Name:​ Saloni Khanna

Course: ​B.Sc. Medical Physiology

Name of Institute: ​Amity Institute of Physiology and Allied Sciences

Objective: ​Diagnosing, assessing and treating female infertility for IVF

Background:
1. Female reproductive system
2. Oocyte Morphology
3. Buffers and Laboratory equipment knowledge

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EMBRYOLOGY Lab Procedures

Step 1: Evaluation Of Female Infertility 

Multiple tests have been proposed for evaluation of female infertility. Some of 
these tests are supported by good evidence, while others are not. This topic will 
provide an evidence-based approach to the evaluation of female infertility. 
 
I. History — The most important points in the history are: 
 
● Duration of infertility and results of previous evaluation and therapy. 
● Menstrual history (cycle length and characteristics), which helps in 
determining ovulatory status.  
● Medical, surgical, and gynecological history.  
● Obstetrical history to assess for events potentially associated with 
subsequent infertility or adverse outcome in a future pregnancy. 
● Sexual history, including sexual dysfunction and frequency of coitus. 
Infrequent or ineffective coitus can be an explanation for infertility. 
● Family history, including family members with infertility, birth defects, 
genetic mutations, or mental retardation. Women with fragile X 
premutation may develop premature ovarian failure. 
● Perso​nal and lifestyle history including age, occupation, exercise, stress, 
dieting/changes in weight, smoking, and alcohol use, all of which can affect 
fertility.  
 
II. Physical examination
 
● The physical examination should assess for signs of potential causes of 
infertility. The patient's body mass index (BMI) should be calculated and fat 
distribution noted, as extremes of BMI are associated with reduced fertility 
and abdominal obesity is associated with insulin resistance. 
 ● Abnormalities of the thyroid gland, galactorrhea, or signs of androgen 
excess. 
● Vaginal/cervical structural abnormalities or discharge. Uterine enlargement, 
irregularity, or lack of mobility.  
 
III. Diagnostic tests 
 
● Assessment of Ovulatory Function 
○ Assessment of ovulatory function is a key component of the 
evaluation of the female partner since ovulatory dysfunction is a 
common cause of infertility.  
○ The treatment of women with ovulatory dysfunction is aimed at 
improving or inducing ovulatory function; a variety of treatment 
strategies are available. 
○ Women who have regular menses approximately every 28 days with 
molimina symptoms prior to menses (breast tenderness, bloating, 
fatigue, etc.) are most likely ovulatory.  
○ In women who do not describe their cycles as such, laboratory 
assessment of ovulation should be performed. 
 
● Assessment of Ovarian Reserve 
○ Diminished ovarian reserve can refer to diminished oocyte quality, 
oocyte quantity, or reproductive potential.  
○ The identification of diminished ovarian reserve is an increasingly 
important component of the initial infertility evaluation as patients 
are presenting for diagnostic evaluation later in their reproductive 
lifespan.  
○ We test ovarian reserve with an AMH level and a day 3 
follicle-stimulating hormone (FSH) and estradiol levels. 
 
● Assessment of Fallopian tube patency 
● Assessment of the uterine cavity 
○ Modalities to assess the uterine cavity include saline infusion 
sonohysterography, three-dimensional sonography, 
hysterosalpingography (HSG), and hysteroscopy.  
 

Step 2: Controlled Ovarian Hyperstimulation 

 
Controlled ovarian hyperstimulation​ is a technique used in assisted reproduction 
involving the use of fertility medications to induce ovulation by multiple ovarian 
follicles.  
Response predictors determine the protocol for ovulation suppression as well as 
dosage of medication used for hyperstimulation. Response prediction based on 
ovarian reserve confers substantially higher live birth rates, lower total costs and more 
safety. 
The definition of "​poor ovarian response"​ is the retrieval of less than 4 oocytes following 
a standard hyperstimulation protocol, that is, following maximal stimulation. 
On the other hand, the term "​hyper response​" refers to the retrieval of more than 15 or 
20 oocytes following a standard hyperstimulation protocol. 
 

Step 3: Oocyte Retrieval and Pick-up  

Oocyte retrieval​ (​OCR​), is a technique used in in vitro fertilization (IVF) in order to 
remove oocytes from the ovary of a woman, enabling fertilization outside the body. 
 
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For extra Information: 
 
● Under ultrasound guidance, the operator inserts a needle through the vaginal 
wall and into an ovarian follicle, taking care not to injure organs located 
between the vaginal wall and the ovary.  
● The other end of the needle is attached to a suction device.  
Once the follicle is entered, suction is gently applied to aspirate follicular fluid 
and with it, hopefully, cellular material including the oocyte.  
● The follicular fluid is delivered to an embryologist in the IVF laboratory to 
identify and quantify the ova.  
● Next, other follicles are aspirated.  
● Once the ovarian follicles have been aspirated on one ovary, the needle is 
withdrawn, and the procedure repeated on the other ovary.  
● It is not unusual to remove 20 oocytes as women are generally hyperstimulated 
in advance of this procedure.  
● After completion, the needle is withdrawn, and hemostasis is achieved.  
● The procedure usually lasts 20–60 minutes. 
 
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Oocyte Pick-up By the Embryologist 
 
Materials Required: 
- 100mm dish 
- Centerwell 
- MHM (Multiple Handling media; HEPES + MOPS) 
- Mineral Oil  
- Bicarbonate culture media 
- 60mm wash dish 
- 1ml Flexipet pipette 
- Mechanical Pipettes 
- Steriosome Microscope 
 
Procedure: 
 
 - Prepare a wash dish by adding MHM to a centerwell, both on the outside 
and inside.  
- Use a 100mm dish for oocyte screening. The follicular fluid is dispelled 
from its vial into the 100mm dish and the fluid is screened under the 
steriosome microscope to look for OCCs (Oocyte cumulus complex). 
- The follicular fluid is pale yellow in colour. It becomes red due to blood 
contamination.  
- From the naked eye, OCCs are perceived as a white collagenous dot.  
- These are retrieved using the flexipet pipette and transferred into the wash 
dish (outer area).  
- The total count of the OCCs retrieved is called out after every follicular fluid 
vial screening.  
- All the OCCs are then further washed in the wash dish (inner area) and using 
the flexipet pipette, the corona radiata is separated out from the cumulus 
complex.  
- A new dish is prepared using another centerwell containing Bicarbonate 
CSC ( Continuous single culture) overlay with mineral oil (in the inner area 
only). 
- The Oocytes are then transferred to this dish containing CSC and stored in 
dry incubators, idly for 2-3 hours before IVF or ICSI procedure.  

Step 4: IVF or ICSI 

➔ For IVF aka. Conventional Insemination 

 
◆ Conventional Insemination involves a specific amount of prepared 
sperm mixed with each oocyte in a culture dish. 
◆ Procedure: 
● Step 1: Preparing culture dish 
○ Use a 60mm dish. 
○ Place three small CSC drops and one large CSC drop in 
the dish. 
○ Overlay with mineral oil.  
○ Transfer the oocytes from the previously dry incubated 
center well dish into the first small drop of culture dish.  
○ Keep washing the oocytes from the first small drop till 
the third using flexi pets to completely be rid of the 
MHM. 
○ Place the oocytes in the large drop.  
● Step 2: Performing IVF 
○ Prepared semen is taken out of the incubator.  
○ A small amount is taken and mixed around the oocytes in 
the culture dish.  
○ Dish is then kept in the dry incubators to be assessed the 
next day.  
● Step 3: Denudation of eggs 
 ○ After Day-1 of the newly formed embryos post 
conventional insemination, the eggs are denuded.  
○ The embryos are mechanically denuded using a 170 or 
175 micrometer syringe.  
○ The culture dish is then refreshed and the embryos are 
stored for further day assessments.  
 
➔ For ICSI 
 
◆ ICSI or Intra-cytoplasmic Sperm Injection involves the direct 
injection of a sperm into the oocytes obtained through IVF 
procedures.  
◆ ICSI should only be used for specific indications. With an apparently 
normal sperm sample, ICSI should not be used in a first cycle even if 
only few oocytes are obtained.  
◆ When there is reason to suspect poor fertilization, ICSI can be used in 
combination with conventional IVF in a split cycle.  
◆ Absolute indications for ICSI include two previous fertilization 
failures with conventional IVF, use of epididymal or testicular sperm 
samples, or when only acrosomeless or immotile spermatozoa are 
available. 
◆ Procedure: 
 
● Step 1: Denudation of Oocytes 
○ Denude the oocytes in the wash dish itself.  
○ Take out the dish from the incubator.  
○ Add Hyaluronidase solution to the media and denude the 
oocytes with the help of a 135 micrometer flaxipit.  
○ This is known as Enzymatic denudation.  
○ Why is enzymatic denudation needed? The acrosome of 
sperm contain hyaluronidase enzyme which is released 
when a sperm fertilizes the egg. The enzyme causes 
breakdown of the cumulus cells (made up of hyaluronic 
acid) around the oocytes and causes easy penetration for 
the sperm through the zona pellucida. 
○ Do not denude the oocytes in hyaluronidase solution for 
more than 2 minutes. Long exposure to Hyaluronidase 
can lead to toxicity and affect the development of 
oocytes.  
○ Transfer the denuded oocytes to HEPES media and 
perform Mechanical denudation.  
○ Why did you transfer to HEPES? Bicarbonate media 
needs CO2 to maintain pH, hence they cannot be used 
outside of incubators. However, HEPES media does not 
change the pH. Oocytes are highly susceptible to every 
minute change in pH in the environment. 
○ Transfer denuded oocytes to the ICSI dish.  
 
 ● Step 2: Preparation of ICSI dish 
○ ICSI dish is always prepared one day prior to OPU.  
○ Using a 60mm dish, place PVP solution in it in a 
lengthwise form in the middle of the dish.  
○ Place one drop of PVP on one end of the line of PVP.  
○ PVP (Polyvinylpyrrolidone) is used to immobilize the 
sperms aka. Causes reduction in sperm motility for easy 
pick-up and morphology screening when performing 
ICSI. 
○ Place multiple drops of HEPES in a line on both sides of 
the PVP solution.  
○ Overlay with mineral oil to avoid evaporation of media 
and temperature fluctuation.  
 
 

 
 
 
 
 
● Step 3: Performing ICSI  
○ The oocytes from the wash dish are transferred to the 
HEPES drops in the ICSI dish after their denudation.  
○ From the prepared semen, a drop from the top layer is 
loaded to the PVP solution for sperm screening.  
○ Before performing ICSI, neutralise the machine. Bring all 
the controls to zero.  
○ Attach the holding pipette and injecting pipette to their 
respective holders.  
○ Bringing them into position, use the injecting pipette to 
aspirate out a morphologically normal sperm.  
○ Use the holding pipette to keep the oocyte in place and 
inject the single sperm into the oocytes with the help of 
the injecting pipette. 
  
● Step 4: Preparing culture dish 
 ○ Use a 60mm dish. 
○ Place three small CSC drops and one large CSC drop in 
the dish. 
○ Overlay with mineral oil. 
○ Transfer the injected oocytes from the ICSI dish into the 
first small drop of culture dish.  
○ Keep washing the oocytes from the first small drop till 
the third using flexi pets to completely be rid of the 
MHM. 
○ Place the oocytes in the large drop and incubate in dry 
incubators for embryo day assessments.  

Step 5: Embryo Culture and Day Assessments 

Fertilization is assessed 16 - 18 hours after insemination or ICSI. The fertilized eggs 
are called zygotes and are cultured in a specially formulated culture medium that 
supports their growth.  
 
➔ DAY 1- Fertilization Check (PN formation; 2 Cell Stage) 
➔ DAY 2- Cleavage Check (Cell Division; 4 cell stage) 
➔ DAY 3- 6 to 8 Cell Stage 
➔ DAY 4- Morula (Compaction) 
➔ DAY 5- Blastocyst stage