation, and increased miscarriage rates. Smoking is strongly correlated with an increased risk of erectile dysfunction.

In females, smoking leads to increased risk of miscarriage, possibly due to damaged eggs, damage to the developing foetus, or unfavorable changes in the uterine lining, making healthy implantation of an embryo less likely.
We have tried to highlight the main factors that would impact an embryo development. It is very important to understand that a lot that goes on inside the IVF lab, depending on both intrinsic and extrinsic factors. The more variables one is able to control, the better will be their lab environment and culturing system.

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