I had the valuable opportunity to participate in a week-long Observership at Prega IVF, Surat, from 28th July to 2nd August 2025. This was my first exposure to the practical application of Clinical Embryology, and it was a profoundly insightful experience. I had the privilege of shadowing the andrologist, embryologist, and gynaecologist at Prega IVF, for which I am extremely grateful.

This report details the various procedures I observed, from initial semen analysis and sample preparation, egg pick-up, artificial fertilization via ICSI, and embryo culture, to the final transfer of the developed embryo. The experience has solidified my interest, and I am eager to one day learn and perform these procedures hands-on.

Andrology Lab

Contains a small LAF (Andro mini), microscope and small incubator (set to a constant 37°C temperature,) both placed inside the LAF, and a centrifuge.

I learnt the importance of correctly labelling everything from semen collection jars, to centrifuge tubes to even droppers. Precision in labeling is key to avoid unfortunate mixups.

As a rule, the wife's name is written first, followed by the husband's name and then their surname beside it. Both the patients’ ages are to be mentioned under their respective names. This is followed by the Abstinence Days (AD) - the number of days the husband has abstained from ejaculation before delivering the sample. The recommended AD is 3 to 5 days. The last piece of information is the time of semen collection, also crucial.

Liquification and Initial testing

After the collection, the semen is allowed to sit for about 30-60 mins, for Liquification. If the sample takes longer than that to liquify, then it is considered to be “viscous”, otherwise it is normal.

Using a 3ml dropper, the volume of the sample is measured. This is followed by allowing it to fall drop by drop to assess the viscosity of the sample. If the sample falls as drops, the sample has liquified normally. However if it falls as a straight line, the sample is considered viscous.

Sperm Count

A drop of the sample is taken into the Sperm Meter (used for sperm enumeration). This does not require a diluting fluid and it is always calibrated to contain 1 micron of sample. It has 100 microscopic boxes, out of which sperms seen in 4-5 rows are counted. The total is divided by the number of rows counted to get a count.

The sperms are of different types based on their motility:

Linear » fast
Moderate » slower, but motile
Mild » move in one place
Non-motile » dead sperms

The count for each type is taken and the percentage of each is calculated by dividing the number of that type by the total and multiplying into 100.

The count is multiplied into the volume.

The linear and moderate sperms are considered to be of a higher quality than the rest, and these are usually selected for further processing.

Sperm Washing - Swim Up

The sample is then made to undergo “Sperm Washing”, which isolates the more motile sperm from the slower/non-motile sperms. This process includes the sample, mixed with sperm wash media to be centrifuged at a standard 1200 RPM for 7 mins (which concentrates the sperm to the pellet), followed by layering 0.5 mL of media on top of the pellet, after carefully discarding the supernatant, making sure it does not disturb the pellet and forms 2 distinct layers. The better the sperm count, the denser the pellet will be. The test tube is tilted at an angle while layering the media. This is then set aside for 5-10 mins, resulting in all the motile sperm “swimming up” into the media. This method is therefore aptly named the “swim up” method. This ensures only the best quality sperms are selected, and it is faster than the Density Gradient method, making it the better choice in labs that have a tight schedule.

Abnormalities

Some samples may have impurities and abnormal sperms, here are some that I got to observe at Prega IVF:

Extra lipid: yellowish thick impurities, made to settle in a test tube and then the rest of the usable sample is pipetted out. These affect the centrifugation and reduce the number of sperm in the pellet.
Pus cells: round structures along with the sperm, possibly a result of some kind of infection in the patient’s system
Abnormal morphology (under ICSI machine): large head, irregular tail/midpiece, etc. These irregularities are caused by tight clothes, improper diet/stress, excess alcohol consumption. They are indicative of genetic defects and are not chosen for ICSI
Azoospermia: no/very less sperms seen under initial observation. Centrifugation of the sample concentrates the live sperms to the pellet, which is mixed with a small amount of supernatant and smeared on a slide to be observed under the higher magnification of the ICSI machine. The number of sperms are checked and a report is generated.

Sperm thawing

Sperm is stored long term in liquid nitrogen at 196°C. It is kept in vials as a mixture of 0.5mL media and 0.5mL semen. Before freezing it is acclimatised to the extreme temperatures by first hanging it on top of the container containing the liquid nitrogen, but not submerging. This prevents heat shock.

In order to thaw the semen sample, the vial is first held under running water, after taking it out of the cold storage. After a few mins it is placed in an incubator at 37°C for 15 mins. This is followed by sperm washing of the thawed sample.

IVF Lab

Contains a LAF, incubators (CO2 and tri-gas), and a magnificent ICSI machine. I was required to wear proper hospital scrubs and wash my hands thoroughly before entering the OT and the lab. Strict protocols for sterility and systematic labelling were followed.

Egg Pickup

This is a beautifully collaborative process between the doctor and nurses in the OT and the embryologists in the IVF Lab. The doctor, with sonography guidance, looks at the follicles of the patient and tries to extract fluid containing the eggs using a suction machine. The fluid is taken into tubes containing media, provided by the lab beforehand. The patient is placed under anaesthesia and an i-gel is inserted to aid respiration.

The OT is connected to the lab via a tiny window, through which the tubes are passed to the embryologists. The first embryologist receives the tube and pours it into petri dishes. The second embryologist looks at the dishes under the microscope, carefully searching for the egg cells by prodding at the mucus using a micropipette. This to me looked like watching a skilled crane searching for its food with its sharp eyes and long beak.

Once he finds an egg, he conveys it to the first embryologist, who calls out the total eggs received to the doctor in the OT. Each egg is precious, and it is crucial for everyone involved to stay vigilant and avoid mistakes to the best of their ability. There are literally lakhs at stake as each IVF procedure costs the patient around 1.2 to 1.5 Lakh Rs.

After each petri dish is thoroughly checked, the fluid is discarded into a jar. Next up is proper denudation of the oocyte using 135 and 175 denudation pipettes. This process removes the cumulus surrounding the egg cells and is done in the presence of HYASE and HEPES media. The andrologist equated it to removing the egg whites from a chicken egg to isolate the yolk. The oocytes are then stored in pre-ICSI media, after determining their growth phase.

The husband's semen is collected at around the same time as the egg pickup.

IUI

Intrauterine insemination is usually done for patients with a lower sperm count. In such cases the semen sample, upon centrifugation yeilds a smaller pellet. This pellet is more concentrated with sperm cells. It is treated with the swim up method and the best, most motile sperms are selected. These are then loded onto a catheter and inserted into the uterus, through the cervix, guided by sonography. The cervix is expanded using metal clamps and is cleaned with saline solution.

ICSI

Intra Cytoplasmic Sperm Injection is the process of artificially introducing a sperm inside the oocyte. It is done in ICSI plates - which have different compartments for the cells, using the ICSI machine (Integra 3). I particularly loved observing this process, and time seemed to fly by so quickly every time I watched it on the screen. It felt as if I was watching a movie, about how the most eager of the sperms - our hero, meets the heroine - the ovum, a love story between the largest and the smallest cell of the human body, directed masterfully by the embryologist.

The microscopic needles of the ICSI machine became extensions of his hands, and he used those to perform the procedure. Using the thinner needle, he would first select and immobilise sperms and “suck” them in. He would then line them up and slowly load his needle with the sperms like ammunition in a gun. Next, he would move to the compartment containing the oocytes and hold the cell in place using the thicker needle. A sperm is brought close to the end of the thin needle and the needle is injected into the cytoplasm of the egg. The needle “sucks” some cytoplasm in, before aspirating it along with the sperm. It is critical that only a single sperm enters the egg, as more than one sperm would cause aneuploidy and a failure to fertilise.

The fertilized cells are then placed in CO2 or tri-gas incubator to facilitate growth. Embryo culture media is usually kept at 6% CO₂ to maintain pH (~7.2 to 7.4). If the CO₂ concentration drops, pH increases (media turns pink) and if it increases, pH decreases (media turns more yellow).

Embryos that fertilize successfully exhibit two pronuclei (2PN) about 16–18 hours after insemination (Day 1). By Day 2, they typically reach the 2–4 cell stage. On Day 3, they develop into 6–8 cell embryos, and by Day 5 or 6, they form a blastocyst, which is then ready to be transferred into the uterus for implantation.

Assisted Hatching

Day 3 embryos are made to hatch by artificially creating a perforation in the zona pellucida using a laser. The plate is placed on the stage and the embryologist drags a line, or clicks the position where the laser is supposed to be fired.

By the time the embryo reaches the blastocyst stage, it emerges out of this perforation, and is ready for implantation. This process improves implantation rates by mimicking natural hatching.

Embryo Thawing

Embryos are stored in “straws/needles” at 196°C in liquid nitrogen, in freezing media. The embryos are thawed by placing them in 3 different media present in the ready made thawing kit:

Thawing Solution (TS): 1 min
Dilution Solution (DS): 3 mins
Washing Solution (WS): 5-6 mins

These media have different concentrations of cryoprotectants (sucrose, etc.) The embryos are incubated in each media for a specific amount of time and great care is taken not to exceed the limit. A stopwatch is used to keep track of the thawing time.

Embryo Transfer

When the embryos have finally developed into blastocysts on Day 5 or 6, it is ready for implantation. The patient is made to lie in the OT, and the doctor inserts a catheter into the cleaned cervix, with sonographic guidance.

When the embryo straw is ready the embryologist brings it into the OT and carefully inserts it into the catheter. The target area is that between the mid-cavity and fundus, which lies at the upper part of the uterus. The embryologist calls out how many centimetres deep the catheter has entered, until the doctor tells him to stop. The embryos are displaced into the uterus through the catheter, which is then taken out and transported back to the lab for checking. The entire process takes about 15 mins to complete.

It is possible (however, rare) for an embryo to stick to the straw and return when it is pulled out of the cervix, therefore the embryologist must see if the straw is “clear.”

The patient is then advised to rest for 15 days, after which her β-HCG levels are screened to confirm the implantation.

I cannot describe the joy I could see in the faces of the staff whenever they received a call confirming the “positive” pregnancy test results of the patient whose embryo they had so meticulously worked to develop and bring to life, but I can assure you that it was contagious. The experience I shared just made me realise how much I love this field, and the difference it makes in people's lives.

Conclusion

I entered Prega IVF as an eager student hoping to witness science in action, and I left with something far more profound - a glimpse into a world where even the tiniest cells carry the weight of someone’s entire hope.

I would like to thank the two embryologists at Prega, and Dr. Ravindra Korat and the staff for giving me this opportunity and helping me throughout my time there.

This observership wasn’t just an academic experience. It was emotional, grounding, and at times, overwhelming in the best way. I watched life unfold under a lens, and I saw how much of it depends not only on expertise but on patience, ethics, and trust.

The field of Clinical Embryology is unlike any other - deeply technical, deeply human. And after this week, I feel more certain than ever that this is the field I want to grow in. I may still be learning, but this experience has planted a seed of clarity, and I intend to nurture it with everything I’ve got.

Inside the Lab: Prega IVF