Managing Egg-Spectations

by Samantha Wake

 

Introducing the Oocyte

In a fetus, there is a pool of around 7 million primordial follicles in the developing ovaries. At birth, this amount drops to 300 – 400,000 and at puberty the size of the pool plateaus around 200,000. At this stage in life, the primordial follicles will start to be recruited into a growing follicular pool that stay in a rested early Meiosis 1 stage of development.

Ovarian Reserve

The transition of primordial follicles into the growing follicular pool is regulated by AMH (anti-müllerian hormone). It is a growth factor secreted by granulosa cells in the developing follicles. AMH peaks at puberty and declines until menopause. It is a good indicator of ‘ovarian reserve’ since it reflects the size of the growing follicular pool. The AMH test is often ordered during infertility investigations and can be used to help diagnose someone’s infertility and/or predict how well individuals will respond to ovarian hyper-stimulation. The results are categorized into high, medium, low, and very low ovarian reserve.

The Menstrual Cycle

At the onset of puberty, cohorts of antral follicles are recruited from this growing follicular pool. During each menstrual cycle around 5-10 antral follicles are recruited in response to increasing FSH (follicle stimulating hormone) levels. Out of this cohort, one is selected to be the dominant follicle; the rest will undergo atresia (die). Around day 14 of the cycle, there is a surge of LH (luteinizing hormone). This causes the ovulatory (dominant) follicle to ovulate and expel one mature egg from the ovary. The remaining follicle structure (granulosa and theca cells) forms a corpus luteum that secretes progesterone to maintain a potential pregnancy by preparing the endometrium. Approximately 400 eggs will be ovulated during a reproductive lifetime.

Stimulated Cycles

Antral follicles are present in the ovary and will continue to grow when sufficiently stimulated with FSH. Therefore, by administering continual doses of gonadotropin medications (e.g GonalF/Puregon/Rekovelle) starting on day 3 of a cycle, the selection of one dominant follicle can be disrupted and multiple follicles can be stimulated to grow instead. This is known as controlled ovarian hyperstimulation.

Once follicles reach a size of 1.7mm or higher, a hCG (human chorionic gonadotropin) trigger can be administered. This medication is used since it shares the same receptors as LH and thus mimics the natural LH surge. The number of expected eggs to be retrieved can be estimated by counting how many follicles are measuring 1.5mm or higher on an ultrasound scan on the day of trigger. Estrogen levels measured on this day can also act as a predictor for egg number; the optimal estrogen:egg ratio is 734-1097 pmol/L per mature follicle.

Oocyte Maturation

Three stages of oocyte maturation are commonly seen in the IVF lab: MII (metaphase II), MI (metaphase I), and GV (germinal vesicle).

A GV is an oocyte where the nucleus is still visible. Through the process of germinal vesicle break down (GVBD), the oocyte becomes an MI stage with no visible nucleus. Maturation continues as the oocyte releases the 1st polar body (a small package of excess DNA) and becomes an MII stage oocyte, resting at the end of Meiosis I. This is the fully mature stage and the oocyte is now ready for fertilization with a sperm cell.

Transition occurs from GV (immature) to MI (partially mature) to MII (fully mature) in response to the mid-cycle surge of LH or the hCG trigger. Timing is important, if a retrieval happens too soon after the trigger shot, the eggs may not have enough time to make it to the MII stage!

Not all eggs retrieved will be mature; on average 85% will be at the appropriate MII stage, 4% will be MI, and 11% will be GVs. This does vary person-to-person depending on their own physiology and the response to the medications.

ICSI

Prior to the ICSI (intra-cytoplasmic sperm injection) procedure, oocytes are denuded or “stripped” which removes the support cells (cumulus) that surrounds them and allows the embryologists to assess the maturation of the eggs. Following that, mature oocytes are injected directly with a single sperm cell. Preferably MIIs are used as they are fully mature and have a higher fertilization rate, but MI may also be inseminated if the number of MIIs retrieved is low. GVs are not injected. Injection occurs approximately 3-4 hours after retrieval to allow the oocytes to “rest” following the stressful collection process and stripping procedure.

IVF

For standard IVF, the oocytes are not stripped prior to insemination. Instead, they are put in a culture dish with a specified amount of prepared sperm and allowed to inseminate overnight. This means that the maturation status of the eggs remain unknown. The cumulus cells are left intact as they assist in fertilization since they attract swimming sperm. The following morning, the possible fertilized eggs are stripped of the cumulus cells and the fertilization status is assessed under the microscope. Oocytes that did not successfully fertilize can be assessed for maturity.

Signs of Fertilization

Fertilization assessments take place 18 hours after the time of insemination (IVF or ICSI). Fertilization is successful when the presence of two pro-nuclei (2PN) are seen and two polar bodies are present. The 2nd polar body is released after the sperm penetrates the egg and the oocyte completes the Meiosis 2 stage, removing the final bit of unnecessary DNA before the two genomes fuse. PNs are formed when the egg and sperm DNA combine to create the correct chromosomal compliment (46 chromosomes). On occasion, the fertilized egg (zygote) may display one pronuclei or three pronuclei; any zygotes showing 1 or 3PNs are abnormal and will be discarded as they have a higher risk of genetic abnormalities.

Average Outcomes

Following IVF or ICSI, approximately 70% and 80% of MII oocytes will successfully fertilize, respectively. These success rates will decrease if there is any reduced quality of the sperm or eggs (e.g. increased age, poor response to medications, abnormal sperm motility/morphology etc.). Around 40% of these fertilized eggs (zygotes) will go on to develop to the blastocyst stage. Of these blastocysts 25–35% will be of good quality (A/B grade) and will be suitable for transfer or freezing for future use.

Conclusion

The purpose of this blog is to give patients insight into the science pertaining to the development of their oocytes and embryos. If you have any questions related to this blog topic, please get in touch with lab@karmaobgyn.com.

KARMA Blog – How does the Ontario Fertility Program Work?

By Sonja Swanson

 In December 2015, the Ontario Ministry of Health began the Ontario Fertility Program (OFP). This covers some of the costs associated with fertility treatments to help Ontarians grow their families. There are some eligibility criteria and some aspects of care that are not covered.

 

IUI Funding

The patient must have a valid OHIP card and there is no limit on the number of IUI cycles that can be done per patient in their lifetime. There is not an age limit for someone wanting to do IUI.

The Funding covers all monitoring (ultrasounds, blood work, doctor/nurse visits) during the IUI cycle and one IUI insemination procedure. However, there is an additional cost for processing the sperm sample and any medication costs are to be paid by the patient.

The IUI funding covers use of partner or donor sperm for IUI. If using frozen donor or partner sperm, the purchase cost or cryopreservation and annual storage fees are not included in the funding program.

 

IVF Funding

The Funding is billed to the person receiving the embryo transfer with the goal of establishing a pregnancy (the Primary Patient); this could be the intended parent (IP) or a surrogate. The Primary Patient must be less than 43 years old at the start of the cycle and have a valid OHIP card. There is one (1) funded cycle per lifetime as a Primary Patient. If the Primary Patient is a surrogate, at least one IP must also have a valid OHIP card and have not received a funded cycle previously.

The Funding covers all monitoring (ultrasounds, blood work, doctor/nurse visits) during the IVF cycle and standard embryology services to inseminate, culture, and cryopreserve the embryos. Fees that are not part of the funding include: annual storage, medication, PGT biopsy (KARMA) and PGT testing (CooperGenomics), and purchase of donor gametes (sperm or eggs).

Any funded cycles must follow the Embryo Transfer Policy, which means in most cases, patients are only allowed to have one embryo transferred at a time.

Milestone 1 involves the creation of embryos (and a possible fresh embryo transfer) in the following ways:

  • Primary Patient undergoes oocyte retrieval

  • Secondary patient (egg donor) undergoes oocyte retrieval

  • Primary Patient uses a previously frozen batch of their own oocytes

  • Primary Patient uses a single batch of frozen donor oocytes

Milestone 2 covers all the subsequent transfer of cryopreserved embryos:

  • Any/all frozen embryos from the Milestone 1 procedure

  • Embryos previously created during a self-pay IVF cycle

  • A single batch of donated frozen embryos

Once the funded IVF cycle is started, there are multiple end-points that indicates completion of the cycle:

  • Two attempts at cycle monitoring were conducted and no retrieval occurred

  • An oocyte retrieval was performed and no viable oocytes were found

  • A retrieval occurred, viable oocytes were found, but no viable embryos developed

  • All oocytes/embryos were thawed and no viable candidates for transfer developed (if using a previous batch of oocytes/embryos)

  • All viable fresh and frozen embryos generated by the funded cycle have been used for transfer (a pregnancy may or may not occur)

  • Primary Patient concludes care in writing (stating they no longer want to continue)

This means that if your IVF cycle “fails” and no oocytes are found, no embryos are viable, or no pregnancy occurs following transfers, that is still considered the funded cycle. At KARMA we do our very best to create embryos that are of good quality and are able to be transferred, but unfortunately, that cannot be guaranteed.

 

If you have any questions or concerns whether your cycle is eligible to be funded, please speak to the nursing team or the clinic manager (sonja.swanson@karmaobgyn.com).

The OFP has helped many families at KARMA and throughout Ontario to grow their families and is a great opportunity for those who may struggle to afford fertility care on their own!

 

The KARMA Network – How to Understand Your Results

As part of the Investigative Cycle, you will have access to the KARMA Network, our online system of viewing your daily results. This is used as an aid to help you better understand your ultrasound and blood test monitoring. If you have any questions, please don’t hesitate to contact the nursing team for further clarification at 519-570-0090 x 2. The KARMA Network is complimentary for the first cycle and then $30 for 6 months of access.

When you first log in you can select Schedule to see all your upcoming appointments, Flowsheet to see your daily results, Messaging to contact the clinic, or Discussion Forum to chat with other patients.

 

Ultrasound

When you click on Flowsheet, the default view is the Ultrasound report.

In the Endometrium column, the ultrasound staff will record the thickness and brightness of the endometrial lining. The lining increases in thickness as you get closer to ovulation to prepare the lining for a potential pregnancy. The lining sheds every month with your menstruation when you are not pregnant. When you are pregnant, this is where the egg implants and the lining will remain complete.

The endometrium is hyper-echoic early in the cycle and is thin and brighter then the surrounding tissue. As the cycle progresses, it will become more hypo-echoic (darker than surroundings) on outer edges of the lining and after ovulation, the uterus will be mostly iso-echoic, or the same darkness as the other tissues as the lining prepares to shed. When assessing the uterus, we can determine if the appearance is typical at each stage of the cycle.

Uterine contractions are also measured. Early in the cycle, the entire uterus can contract from the cervix to the fundus (top of the uterus). This type of movement is beneficial as it can help draw sperm upwards and into the fallopian tubes. We may also identify the endometrium moving back and forth or “to and fro” as it prepares for implantation. If a pregnancy does not occur the endometrium tends to contract from the fundus toward the cervix as it prepares to be sloughed off.

“Ut art avg PI” measures the resistance to flow of the uterine artery that feeds blood to the uterus. A measurement less than 3 is optimal for this arterial pressure. If it is greater than 3, it could indicate decreased blood flow to the uterus, which may cause the endometrial lining to be affected for implantation of a fertilized egg. The doctor or nursing team will discuss treatment for this if the value remains to be greater than 3 through your investigative cycle.

Right and Left Side Follicle columns show the number of follicles each ovary has. At the beginning of the cycle, there will be small (<0.5cm) follicles called Antral Follicles, that are not currently growing but have the potential to begin maturing. The Antral Follicle Count (AFC) can vary with each cycle and the significance of how many are seen on Cycle Day 3 can be discussed with the nurse or doctor. Usually, one follicle will grow and mature each cycle; this follicle is usually detectable by Cycle Day 9 but may be variable depending on your average cycle length. The follicle tends to grow between 0.1 and 0.2cm daily, but this is variable as well. How the follicle develops will be discussed with the doctor at your review appointment.

There is also a column for Right and Left Cysts, if you have a measurable cyst it will be listed here. Cysts are quite common and usually unremarkable. If there is any concern, this will be discussed at your review appointment with the doctor.

Free Fluid refers to fluid that is within the pelvic cavity; a small amount of fluid in the pelvis can be normal. It has been documented that when ovulation occurs, a larger amount of fluid can be seen because when the follicle ruptures, the released follicular fluid accumulates in the pelvis. This can be helpful when trying to determine if ovulation has occurred.

 

Blood Results

Oocytes (eggs) are too small for ultrasound to see. The blood work we draw helps us determine the presence and the growth of an egg inside the follicle(s). To see your blood results on the Flowsheet, select “Lab” in the Cycle Type drop-down menu and click update view.

LH – Luteinizing Hormone – The final trigger for the maturation of a follicle. The level will generally be steady throughout the cycle and then “surge,” or approximately double in value, one day and the egg will mature and ovulate 36 - 42 hours later.

E2 – Estradiol – This is a type of estrogen that is produced by the follicle, this level can indicate if a growing follicle has a maturing egg inside. As a follicle grows larger or more follicles develop, the E2 level should rise and peak at ovulation where it will then drop after the egg(s) are released. If a pregnancy occurs, E2 will rise again and stay elevated, if no pregnancy occurs, the level drops and menstruation will occur.

Prog – Progesterone – This hormone is responsible for maintaining the lining of the uterus for implantation of the embryo. Like E2, it will rise after ovulation and stay high if you are pregnant but will drop if you are not pregnant.

FSH – Follicle stimulating hormone – Measured on Cycle Day 3, this hormone is sent from the brain to the ovaries, telling them to start producing oocytes. A higher number means that your body has to work harder to get eggs to develop; a value of less than 10 is ideal. This level will be discussed at your review appointment with the doctor.

PRL – Prolactin – This is the hormone that stimulates milk production during breastfeeding. It has fertility-inhibiting properties and can prevent ovulation. It can be elevated due to other reasons such as stress or a physical abnormality of the pituitary gland (where it is produced). If your level is high, treatment would be discussed at your review appointment.

 

Medication

To see your medication on the Flowsheet, select “Medication” from the Cycle Type drop-down menu and click update view.

Verbally Ordered and Ordered is the record of what the doctor instructed you to take that day (either told to the nurse or written down in your chart). Dispensed is the medication that was given to you by the nurse to take home. Administered is the medication that the nurse gave to you directly while you were at the clinic (usually an injection).

Depending on your daily ultrasound and blood work results, the doctor may order more or less medication each day. Depending on the treatment type, they may also trigger ovulation and schedule a procedure.

If you ever have any questions about the type of medication you are given or how much medication to take, call the nursing team at 519-570-0090 x 2.