Live birth with healthy child is the ultimate goal of ART cycles. There is a complexity of interlinked events that decides the final outcome of ART. This event can be categorised as,
- Clinical (stimulation protocol, opu, embryo transfer, pregnancy rate etc),
- Patient related (age, diseases) and
- Laboratory (ART Lab- oocyte, sperm, embryo, media, etc).
There cannot be any single thumb rule for success in any ART cycle due to numerous variations in each categories. However by setting up Key Performance Indicators (KPI) and interpreting the data statistically we can have a better understanding of our success combinations as well as flaws.
In this topic we will restrict our discussion upto laboratory category. ART lab and equipments are designed to mimic intra-uterine/ intra- fallopian tube environment. Thus by giving minimum stress to gametes a better embryo or blastocyst can be achieved which may result in healthy pregnancy. In order to have significant pregnancy rate various studies have been performed keeping in mind variables like, co2, pH, temperature, media etc. To have an insight of entire process majority of KPI are directed towards maintaining the above controls and also the clinical aspect of patient. This KPI gives us entire picture from patient age, stimulation protocols, drugs, opu, gametes, zygotes, embryos as well as record of temperature of work stations, room temperature, humidity, incubators conditions, ph, air quality , infection control policy.
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Thus KPI makes us more vigilant and observes the pattern of success or failure of our lab. By using our KPI we can have customised settings of our monitors and set protocol for a cycle. This Gametes and embryos are in artificial environment mimicking natural environment a stricter control on ph, temperature, air are perhaps most crucial. It is believed and observed that a compromise in any of this parameters does cause detrimental effect on embryos.
Usually laboratories have protocols of checking the incubator settings, media, temperature daily. We do observe success rate to vary routinely. Where a drop in preganacy rate is to be considered more seriously , this makes us ponder over our KPI which should reflect the cause. We have observed many instances but we discuss about three such scenarios which caused damage to our pregnancy rates and KPI helped us.
First Scenario- opening incubator door frequently
On one occasion we observed more number of patients than usual in a batch of ART, while other KPI parameters were within accepted range. This observation lead to the fact that increased number of patient resulted in increase in number of oocytes.. This means that the load on incubator was much higher than usual. This load can be described by frequent opening of door to make fresh plates , transfer embryos, change media, observe growth, and embryo transfer. We used a conventional front load door incubator. Our KPI did not have a count or time period of incubator door openings, though it was handled to be bare necessity. To establish this assumption few studies observed that, there is a difference in temperature throughout the incubator where front and upper shelf had temperature nearer to 37 c as compared to other locations. Temperature displayed on screen of incubator was just an average. Temperature probe recorded average temperature of inside the incubator and hence KPI recordings were quite stable . However an important study observed that incubator door opened for 5 seconds takes a temperature recovery time of around 20 minutes whereas dishes took 30 minutes to come back to 37c. This can be explained due to cooler air from room entering the incubator and also cold co2 gas being pumped in due to decrease in co2 concentrations. IVF work station also has heating mechanism adjusted to 37 c but even that is average temp. And we do see different tempertaure in different parts of table. Further work station is also cooled by laminar air flow .
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Studies have proven that prolonged temperature variations impacts fertilisation especially high temperature affects cytokinesis of embryos. However we did not record absolutely mass failed fertilisation or mass embryo arrest .Though the number was less but the embryos progressed to blastocyst formation and also gave pregnancies. This leads to debate of optimal temperature required for embryo culture. Researchers have proved that core body temperature of reproductive axis to be 1c less than 37c. Interestingly a study observed difference in blastocyst formation from culturing the embryos at 36c .however fertilisation rate and implantation rate at both 36 c and 37 c were similar. With the concept of mimicking natural environment it was observed that preovulatory follicle had 2.3c less temperature than 36c. This could explain why even after temperature fluctuation we could still get good blastocyst as well as implantation. Another possible theory is a temperature regulating mechanism in embryo which remains an interesting research. Temperature fluctuations have an impact on pH also . Considering the importance of temperature and as most study design are carried out at 37c and better success rate at this temperature makes us accept cell culture to be at 37c.
In order to set things right measures were initiated to reduce the number of patients or oocyte in one incubator. On an average not more than 2/3 patient with 6-8 dishes were kept in an incubator. Open culture system was changed to closed culture. Procedures like Dish equilibration, sperm swim up where carried out in mini incubator rather than routine incubators. Another way of controlling this could be use of embryo scope or using single step media or bench top incubators. But we preferred the older method to be cost effective.
Second scenario- pH of media, calibration of instruments.
Our KPI includes routine CO2 check by CO2 analyser and they were fairly within accepted range and settings were adjusted depending upon observed value to maintain co2 at 5 % or 6% . this percentage varied according to media manufacturing company like, SAGE needs equilibration at 5 % while vitrolife needs it at 6% . As co2 is used to maintain Ph . Despite being vigilant there was drop in overall blastocyst formation and implantation rate. A study of KPI statistics revealed more or less steady co2 and temperature with no changes in protocols . A detailed study questioned co2 analyser observation and it was found that co2 analyser was not serviced or calibrated over 6 months.
Investigations confirmed Co2 analyser reading to be faulty. This scenario explains a lot about calibration of our instrument which are meant to check and control our functioning instruments like incubator.Routinely a calibration tolerance of 0.1 was accepted by us. We were using colourless media( vitrolife) rather than “pink” media ( phenol) hence changes in ph of media were unnoticed. literature supports ph to be most crucial for gamates. Routinely a ph of media scaling of 7.2-7.4 is accepted for cell culture. ph of media is also very dynamic and influenced by temperature,co2, volume of media. This leads to debate of optimal ph of media. Studies have revealed variable ph,that is alkaline in oviduct and acidic in uterus. Oocytes also showed varied ph from Germinal vesical stage ( 7.04 +/- 0.07) to MII stage ( 6.98 +/- 0.010). while amphibian oocytes shows rise in pH during fertilisation but unseen in humans. This opens a theory of intracellular ph regulatory system to maintain optimal Ph as required . This balance of CO2 and HCO3 in media sets the extracellular ph(phe) while intracelluarly (pHi) HCO3–/Cl– ad Na+/H+ regulatory systems maintain stability in cell. This self regulatory system of embryos seems to maintain the balance inspite of fluctuations and hence we see blastocyst formation and even implantation rates. Oocytes maintain their pHi through combination of follicular fluid, granulose cells. Hence denuded oocyte are more vulnearble and depends only up on pHe of media. Studies observed that raising ph to 7.4 or lowering to 6.8 affected the embryo metabolism where alkalinine media caused more damage. Thus ph around 7.2 seems to be more stabilising. Studies have observed ph irregularities to affect actin, cytoskeletal elements, meiotic spindle, cell apoptosis, mitochondria position in embryo.
This problem was addressed by calibrating our co2 analyser every 3 months. Media tubes should be capped off immediately after opening to prevent evaporation which can cause ph fluctuation.KPI included servicing dates of each instruments with due date notifications.” Pink media” was not used considering toxicity of phenol and above all in subsequent cycles pregnancy rates were up with above correction.
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