LEARN INSURANCE

This is a guest post by Saiprasad Gundeti, Senior Embryologist, Malpani Infertility Clinic.

As an embryologist, I help in making the dreams of infertile couples come true ! Most couples do not know what happens in an IVF lab, so I am happy to describe a day in my life !

This is the daily routine we follow at our centre :

First thing in the morning –

Cleaning

Cleanliness is a very important factor in IVF Lab. We need to make sure everything is sterile ! Because I work alone in our lab, it's much easier for me to ensure that everything is clean !

• Hood and bench work surfaces (including microscope working areas, heat baths, petridish warmer) are cleaned and wiped down with 6% Hydrogen Peroxide.
• Once a week centrifuge rotors and carriers as well as outside area of the centrifuge are cleaned and disinfected using 6 % Hydrogen peroxide
• Floor is nicely cleaned with 6 % Hydrogen Peroxide.

Monitoring and Maintenance of All Equipments

Your embryos can be quite demanding, and we need to keep them happy in the IVF lab ! We do this by ensuring that the conditions in the lab are just right for your embryos !

• The Incubator CO2 % is confirmed with the help of CO2 analyzer. Calibrated if any anomaly seen in the readings. Temperature is measured with the help of digital thermometer. Calibration is done in case of any temperature variation seen.
• Temperature is measured for all heating baths, heating plates of all microscopes and petridish warmer.
• Gas cylinders connected to CO2 incubators are checked. Cylinders are changed if they are found half empty.

Monitoring of embryo development

• Fertilization assessment of each patients eggs on which ICSI was performed a day before.
• Stripping (denudation) of eggs on which IVF was preferred.
• Transferring fertilized eggs to new dishes (prepared, labeled with patients name and incubated a day before)
• Assessment of embryo development grading of day2, day3, day4, day 5, day 6 embryos.
• Documentation in IVF file as hard copy and IVYSITE ( our electronic medical record) as soft copy.
• We routinely give all our patients photos of their embryos. We take pride on our work, and good quality embryos are a testimony to the fact that we are providing a high quality service to our patients !

Preparation for the Cases

• Preparation for ICSI procedure -
1. Preparation of ICSI dishes

1. Preparation of Hylase dishes (for stripping eggs before ICSI procedure)

EMBRYO TRANSFER PROCEDURE

This procedure involves the transfer / placement of day-2 or day-3 embryos to the patient's uterus. It is performed transcervically with or without anesthesia.

Showing embryos to the patient
We at our centre show every patient their embryos before transferring them to their uterus.

• The nurse in charge brings the patient couple to the lab and introduces them to me.
• I walk them through their cycle, giving them details about –
• Number of eggs retrieved
• Procedure done (IVF/ICSI)
• Number of mature eggs
• Details about husband's sperms (fresh/frozen/epidydimal/testicular)
• Number of eggs fertilized
• Quality of embryos
• Number of top quality embryos
• Number of embryos that will be transferred
• Number of embryos that will be cryopreserved

• I show them the embryos and grade them in front of the patient couple. Many women get quite emotional on seeing their embryos !
• Guide them to the operation theatre.
• I select the best embryos and transfer them to "Embryo transfer" dish.
• When the OT is ready, I confirm with the doctor, as to which catheter to be used.
• I load the embryos into the catheter. A nurse stands as a witness while loading.
• I hand over the catheter to the doctor, by verbally confirming the name of the patient.
• The doctor gently guides the catheter through the cervix and gently pushes the embryos into the uterus.

EMBRYO CRYOPRESERVATION PROCEDURE

This procedure involves freezing of supernumerary embryos after transferring the best embryos.
The procedure is done only after taking patient couple's consent.

• This procedure is done at room temperature. So temperature of microscope stage is brought down to room temperature.
• Vitrification (freezing) medium is taken out. Freezing dish is prepared by placing droplets on the dish.
• Cryolocks are labeled with patients name, date of freezing, number of embryos per cryolock.
• After the embryos are frozen, the cryolocks are stored in cryocanisters.
• Details are documented in the register.

COLLECTION OF SEMEN SAMPLE FOR HUSBAND OF PATIENT UNDERGOING OVUM PICK UP PROCEDURE

• The patient is given a pot labeled with his name to collect semen sample.
• Once the sample is collected, it is brought to lab along with the file.
• Sample is processed for the procedure (IVF/ICSI)

PREPARATION FOR OVUM PICK UP ( OPU) PROCEDURE

• Temperature of instruments are checked.
• Patient's ID is checked with doctor/ nurse in charge.
• Petri dishes are arranged on a petridish warmer
• Remind clinician to collect clean follicular fluid (~3-4 ml). I Pour this in warm petri dish.
• subsequent follicular fluid is poured into a fresh warm petridish.
• As soon as egg (Oocyte Cumulus Complex) is located I take clean follicular fluid in to Pasteur pipette and pour 2-3 drops over OCC and then take OCC into the Pasteur pipette and deliver in the dish at 12 O'clock position.
• Inform the clinician by loudly saying "EGG" when the egg is identified.
• I inform Dr. Anjali about presence of egg in the tube by loudly saying "EGG".
• If there is no egg in the tube I inform by loudly saying "NOTHING" and I do inform about the presence of Granulosa cells in the fluid.
• Similarly I identify and collect all the eggs at 12 O' clock position in 60mm petridish.
• I Take out OPU dish labeled with ( Patient Name) and ( Egg Wash)-prepared one day earlier.
• I Rinse all the OCC's in the outer well containing Flushing medium and transfer them in to the centre well of the same dish containing Fertilisation medium
• Following OPU, OCC's are cultured in fertilisation medium in CO2 incubator till IVF or ICSI being done on them.

Continuing medical education

IVF is a field in which technological advances occur very rapidly. I read the journals, Human Reproduction and Fertility Sterility, to ensure that I am uptodate !

Preparation for the Next Day

• I go through the files of all patients that are scheduled for next day.
• Preparation depends on what procedure patient is undergoing.

1. Fertilisation check dishes:
   dishes for each patient



2. Dishes / medium for OVUM PICK UP :


1 tube with Oil (6ml)
1 tube with Flushing medium (6ml)

3. Dishes for IVF (No. of Dishes dependent on no. of follicles):



4. Dishes for ICSI (3 Dishes for each patient):

1. Dishes for Frozen Thaw (2 Dishes for each patient) :
   
   
   
   

   
   

2. Medium for Embryo Transfer :

1 ml for each patient

HOW I IMPROVED MY VITRIFICATION TECHNIQUE !

SAIPRASAD GUNDETI, SENIOR EMBRYOLOGIST, MALPANI INFERTILITY CLINIC PVT. LTD.

Vitrification involves freezing the embryo about 600 times faster than ever before. This ultrarapid process is so fast that it literally allows no time for intracellular ice to form. As a result, vitrification avoids trauma to the embryo.
In conventional (slow) freezing, 20-30% of embryos do not survive the freeze-thaw, and those that do survive have less than half the likelihood of generating a pregnancy as do fresh embryos. In contrast, vitrified embryos have a better than 95% freeze-thaw survival rate, and a pregnancy generating potential that is comparable to fresh embryos.
Vitrification is now regarded as potential alternative to conventional (slow)freezing.

Major advantages of Vitrifiction over Slow freezing are :

• Its prevents ice crystal formation within the cells, which can damage the embryo.
• It eliminates use of expensive freezing unit(required for slow freezing).
• High survival rate post thaw.
• High pregnancy rate following transfer of thawed vitrified embryos.

At Malpani infertility clinic, we have been using vitrification for a number of years to cryopreserve both embryos and eggs. When it comes to embryo cryopreservation, we freeze all stages of embryos i.e. Day 1, Day 2, Day 3, Day 5 and Day 6.

Steep Learning Curve :

To avoid embryo exposure to high concentrations of toxic cryoprotectants for extended periods, vitrification protocols generally require that embryos remain in the vitrification solution for a fixed time. This can be difficult, especially since vitrification technique have an extensive learning curve before the technique is mastered. Even with practice, one may not be able to quickly pick up embryos and vitrify within the time required.

Therefore it's critical that we determine the length of time the embryos can safely reside in vitrification solution, so that we don't overexpose embryos to toxic cryoprotectants, which can kill them or lead to very poor post thaw survival.

How difficult is it to master the technique?

Vitrification technique is a difficult art to master:

1. Due to high concentrations of cryoprotectants, vitrification medium is very viscous. High viscocity makes it very difficult to handle embryos, while they are in the medium. Needs lot of practice in order to get efficient.
2. One needs to be quick while handling embryos in vitrification medium, as long exposure of embryos to cryoprotectants can kill embryos or lead to poor survival rate post thaw.
3. Loading embryos onto cryolock is the most difficult part. It requires lot of practice. Improper loading can also affect the survival rate.
4. Thawing procedure (called Vitrification Warming) is as important as the Freezing. Improper thawing may lead to poor survival. So one needs to thaw embryos carefully in order to ensure good survival of embryos.

How do I vitrify your embryos and eggs?

VITRIFICATION FREEZING

We use Quinn's Vitrification Medium at our centre for freezing Embryos and Eggs.
Quinn's Vitrification Medium contains 2 media, "Equilibration Medium" and "Vitrification Medium"

Equilibration Medium contains Base Medium(Hepes buffered Medium) + 7.5 % ethylene glycol + 7.5 % DMSO (Dymethyle sulfoxide) + 20 % Protein supplement

Vitrification Medium contains Base Medium(Hepes buffered Medium) + 7.5 % ethylene glycol + 7.5 % DMSO (Dymethyle sulfoxide) + 0.5 M Sucrose + 20 % Protein supplement

Preparation of Vitrification Dish :

Place 1 drop of 0.1 ml of "Equilibration Medium" and 4 drops each of 0.05 ml of "Vitrification Medium" as shown in the figure.

The entire Freezing procedure is performed at room temperature.

We need to leave the Petri dish in the workstation for 15 min, for the medium to recover to room temperature.

Equilibration

• Place embryos or eggs to vitrify in equilibration medium. (This will be number of embryos or eggs that will be placed on 1 cryolock). We don't put more than 3 embryos or eggs on 1 cryolock.
• Once embryos or eggs are placed in Equilibration Medium, they spontaneously begin to shrink . This is when the intracellular water comes out.

Picture of shrunken embryo, after placing it in the equilibration drop.

• The shrunken embryo or egg will gradually recover back to its original size. This takes approximately 7 to 8 minutes. This may be less or more than 7 to 8 minutes in some embryos and eggs.

Vitrification

• Once the embryo or Egg recovers back to its original Size, its ready to be vitrified. I generally vitrify the embryo or egg once it recovers to about 80% of its original size.
• Pick up the embryo or eggs from equilibration drop with a flexipet and place them in the 1st drop of vitrification medium.
• Once the embryo or egg is placed in the vitrification drop, it again starts shrinking. Keep the embryo or egg in the 1st vitrification drop for 10 sec. This is basically to get rid of Equilibration medium, which is carried along with the flexipet while transferring them from equilibration medium.
• While the embryo/egg is in the first drop of vitrification medium for 10 sec, rinse the flexipet by pipetting the vitrificaition medium to get rid of equilibration medium.
• After 10 sec, pick up the embryos/eggs from the 1st vitrification drop and transfer them to series of 3 drops of vitrification medium one after other.
• Quickly load the embryos onto cryolock with very little medium. Make sure to gently remove excess medium in case a bigger drop is made on the cryolock.
• immediately plunge the cryolock in Liquid Nitrogen. Now the embryos are vitrified.
• Slowly put the cap to the cryolock, while it is immersed in the liquid nitrogen.
• Put all the cryolocks in one visitube.
• Put the visitube into canister.

Vitrification Warming (thawing)

Quinn's vitrification warming medium contains 3 media, "Thawing Solution", "Dilution Solution" and "Washing Solution"

Thawing Solution (TS) contains Base media (Hepes buffered medium) + 1M Sucrose + 20% protein supplement.

Dilution Solution (DS) contains Base media (Hepes buffered medium) + 0.5M Sucrose + 20% protein supplement.

Dilution Solution (DS) contains Base media (Hepes buffered medium) + 20% protein supplement.

Preparation of dishes :

• Place a drop of 0.2 ml of Thawing solution (TS) in one dish as shown in the figure.
• Keep the dish for warming at 37 deg. C on a warming plate for about 15 min.
• Place 1 drop of 0.1 ml of Dilution Solution (DS) and 2 drops each of 0.2 ml of Washing solution in another dish as shown in the figure.
• Keep the dish at room temperature.

0.2 ml of Thawing Solution at 37 deg. C.

• Identify the cryolock (embryos we want to thaw) and put in the box containing Liquid Nitrogen.
• Remove the cap of cryolock, while it is immersed in Liquid nitrogen.
• Quickly submerge the cryolock tip into thawing solution under stereomicroscope view.
• The embryo will automatically dispel.
• Leave the dish at 37 deg. C for 1 minute.
• After 1 min, carefully collect the embryos from the thawing solution to Dilution Solution at room temperature making sure carrying forward minimal amount of thawing solution along with the embryos.
• After 3 min. pick up the embryos and transfer them to 1st washing solution. The embryos will recover back to its original size approx after 2 min.
• After 3 min. transfer the embryos to second washing solution.
• after 3 min transfer the embryos to culture dish.
• Incubate the embryos for 2 hours before transferring them to patient's uterus.

What mistakes can a beginner do while vitrifying, that can lead to a poor survival rate?

Preparation of vitrification dish

Mistake :

Placing only 1 drop of vitrification medium, trying to cut down on the amount of medium consumed.

Reasons :

• Using only 1 drop of Vitrification medium doesn't allow us to get rid of Equilibration medium before actually vitrifying the embryo/egg.
• The Equilibration Medium is not very effective when it comes to cryoprotecting , as compared to Vitrification Medium.

Solution :

• Use 4 drops of vitrification medium.
• Serially transfer embryos/eggs through the series of these 4 drops of vitrification medium. This allows us to get rid of the equilibration medium effectively.
• This will definitely ensure better survival rate.

Equilibration

Mistake :

• Keeping the embryos in Equilibration medium for a short period of period before transferring them to Vitrificaition medium.
• This happens when we rely on fixed time period for each medium.

Reasons :

• Once the embryo/egg is placed in Equilibration medium, it will shrink spontaneously and slowly recover back to its original size. The recovery period varies for different embryos.
• When we rely on fixed time period for e.g. 5 min in Equilibration medium, it may not be enough for the embryo/egg to recover to its original size.
• If we transfer the embryo/egg to vitrification medium before it recovers back to its original size, it doesn't vitrify properly, which leads to extremely poor survival rate post thaw.

Solution :

• Rather than relying on a fixed time, it's best to individualise the time, based on the shrinkage and recovery of each individual embryo/egg , to ensure better vitrification.
• The embryo/egg can take approximately 7 to 8 min. to recover back to its original size.
• In some embryos it can be less or more than 7 to 8 min.

Vitrification :

Mistake :

• Making a large drop of vitrification medium along with embryo/egg while loading it on cryolock.

Reasons :

• A large volume of vitrification medium on cryolock will not allow proper vitrification of the embryo/egg.
• If the embryo is not vitrified properly, it leads to poor survival.

Solution :

• Make a flat small droplet of vitrification medium.
• Even if the drop is large, get rid of the excess medium by aspirating it gently.
• An embryo placed in a minimal amount of vitrification medium on cryolock vitrifies perfectly.

Many IVF doctors in India travel extensively. They have many clinics all over the city ( and in some cases, all over the country). Naive patients get impressed by such doctors - wow - he is so busy and so much in demand that he needs to travel all over the place. In fact, some doctors even go to Dubai and Africa ! While this seems very impressive, in reality this means that the quality of care they provide to their patients leaves a lot to be desired. Let me explain.

The personal goal for these doctors seems to be to maximise the number of patients they treat, so they can maximise their throughput and their revenue. While it's very good for an IVF clinic to be busy, it's also important that the quality of care provided to their patients not be compromised in the quest for quantity !

How do these doctors manage to do IVF in so many locations ? They usually do so by batching groups of patients together, so they treat lots of patients within 1-2 days. After they finish one batch of patients at one site , they then move on to the next site ! While it's medically quite feasible to program IVF cycles, the truth is that ensuring that all patients grow eggs at exactly the same time so that they will all mature on the same day for egg retrieval is simply not possible. The doctor is forced to cut corners for some patients because he's trying to force a square peg into a round hole - and this is especially harmful for older patients ( with poor ovarian reserve); challenging patients ( who have failed many IVF cycles in the past) ; or patients with PCOD ( who may end up hyperstimulating), because it's not possible to individualise their superovulation protocol. Such a rigid travel schedule means the doctor cannot afford to be flexible or cater properly to the "outliers".

So what happens to these patients ? They fail to get pregnant - and unfortunately they are not sophisticated enough to realise that the failure is not because there is a problem with them, but rather because they are not good candidates for "programmed" IVF ! They need a more focussed clinic which can spend the time and energy needed to optimise their chances. However, doctors who are focussed on the bottom line are really not very interested in treating these challenging patients. They would rather treat lots of young patients and improve their turnover, rather than take pride in getting the difficult patients pregnant !

The other major problem with this kind of itinerant IVF is that it's not possible to develop a relationship with your IVF doctor. In fact, you may not even see the IVF specialist for more than a few minutes, because this "big-shot, big-name" doctor may just breeze in, do the embryo transfer, and then breeze out ! If you have questions, you will never know whom to get the answers from, because all the juniors are uncoordinated and cannot make decisions, because they do not have the expertise or the experience to do so !

Now there's nothing wrong with this McDonald approach - after all, it seems to work well for these doctors ! This is because in each batch, the young patients who are good ovarian responders , will get pregnant ( though the success rate will not be as high as it could be in a full-service IVF clinic). But this approach is obviously not right for all patients ! If you've failed an IVF cycle; or have poor ovarian reserve; or need TESE or have PCOD, then you should rather select a clinic which provides all its services all the time in one location !

This is why all the world's leading IVF clinic all operate from a single location. They understand that IVF is a very labour intensive process, which needs close monitoring and extensive tweaking to optimise outcomes for their patients. Good doctors would rather focus on each individual patient, and are confident that if they are good, patients will come to them, rather that their having to go to other cities to find more patients to treat !

If you need a second IVF cycle, and if your IVF doctor is not present in the clinic all the time, you should be very worried about the quality of the care you will get ! If you want to maximise your chances of getting pregnant, send me your medical details by filling in the free second opinion form at www.drmalpani.com/malpaniform.htm and I'll be happy to help !

Recently I witnessed an infertile couple who had over three heavy files bulging along with medical tests and reviews. They may have visited more than ten infertility experts who tried every single possible examination under the sun to attempt to discover what their issue was. The lady had been examined for antisperm antibodies, NK cells , hostile cervical mucus, tuberculosis, chlamydia, sperm DNA fragmentation… you name it, and she had had the examination done. Every single gynaecologist had repeated test after test, however every result had been normal. They were at their wit’s end. “Why can’t I get pregnant , doctor , if things are all normal ?” was her plaintive cry.

I was surprised about how little she had learned after going to numerous doctors. Rather than understanding from her experience, she was still asking the wrong question ! She expected me to order extra medical tests, or utilize some modern device in order to probe her insides and pinpoint the elusive issue.

I needed to advise her that the quality of the response we get is determined by the standard of the actual question we ask. Nobody cares about issues - folks just worry about solutions. Rather than continue asking “What’s the issue ? Why aren’t I getting pregnant ?”, she need to preferably be asking , “What can I do in order to get pregnant ?”

Ask a better question , therefore you get a far better answer ! It was an entirely new perspective for her, and I had to walk her through this viewpoint. As I told her, it barely mattered what are the reasons for her inability to conceive had been - the only thing which mattered was how all of us can fix the problem.

Everybody knows: GIGO - garbage in, garbage out. In case you continue asking “What’s the problem ?” you will end up throwing away a lot of time, money and energy in trying to discover it. Sadly, several of these diagnostic labels are incorrect, unreliable or simply completely wrong. For instance, a couple of years ago, numerous patients had been identified as having an “inadequate luteal phase” - and perhaps treated for this issue ! We now realize, with hindsight, that there's absolutely no such situation, and we did a significant disservice to our patients by coining this term and doing such a (mis)diagnosis.

This breaks my heart when I notice how much time and money infertile folks have lost in seeking a diagnosis. Is it the endo , doc ? Could I be possessing antisperm antibodies ? These are all the completely wrong concerns, but we see this all the time.

Hence, after a unsuccessful IVF cycle , the first question patients ask is - Why didn’t I get pregnant ? (Interestingly, patients who conceive after IVF (In Vitro Fertilization) never ask - Why did I get pregnant? The truth is that we can't answer either of these questions - and quite often it’s only a matter of chance!

The correct question patients should be asking after a unsuccessful IVF cycle is - What can I do next? Usually, the best solution would be to repeat the IVF (In Vitro Fertilization) cycle. It’s a lot like purchasing a lottery ticket - there’s absolutely no compulsion to do so, but if you don’t buy, you don’t win! And the oftener you purchase, the greater your chances of winning!

That's why, for many infertile patients, it’s better to look ahead instead of to look backwards. The best question is - What’s the next thing? Its best if you make a list of all feasible treatment alternatives - ranging from: stopping treatment and remaining child-free; adopting; repeating the IVF cycle; doing the IVF cycle at a new medical center; or utilizing third party reproduction. After that you can eliminate the alternatives that you will not consider, so that you are then left with the alternatives you are ready to explore. After this you ought to concentrate on each one of these, until you discover one which is best for you.

Usually, if you are using this technique, you might find that the absence of alternatives makes decision making easy , and you can prepare your next step and navigate your path through the infertility maze with much less effort !