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All infertile couples know that the sperm plays a vitally important role in fertility. However, there's still a lot of confusion ! If we need only one sperm to fertilise an egg during ICSI, does the sperm count and motility really matter ? And if it does, how and why ?

The fact that the WHO has kept on changing the definition of what a normal sperm count just testifies to the fact that experts are as confused as patients are. This is especially true when we consider 3 contentious areas. While it's true that the sperm provides 50% of the child's DNA, can it be responsible for:
failed fertilisation after ICSI ?
poor qualty embryos ?
miscarriages ?

In order to drill down further into when the sperm can be responsible for reproductive problems, researchers have developed sophisticated tests to analyse whether the sperm are "normal " or not. In the past, the only tests we had available were the sperm count, motility and morphology. These are admittedly crude tests, and the hope was that newer tests which could directly check the functional status of the sperm's DNA would give us more useful information. Logically, since the man's DNA contributes half of the offspring’s genetic material , it is reasonable to assume that abnormal DNA in the form of fragmented DNA ( when excessive strand breaks are present ) may lead to derangements in the reproductive process.

Let's look at some of these tests.

The tests used for the assessment of sperm DNA integrity can be distinguished into direct and indirect. Direct assays try to detect the actual DNA breaks, while indirect assays quantify the susceptibility of sperm DNA to break after an external insult, such as acid treatment. The most commonly used direct assays are; Terminal Deoxynucleotidyl Transferase-mediated Nick End Labeling (TUNEL), Single Cell Gel Electrophoresis (COMET) and In-Situ Nick Translation (NT) assay. The most common indirect assays are; Flow flow cytometric acridine orange assay, Acridine Orange test (AO), DNA Break Detection-Fluorescence In Situ Hybridization (DBD-FISH) and Sperm Chromatin Dispertion test (SCD).

The very fact that it's such a long list is a testimony to the fact that we really do not understand what the results signify in real life. For example, breaks affecting genes in “silent” areas of the genome are unlikely to have any clinical importance, but no assay can evaluate this factor yet.

The truth is that for the present, there is no differentiation between clinically significant and insignificant fragmentation. While it's true that many studies using a variety of assays have shown statistically significant differences in sperm DNA fragmentation between fertile and infertile men, remember that these refer only to the mean or median . In reality, there is extensive overlap between the values found in fertile and infertile men.

Because these tests are so new, they've not been standardised. Clear reference values have still not been established, just adding to the confusion. Just like conventional semen parameters have been proven to be disappointing at predicting the outcome of IVF, sperm DNA fragmentation has been equally disappointing in predicting pregnancy rates after standard IVF and ICSI.

If you have poor quality embryos and your test shows you have increased sperm DNA fragmentation , it's very tempting to conclude that it's the sperm DNA fragmentation which is responsible for the fragmented embryos. However, this has never been proven ; and please remember this is not necessarily cause and effect. Men with higher sperm DNA fragmentation have had completely healthy and normal babies in their bedroom !

DNA fragmentation is a new parameter for the evaluation of male factor infertility . However, just because it is new does not automatically mean it is better ! In fact, at present it just seems to add to the confusion, rather than clarify it !

Unfortunately, most patients doing IVF treatment are quite clueless about the most important end-result of the IVF cycle - the embryos ! This atlas will help you understand what your eggs and embryos should look like, so you have a better understanding of what happens in the IVF lab !

DAY 0 ( the day of egg collection)

MATURE OOCYTE CUMULUS COMPLEXES

THESE ARE MATURE OOCYTE CUMULUS COMPLEXES, IDENTIFIED IN THE FOLLICULAR FLUID ASPIRATED DURING EGG COLLECTION.

POST MATURE OCCYTE CUMULUS COMPLEX

STRIPPED EGGS
MATURE EGGS :


THIS IS A MATURE EGG (SURROUNDING CUMULUS CELLS HAVE BEEN STRIPPED OFF)

POLAR BODY AT 12 O CLOCK POSITION INDICATES THAT THIS IS A MATURE EGG AT METAPHASE II
STRIPPING IS MANDATORY FOR PERFORMING ICSI ON THEM.
CUMULUS NEED NOT BE STRIPPED FOR CONVENTIONAL IVF.

THIS EGG IS NOT A MATURE EGG. BUT A TINY POLAR BODY WHICH IS PROTRUDING OUT AT 12 O CLOCK POSITION, INDICATES THAT IT WILL SOON BECOME MATURE.

IMMATURE EGGS

Metaphase I egg

We can perform ICSI on Metaphase I eggs. The fertilization rate for Metaphase I eggs is 50 %.


Egg with Germinal Vescicle

THIS EGG HAS A CLEAR GERMINAL VESCICLE IN IT, WHICH INDICATES IT IS AN IMMATURE EGG. WE CANNOT DO ICSI ON THIS EGG. WE CAN USE IVM ( IN VITRO MATURATION) FOR THESE EGGS. AFTER 24 HOURS, SOME OF THEM MAY MATURE AND BECOME M2 ( METAPHASE II) AT WHICH TIME WE CAN DO ICSI FOR THEM

ABNORMAL EGGS


EGG WITH ABNORMAL ZONA EGG WITH ABNORMAL CYTOPLASM.

EGG WITH 2 POLAR BODIES.

POOR QUALITY EGGS

EGGS WITH DARK CENTRAL GRANULATION IN CYTOPLASM.

EGG WITH VACUOLATION IN CYTOPLASM

UNFORTUNATELY, THERE IS NO WAY WE CAN PREDICT THE QUALITY OF EGGS BEFORE WE STRIP THEM.
THIS IS WHY STRIPPING THE EGGS FOR ICSI PROVIDES US WITH VALUABLE INFORMATION ABOUT EGG QUALITY !

DAY 1
DAY 1 EMBRYOS ( ALSO CALLED ZYGOTES) ARE SCORED CONSIDERING

• THE PRESCENCE OF CYTOPLASMIC HALO
• NUCLEAR SIZE AND ALIGNMENT, NUCLEOLI NUMBER AND DISTRIBUTION.

• PRESCENCE OF CYTOPLASMIC HALO

PICTURE A – HALO POSITIVE ZYGOTE PICTURE B – HALO NEGATIVE ZYGOTE

2) NUCLEOLI NUMBER AND DISTRIBUTION

ZYGOTES ARE SCORED ACCORDING TO THE Z-SCORING SYSTEM.

• PICTURE A IS Z1 ZYGOTE WITH EQUAL NUMBER OF NUCLEOLI, ALIGNED AT THE PRONUCLEAR JUNCTION.
• PICTURE B IS A Z2 ZYGOTE WITH EQUAL NUMBER OF NUCLEOLI, SCATTERED IN THE TWO NUCLEI.
• PICTURE C IS A Z3 ZYGOTE WITH UNEQUAL NUMBER (DIFFERENCE OF MORE THAN 2 NUCLEUS) AND / OR SIZES OF NUCLEOLI.
• PICTURE D IS A Z4 ZYGOTE WITH SEPERATED PRONUCLEI AND UNEQUAL NUMBER OF NUCLEOLI SCATTERED IN THE TWO NUCLEI.

NORMAL ZYGOTE


Z1 EMBRYO

POOR QUALITY ZYGOTE

Z3 EMBRYO

ABNORMAL ZYGOTES

ZYGOTE WITH 3 PRONUCLEI

ZYGOTE WITH 2 PRONUCLEI WITHOUT NUCLEOLI

ZYGOTE WITH 1 PRONUCLEUS

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

My patient was at her wit's end and was sobbing her heart out. I have done 5 IVF cycles all over the world, Dr Malpani - and they've all failed. What do I do next ?

This is always a complex question - and there's no easy answer. You need to be analytical and logical, so we know what to do next. The trick is not to waste time looking for problems, but rather to focus on solutions which will allow us to bypass the problems !

We need to ask ourselves - what have we learned from these failures ? What can we do differently the next time ? What can we change to increase the chances of success ?


If you do need to change something, remember that there are only 5 things we can change in any IVF treatment

1. The IVF treatment protocol
2. The clinic
3. The sperm
4. The eggs
5. The uterus

Let's think through this process logically , so we can prepare a plan of action for the next cycle.
This is a very useful framework, which helps you to analyse the failure; learn from it; and then move on !

First, I need to emphasise that sometimes there's really no need to change anything at all. Not every IVF cycle is going to result in a baby, even it it's text book perfect. Sometimes, all you need is to be patient - and a little bit of luck !

But what if 3 cycles have failed ? Then what ? Let's look at what we can change, step by step.

1. The IVF Treatment protocol. While the IVF treatment protocol is pretty standard, every woman's body is different, and every patient responds differently to IVF medication ! Each cycle teaches us how your body responds to superovulation - and a lot of the art of IVF and the skill of a good IVF doctor consists of superovulating you with just the right dose of medications, to get you to produce an optimal number of high quality eggs. While most young patients with normal ovarian reserve grow well in response to a standard protocol, patients with poor ovarian reserve and those with PCOD need a lot of extra attention and closer monitoring. We may need to use additional alternative medicine supplements such as DHEA for these patients; while others may need higher doses of HMG. Some of this is trial and error - and you must keep careful records and learn from each cycle , until we can customise the perfect protocol for you !

2. The clinic. If you find your doctor is very rigid and is not willing to make any changes inspite of repeated failures, then it's a good idea to get a second opinion, to confirm you are on the right track. Getting a fresh brain to look at the problem can be very helpful and can offer new insights ! While the basics of IVF are the same all over the world, there is a great deal of difference in the quality of the services IVF clinics provide - and it's a good idea to keep an open mind and explore alternatives if you feel your present clinic is stuck in a rut; or refuses to do anything different. This is especially true if your doctor batches patients; does IVF on a part-time basis; refuses to share medical information with you; or does not provide you with photos of your embryos ! A word of warning here - doctor shopping is not usually a smart thing to do - and if you have a competent doctor with whom you have a good relationship, then it's best to preserve this, and ask him to offer additional options to you, rather than to establish a new relationship with a new doctor. However, when you do go from one IVF clinic to another, you are quite likely to get VIP care, because IVF doctors are quite competitive, and it's often a matter of pride for them to get a patient pregnant when the earlier IVF doctor has failed to do so !

3. The sperm. If you have failed fertilisation in an IVF cycle, then you need to do ICSI in your next cycle. This will ensure fertilisation and is a simple and effective solution ! Using ICSI allows us to work with practically any man's sperm, no matter how low the count or poor the motility and morphology . I do not believe DNA fragmentation affects success rates if ICSI is being used if the embryologist is competent ; and I believe that if we have poor quality embryos after ICSI even in men with severe oligoasthenospermia, the problem is usually because of the egg or the lab, and not because of the sperm. ( I know that many clinics will blame the poor sperm quality when they get poor quality embryos after ICSI. This is rubbish - it's usually just a poor quality lab which is to blame ! However, men with a low sperm count have low self-esteem - and they are quite willing to accept this flawed conclusion - and use donor sperm for the next cycle ! ) . It's possible that in a very very few men who have persistently poor embryos after ICSI, it could be the poor quality sperm which are responsible. The only way to prove this is by split crossover testing ( fertilising the wife's eggs with donor sperm; and fertilising donor eggs with the husband's sperm, and then comparing the embryo quality ), but as you can imagine, this can be very hard to do in real life !

4. The eggs. When you remember that the egg is a thousand times bigger than the sperm; and that the energy for cell division during embryo cleavage comes from the mitochondria in the egg's cytoplasm, it's hardly surprising that the vast majority of time embryos fail to implant is because of "egg problems". The problem is that it's very hard to make this diagnosis, as eggs are just spherical blobs, and we simply do not have the technology to assess egg quality or egg function. However, if there is a problem with embryo quality in a good IVF lab, 9 times out of 10 the problem is because of the eggs. While it's sometimes possible to correct this problem in the next cycle by using various measures to optimise egg quality ( such as DHEA or a more aggressive superovulation procotol), most of the time the most effective solution is to use donor eggs. This option has a very high success rate - but it can be quite hard for some women to accept - especially those who are young and have a good ovarian response, with a normal AMH level and a normal antral follicle count. It can be hard for them to come to terms with the possibility that their eggs may be flawed - and the idea of using foreign genetic material can be unpalatable to many women ( and their partners).

5. The uterus. Surrogacy has become very popular recently because of the disproportionate amount of media attention it attracts, and many women feel that the best medical solution for them after many failed IVF cycles is surrogacy. After all, the fact that the embryos are not implanting means the uterus must be " defective" , so doesn't it make sense to use a fertile woman's uterus as an incubator for 9 months ? However, the truth is that surrogacy is an expensive and complex treatment option, which is best reserved for women without a uterus. Research shows that the reason for failed implantation is much more likely to be genetically abnormal embryos ( because of poor quality eggs), rather than a uterine problem. However, because it's very profitable to offer surrogacy , many IVF doctors are keen to push their patients towards this option . For a woman who has failed 5 IVF cycles and is fed up and frustrated, surrogacy does seem to be a very attractive option - no hassles with the dreaded 2 week wait - someone else can do all the hard work ! However, the success rate for surrogacy for these women is very poor, because it's not their uterus which was the problem ; which means that using another woman's normal uterus is not going to help at all !

I remind patients who have failed IVF to remember the Serenity Prayer -
God grant me the serenity to accept the things I cannot change;
the courage to change the things I can;
and the wisdom to know the difference. Outcomes are always uncertain, but if you take well-informed decisions on a logical basis, you will have peace of mind you did your best !

Most IVF clinics are very good at making embryos in vitro, which means that for most patients, each IVF cycle results in the formation of embryos which they can admire.
( Tip: If you are doing IVF treatment, insist that the clinic give you photos of your embryos. This is the best documentation that you have received good quality medical care. You have a legal right to your medical records - every hospital has to provide them by law ! Please make a request for this in writing ! Poor quality documentation without photos of your embryos suggests you have received poor quality medical care !)

However, sometimes there is complete failure of fertilisation, as a result of which no embryos are formed. This can be a rude shock to the patient, and causes major heartburn !

Why do the sperm fail to fertiise the eggs in vitro in these patients ?

There are 3 possible sources of this problem: the eggs; the sperm ; and the Lab. Let's look at these one by one.

The commonest reason could be an egg problem. Poor quality, "tired" eggs will often fail to fertilise. This is true in older women ; and in those who have entered the oopause. Even though the structure of the eggs looks fine ( which means that under the microscope, their morphology is normal), they simply do not have the competence to fertilise and form embryos. Remember that the energy for fertilisation and cleavage comes from the mitochondria ( the power houses of the cells); and in older women, sometimes the mitochondria are just not able to provide the energy which allows the formation of the cell spindle.

We also see this in younger women who have PCOD. Doctors are often so scared of OHSS, that they end up mistiming the HCG injection, as a result of which they get few eggs and poor quality embryos.

What about sperm problems ? There are men who have very good sperm counts and motility, but whose sperm are not functionally competent; and cannot fertilise the eggs in vitro. Unfortunately, there is no way of predicting this in advance - and total failed fertilisation provides important diagnostic information that the " unexplained infertility " in this case was because of an undiagnosed male factor. The good news is that it's easy to treat this problem ( but only in the next treatment cycle) by doing ICSI. While it is possible to attempt rescue ICSI with these unfertilised eggs in the same treatment cycle, but the success rates with this are poor.

The third group of reasons is a poor quality lab. Thus, fungal contamination or bacterial infection in the IVF culture medium can kill all the eggs. A poorly equipped lab ( for example, one with malfunctioning incubators) will also have poor fertilisation rates. This can occur because of various other technical problems as well - such as electricity failure ; or the use of expired IVF culture medium. Unfortunately, it's very hard for a patient to find out if the failed fertilisation is because of a lab problem, because it's hard to get this information from the doctor.

Usually, if it's a lab problem, it should affect all the embryos of all the patients on that day. An important question to ask is - What happened to the other patient's embryos ? ( though this information may not be readily available ! ) The good news is that if the failed fertilisation is because of poor lab conditions, it's easy to fix this problem by finding a better IVF clinic for your next cycle.

A close examination of the eggs and sperm can give the doctor valuable information, which can help him to diagnose the problem and find the right solution . Are the eggs in the dish all immature ? Are the sperm in the IVF petri dish still motile after 24 hours ? ( If the sperm are all immotile, this suggests a problem with the IVF lab conditions). You should ask for photos of the unfertilised eggs. If they are dark, this suggests they have degenerated, which could be because of an infection.

What are the possible solutions ? If IVF was done, it's possible to do a rescue ICSI on the same day , though the success rates with this are not always good. The important thing is to use this hard-earned information to help in making decisions about the next treatment cycle !

You options include:
1. change the treatment. Instead of doing IVF, ICSI may be a better option, if the failed fertilisation was because of dysfunctional sperm
2. change the eggs. You might want to use donor eggs, if you have few poor quality eggs
3. change the clinic. This is often your best choice, as a new IVF expert will be able to look at the problem dispassionately and hopefully do a better job !