First published online on 25 August 2023

Given the threat posed by cancer treatments , such as chemotherapy and pelvic radiotherapy , to the ovarian oocyte pool , oocyte cryopreservation is now a routine fertility preservation option for women of reproductive age diagnosed with cancer . Aside from medical oocyte cryopreservation as an adjunct to cancer treatment , increasing numbers of women are freezing oocytes to combat the effects of natural ageing ( so-called elective or non-medical oocyte cryopreservation ). Illustrating this , there has been a staggering 311 % increase in oocyte cryopreservation cycles seen in Australia and New Zealand between 2010 and 2015 . 1

This article will review the role and processes involved in oocyte cryopreservation .

The importance of quality

Oocytes contribute virtually all the building

blocks required by embryos . This means that oocyte quality is rate limiting for pregnancy success . 2 From a pregnancy perspective , therefore , a decline in oocyte quality underpins the inverse relationship between fertility potential and female age . It is important to appreciate that , at any given time , the ovary contains a mixture of good- and poor-quality oocytes — albeit this is an oversimplification , as quality is a continuum . The term ‘ oocyte quality ’ therefore refers to the proportion of good-quality oocytes . 2

The best-defined downstream consequence of poor oocyte quality is chromosomal abnormality ( aneuploidy ) in embryos . The rate of embryonic aneuploidy is inversely related to the proportion of good-quality oocytes . Thus , for 30-yearold women , embryonic aneuploidy rates are around 20 %. This rises steeply to around 60 % for 40-year-olds , reflecting the marked decline in oocyte quality during this decade of life . 3 Given that ovulated oocytes are only viable during a narrow two-day window per menstrual cycle , this further illustrates why female age exerts such a marked impact on time to conception .

The marked deterioration in oocyte quality occurs surprisingly early , during the 30s , when women are still physiologically young , and decades before other organ systems start showing any appreciable decline in many women . Consequently , the erosion of fertility potential occurs silently and , unfortunately , for many , only comes to light after the deterioration is well advanced .

This would not be a major problem if there were effective interventions for reversing poor oocyte quality . Despite feverish research in this space , however , there are currently no such interventions . Research , by the author and others , using animal models has uncovered agents that are very promising , such as those that restore levels of nicotinamide adenine dinucleotide ( NAD +) — an essential metabolic cofactor for a number of critical cellular processes . 4 However , these await validation in human trials . In the absence of being able to reliably rejuvenate oocytes , the alternative is to cryopreserve oocytes when their quality is good .

Technological advances

Sperm banking via freezing has been in use

for several decades . In contrast , it has been considerably more challenging to develop effective oocyte cryopreservation techniques . The American Society for Reproductive Medicine only lifted its experimental label relatively in 2013 . 5

Increasing numbers of women are freezing oocytes to combat the effects of natural ageing .

Additionally , oocytes pose unique challenges to cryopreservation : one being their very large volume and water content . Oocytes are the largest cells in the body , with a volume more than 100 times greater than a typical somatic cell . The other major challenge is that mature oocytes possess a fragile structure , known as a spindle , which delicately balances chromosomes at its equatorial region and is responsible for executing the second round of meiotic chromosome separation following fertilisation . Intriguingly , therefore , and again unlike sperm , mature oocytes have not yet completed the process of chromosome halving ; this only occurs after sperm entry .

Initial attempts to freeze oocytes using slow-freezing methodology were thwarted by the production of ice crystals , which damaged the spindle and compromised post-thaw survival and development . More recently , newer vitrification technology has been developed based on ultra-rapid cooling and solidification of the oocyte into a glasslike state . 5 Significantly , vitrification is not plagued by ice crystal formation , markedly improving post-thaw performance and the pregnancy potential of vitrified oocytes .

Ovarian reserve

Because oocytes reside within the ovary ,

which is itself located deep in the pelvis ,

obtaining oocytes for freezing requires an IVF-type treatment cycle involving FSHbased ovarian stimulation . The subpopulation of follicles at the antral stage are most responsive to the FSH preparations used routinely in IVF . The number of oocytes that can be retrieved and frozen is thus limited by the ovarian antral follicle count . A good ovarian reserve is represented by a total antral follicle count of 10-20 . Polycystic ovarian morphology is an example of very high ovarian reserve and refers to an antral follicle count of at least 20 per ovary .

Anti-Müllerian hormone ( AMH ) is a glycoprotein hormone produced by ovarian granulosa cells . Since the bulk of circulating AMH is produced by early antral follicles , AMH levels also provide a guide as to the potential yield of oocytes following ovarian stimulation . 6 Since more eggs equate with greater cumulative probability of success , women with polycystic ovarian morphology have higher per-cycle prospects than women with low ovarian reserve . It is important to note that , while AMH is a measure of oocyte

numbers , it is not a measure of quality . AMH does not therefore provide a readout of natural fertility potential .

The process of egg collection

Elective oocyte cryopreservation cycles

usually commence FSH administration via subcutaneous injection from day 2-3 of the menstrual cycle and typically continue for a total of around 10-12 days . In urgent cases , such as pre-chemotherapy , FSH treatment can be commenced immediately at any stage of the cycle using so-called random-start protocols . Furthermore , two rounds of follicle stimulation can be conducted sequentially within one menstrual cycle ( termed DuoStim ) to maximise egg yield when oocyte-toxic treatment is planned and time is limited .

The supraphysiological FSH stimulation used in IVF overrides single follicle selection and dominance that occurs naturally , thereby inducing multiple antral follicles to grow . FSH dose is determined based on age , BMI and , importantly , ovarian reserve ( predicted from antral follicle count and / or AMH ). During FSH treatment , premature ovulation is prevented by suppressing the endogenous LH surge using either gonadotrophin-releasing hormone ( GnRH ) antagonists or , less commonly , GnRH agonists .

Ovarian follicular response is monitored

Advances in technology mean the chances of conception using frozen eggs have improved substantially , but oocyte quality — largely predicted by maternal age — remains a significant rate-limiting step to success .

There are many promising areas of research that may help determine how best to target oocyte cryopreservation , as well as measures that may lessen some of the impacts of ageing on the oocyte .

using transvaginal ultrasound scanning and hormonal blood measurements . Once the majority of antral follicles have attained the appropriate size (~ 17 mm ) at which they express adequate LH receptors , a bolus ( or trigger ) of recombinant human chorionic gonadotrophin is given to induce oocyte maturation . For high-responder patients , such as those with PCOS , a milder GnRH agonist trigger ( which induces the release of endogenous LH from the anterior pituitary ) is used to limit the risk of ovarian hyperstimulation syndrome .

Oocyte retrieval is then typically performed around 35-37 hours post-trigger under either sedation or general anaesthetic . Oocyte retrieval involves the passage of a 17-G or 18-G single- or double-lumen needle under transvaginal ultrasound guidance through each vaginal fornix and into each ovary . The main risks associated with the collection procedure are bleeding , infection and damage to viscera , such as the bowel . In practice , significant complications very rarely occur ( fewer than 1 % of procedures ). Within a couple of hours of retrieval , retrieved oocytes are denuded of their surrounding follicular cells to determine maturity ( roughly 75-90 % of retrieved oocytes are expected to be mature ), and the mature eggs are vitrified .

How many eggs are enough ?

A critically important question is what constitutes

a good number of oocytes to be frozen — that is , what number of oocytes would be required to provide a reasonable chance of one or more live births , depending on family aspirations . Answering this question requires consideration of several variables .

The crucial variables are oocyte survival post-thaw , fertilisation rates of thawed oocytes , the efficiency of subsequent embryo development to the blastocyst stage ( the embryo stage that develops 5-6 days