Antral Folliculogenesis

Question 1

What is the crucial aspect in the transition from preantral to antral follicles?

Answer 1

• The theca needs to have been formed!
• Studies in 1970s showed that when radio-labelled LH/hCG (both bind to LH receptor) injected into adult female rats, was localized specifically to the theca layer of small preantral, antral and pre-ovulatory follicles but not primordial follicles. The studies showed that the LH receptor is only found on the theca and that the theca was not present on primordial follicles (starts to develop at some point along the preantral follicle stages
• Theca starts to be formed at the secondary follicle stage (when there are two layers of granulosa cells)
• Formation and differentiation of theca extremely important for preantral to antral progression.
  1) GDF9 k/o mice (& GDF9 mutations in human & sheep) fail to develop theca layer and follicles arrest → oocyte-derived GDF9 regulating formation of theca cell layer.
  2) Neo-angiogenesis, hence follicle interaction with systemic endocrine factors
  3) Acquisition of steroidogenic function
• For all of these reasons, formation of the theca is crucial to allow the preantral follicles to continue progressing. It is even more important for antral follicle growth progression and actual formation of the antrum.

Question 2

What is the theca of a follicle?

Answer 2

• Theca of follicle is an envelope of connective tissue → differentiates into theca interna & externa containing vascular tissue, immune cells and matrix factors
• Theca is critical for maintaining structural integrity of follicle and delivering nutrient to avascular GC layer
• Theca starts to be formed at the secondary follicle stage (when there are two layers of granulosa cells)
• Formation and differentiation of theca extremely important for preantral to antral progression.
  1) GDF9 k/o mice (& GDF9 mutations in human & sheep) fail to develop theca layer and follicles arrest → oocyte-derived GDF9 regulating formation of theca cell layer.
  2) Neo-angiogenesis, hence follicle interaction with systemic endocrine factors
  3) Acquisition of steroidogenic function
• For all of these reasons, formation of the theca is crucial to allow the preantral follicles to continue progressing. It is even more important for antral follicle growth progression and actual formation of the antrum.

Question 3

Why is the formation and differentiation of theca extremely important for preantral to antral progression?

Answer 3

1) GDF9 k/o mice (& GDF9 mutations in human & sheep) fail to develop theca layer and follicles arrest → oocyte-derived GDF9 regulating formation of theca cell layer. It regulates formation of theca cell layer but not sure if it’s directly/indirectly via other signalling pathways. GDF9 secreted from the oocyte enters the granulosa cells and attracts the presumptive theca layer towards it (regulates formation of the theca cell layer). It is this constant interaction between signals coming from the oocyte directing these events; quite complex.
2) Neo-angiogenesis; The theca is crucial to the whole follicle because it has blood vessels. Formation of the new blood vessels allow the follicle to interact with systemic endocrine factors. Any nutrients and bloodborne factors have to be delivered, as the follicle grows into bigger stages, via the blood from the theca. Gonadotrophins secreted by the pituitary have to travel through the blood to reach the ovary. A blood supply is needed for access.

3) Acquisition of steroidogenic function
- The theca cells are also steroidogenic; crucial for the whole steroidogenic function of the follicle, because the substrate for aromatase activity (found in granulosa cells) is androgens coming from the theca. Oestrogen can’t be made without androgens (and aromatase present in the granulosa cells).

• For all of these reasons, formation of the theca is crucial to allow the preantral follicles to continue progressing. It is even more important for antral follicle growth progression and actual formation of the antrum. GDF9 secreted from the oocyte enters the granulosa cells and attracts the presumptive theca layer towards it (regulates formation of the theca cell layer). It is this constant interaction between signals coming from the oocyte directing these events; quite complex.

Question 4

What are the two different sources that theca cells are derived from in the embryonic gonad?

Answer 4

• Theca is comprised of the interna and externa.
• Theca cells derived from 2 different sources in the embryonic gonad:
  1) Mesenchymal cells migrating into the ovary from the mesonephros region become the steroidogenic cells
  2) Stromal cells surrounding the follicles and in the ovary (WT1+ stromal cells indigenous to the embryonic ovarian medullary region) variously become fibroblasts, perivascular smooth muscle cells and interstitial ovarian tissue, respectively, in the adult ovary.

Question 5

How does the antrum form in the preantral to antral follicle transition?

Answer 5

• One reason the theca is needed is to allow for antrum formation.
• When the follicle reaches a diameter of 200-400µm, surrounded by a vascularized theca, hence subject to circulating influences.
• Fluid-filled spaces appear between the granulosa cells which soon coalesce together to form a single, large, fluid-filled cavity or “antrum”. Contains fluid formed as exudate of plasma containing secretory products of oocyte & GC = Known as follicular fluid. KL and Cx37 essential for antrum formation in lab animals (at least in mice) – as k/o of these genes result in no antral follicles at all. Cx37 is responsible for gap junction formation.
• Follicular fluid formed by filtration of thecal blood, composition different from plasma as contains secretory products of oocyte and gc
• First, the fluid-filled spaces individually coalesce (join up) to form one single, large space and gradually begin to push the oocyte to one end. The granulosa cells are pushed to the edges and there is differentiation of the granulosa cells around the oocyte. They become the cumulus granulomas cells, and those that get pushed to the edge will be the mural GC. Can see that the size considerably changes as the fluid-filled space (fluid volume) increases.
• As it forms the follicular fluid within that antral space, it changes in nature and becomes different to plasma (start to get secretory products of both the oocyte and the granulosa cells also entering into that space).
• Exudate means fluid leaking from blood vessels
• As the fluid volume increases, the follicle continues to expand greatly in size.
• Antral follicles can range in size from 0.4-25mm diameter

Question 6

How does the the follicular fluid volume change when the number of granulosa cells increase during antral follicle development?

Answer 6

• As the number of granulosa cells (x 10^-6) start to increase (always multiplying), it increases the size of the antral follicle and causes an increase in follicular fluid volume during early antral stages. With the late antral stages, an exponential increase can be seen in fluid volume (ml). Corresponds with a big increase in the number of granulosa cells that are multiplying.

Question 7

Describe the structure of the ovarian follicle.

Answer 7

1) Theca externa - Concentrically arranged smooth muscle cells; innervated by autonomic nerves; lymphatic vessels; important during ovulation. Lymphatic vessels are important during ovulation as they allow inflammatory markers to be brought.
2) Theca interna = Steroid-producing cells; contain LH-r & Insulin-r; richly vascularized (good blood supply)
3) Granulosa cells = GC differentiate into 2 mature cell lineages: mural and cumulus cells.
- Mural Granulosa = found around the edge (adjacent to the basal lamina) and the cumulus cells are found around the oocyte. They are involved in endocrine feedback control via the production of oestrogen; express FSHr, P450arom (aromatase) and eventually LHr in the dominant follicle.
- Cumulus oophorus = Remain in contact with oocyte (surround it) & interact with oocyte via gap junctions; mitotically active; no LHr (even if it becomes selected to be the dominant follicle).
4) Basal lamina
5) Antrum filled with follicular fluid. Follicle antrum is a fluid-filled space.
6) Oocyte has a thick ZP.

Question 8

How do cumulus cells respond so rapidly after the LH surge if there are no LH receptors on cumulus granulosa cells?

Answer 8

• Cumulus cells do not contain LHr, however they respond very quickly after the LH surge to become expanded and have a sticky, mucified nature so they can be picked up after ovulation by the oviduct.
• These granulosa cells produce EGF-like (epidermal growth factor) ligands that bind LH and allow for secretion of hyaluronan and a complex of hyaluronan cross-linking proteins that cause expansion of the cumulus-oocyte complex (COC). Those mural granulosa cells around it and the cumulus cells secrete the hyaluronan to cause expansion of the COCs.

Question 9

What is the role of FSH in progression of antral follicles?

Answer 9

• Inter-cycle rise in FSH is crucial for recruitment of AF (right stage at the right time) into the menstrual cycle
• Leads to:
  1) Progression of antral follicles
  2) Selection of dominant follicle
  3) Fate of remaining AF

Question 10

What is the Two-cell, Two-Gonadotrophin concept?

Answer 10

• The HPG axis acts to control antral follicle growth at this stage (very extra-ovarian).
• In response to LH, theca expresses key steroidogenic enzymes to make androgens from cholesterol. Suggests that LH binds to LHr on theca cells, sets up downstream signalling to express key enzymes that are responsible for the conversion of cholesterol into progesterones and androgens. Those key steroidogenic enzymes are crucial to this process. The androgens that are formed diffuse across the basement membrane into the GC.
• Likewise granulosa cells respond to FSH by up-regulating aromatase (CYP19A1) and 17β-HSD to make oestrogens. Under the influence of FSH (binds to its receptor), the androgens that diffused in will stimulate aromatase expression and activity. Aromatase converts the androgens (androstenedione) into oestrone. 17-beta-HSD1 converts oestrone into oestradiol. Those oestrogens then diffuse into blood, circulate round and exert feedback effects of the hypothalamus and pituitary.

Question 11

There are thousands of primordial follicles, but only a few make it into the menstrual cycle.

What determines survival?

Answer 11

• Although activation of many thousands of primordial follicles can occur, not all of them will survive (99.999% of them will die through atresia).
• Of those that survive, they need FSH. Even from the antral follicles that do receive FSH and grow, only one will be selected to become the dominant follicle and go on to ovulate. In other words, the majority of follicles will die off but FSH is crucial to maintain the survival of those select few that will grow in the menstrual cycle and the ones that become the DF.
• Many follicles to one

Question 12

What is the role of FSH in recruited antral follicles?

Answer 12

1) FSH increases granulosa cell proliferation; increases mitotic potential of the GC to increase in numbers.
2) Increases aromatase for conversion of androgens from the theca into oestrogen
3) ↑ induce and maintain FSHr = Allows FSHr to continue binding to FSH. Activin comes from the GC and acts on them to also induce and maintain FSHr (autocrine effect). Has an effect at the level of the pituitary to stimulate FSH too.
4) FSH is also crucial to be able to induce expression of LH receptors and maintain them in the selected follicle that becomes the DF.
5) There are also other factors produced by the follicle (paracrine factors) which interact with FSH. These include AMH and inhibin (involved with pituitary feedback).

Question 13

How does autocrine control of FSHr expression work?

Answer 13

• In order to get effective action of FSH, FSHr expression is required. FSHr is a G protein-coupled receptor; there is a downstream signalling cascade which produces cyclic AMP to activate PKA. Signalling of Gs protein-coupled receptors (GsPCRs) is accomplished by stimulation of adenylyl cyclase, causing an increase of the intracellular cAMP concentration, activation of the intracellular cAMP effectors protein kinase A (PKA).
• The effects of PKA include differentiation of the various GC, increased follicular fluid, gap junctions, induction of LH receptors, increased FSH receptor (maintaining and inducing), increased aromatase.
• FSH activated the receptor in a feedforward loop (keeps stimulating the FSH receptor gene to keep producing FSH). Aided in some degree by activin.
• Interactions with AMH and other factors like androgens?

Question 14

How is a dominant follicle selected from the cohort of antral follicles? What is involved in selection of the DF?

Answer 14

• As the antral follicles grow, they increase oestradiol production which exerts negative feedback effect on hypothalamus and pituitary to result in a drop in FSH levels. As the FSH levels drop, the antral follicles will die off (except for the one that becomes the DF).
• Thought to be due to the threshold to FSH within the microenvironment of that follicle. In other words, the selected follicle is the one that needs the least FSH to be recruited (important consideration).
• Serial ultrasound scans (bringing women in and scanning them every day in the follicular phase of the menstrual cycle) have shown it is not always the largest follicle that becomes the DF.
• It is currently believed that the DF has the lowest threshold because it has the most FSHr. As FSH levels are decreasing, the increased FSHr are still able to bind to any FSH ligand available (even with low levels). It is also believed that the FSHr in the DF are able to exert actions of FSH more effectively as they are coupled more effectively to downstream signalling.
• These actions include increasing GC numbers and exponential growth in size. Selected DF will produce large amounts of oestrogen due to increased aromatase.
• The increased number of blood vessels in the DF means it is more open to circulating influences, e.g. insulin, insulin-like growth factors, EGFs and gonadotrophins.
  1) FSH receptors
• Increased numbers
• Coupled more effectively to down-stream signalling
  2) Growth and oestradiol production
• increased cell division (2-5 million GC in EFP and 50-100 million at ovulation)
• Size ~5.5-8.2mm in EFP and 18-20mm in LFP
• increased aromatase (200x more E2 than other follicles)
  3 and 4) Androgens & Oestrogens and Intra-follicular cAMP (All act within the microenvironment to allow the follicle to be selected at the DF)
  5) Increased area of theca vasculature

Question 15

How are LHr acquired in the DF?

Answer 15

• FSH acquisition of LHR in GC of selected DF
• FSH binds to its receptor, sets up all the downstream signalling to result in increased proliferation of GC, increased expression and activity of aromatase resulting in increase of oestradiol.
• Crucially, it also switches on the LH receptor gene in that selected follicle to make LH receptor. This is a very important and crucial aspect of this whole process.

Question 16

How does the dominant follicle survive the fall in FSH?

Answer 16

1) Increased sensitivity to FSH = increased FSH receptors. Even if FSH levels are falling, there are many more receptors to bind to the limited FSH available. Probably very effectively coupled to downstream signalling.
2) Increased numbers of granulosa cells. The increased number of granulosa cells also results in increased amount of oestradiol due to increased aromatase.
3) Acquisition of LH receptors = the LHR gene is switched on by FSH
4) possible involvement of insulin-like growth factors 1&2 (IGF-2 particularly important in humans)

Question 17

What is the role of IGF-2 produced by antral follicles?

Answer 17

• IGF-2 enhances FSH effects; stimulates androgen output and hence oestrogen production
• IGF activity itself suppressed by IGFBP (IGF-binding protein)
• IGF cleaved from IGFBP by - PAPP-A (pregnancy-associated plasma protein A)
• PAPP-A expression high in DF
• Thought that other AF in cohort may have higher levels of IGFBP hence preventing co-stimulatory effect of IGF & FSH
• IGFBP will bind to IGF and prevent it from acting. IGF must be cleaved to be released from IGFBP. This allows it to exert its effects to upregulate FSH activity.
• If the other antral follicles in the cohort have higher levels of IGFBP, free IGF will be prevented from having a co-stimulatory effect with FSH.

Question 18

How does the size of the dominant follicle compare to the late antral stage?

Answer 18

• Note that the dominant follicle at ovulation measures ~25mm in diameter and contains ~50 million granulosa cells and 7 ml of follicular fluid. Vast amount compared to late antral stage.
  (McNatty KP: Hormonal correlates of follicular development in the human ovary. Aust J Biol Sci 34:249, 1981)
• Focusing on the preovulatory stage, the dominant follicle has grown exponentially and is just ready for ovulation.
• Preovulatory follicle has greatly increased in size compared to late antral stage.

Question 19

What happens to the rest of the growing antral follicles in the ovary?

Answer 19

• Die off by atresia
• Atretic follicle has thin walls and an irregular shape
• Contrast to the polycystic ovary

Question 20

What evidence suggests the importance of LH in folliculogenesis?

Answer 20

1) Inactivating mutations of LH receptor = normal *EFP E2 (early follicular phase, oestradiol) levels, but they don’t ovulate (anovulatory) and they have multiple cysts because the follicles hang around. They also have morphologically normal antral follicles = a whole cohort of large follicles that do not ovulate and become cystic (Toledo, 1996). Can look at the phenotypic response to naturally occurring mutations in particular genes.
2) Hypogonadotrophic women = FSH treatment effective as long as some LH present. This is because substrate for oestrogen needs to be considered. E2 is significantly reduced but detectable. This is because some degree of LH is needed to result in stimulation of androgens from the theca to allow for formation of LH. It is also thought that maybe these women could access some androgens from the adrenal. However, if some LH is present, that should be enough to stimulate androgen secretion from the theca to form oestradiol levels.
3) LH k/o mice = Progress through the preantral stages, Antral stage growth blocked

Question 21

What is the importance of LH in antral follicles?

Answer 21

1) LH acts on the theca and also on the granulosa once the follicle has been selected to be the DF and FSH switches on LH receptors in the DF GC.
2) It increases theca function of the steroidogenic enzymes (CYP11a, CYP17) which are responsible for production of progesterones and androgens.
3) Increases growth and steroidogenesis in dominant follicle; increasing vasculature to allow it to grow big.
4) During ovulation, LH has a role in maturation of the oocyte, resumption of meiosis, withdrawal of gap junctions between GC and oocyte
5) Expansion of COC
6) Ovulation & luteinization = Ovulation and luteinization of both theca and GC; when it causes luteinsation of GC, this induces progesterone receptor formation in the GC. There is still production of inhibins, this time inhibin A in the luteal phase, which feeds back.

Question 22

How does LH signal in the theca interna?

Answer 22

• As seen with FSH, LH signalling in the theca interna is also via binding to its LHr (a GPCR). This results in production of cAMP and PKA. Then triggers downstream signalling events (formation of various steroids from cholesterol precursor). LH is crucially important for activation of these enzymes which catalyse these reactions.
• Insulin was included in this diagram because it binds to its receptor on theca and can augment the actions of LH. It plays an important role in doing this and is important to consider in women who have PCOS (often develop insulin resistance and hyperinsulinaemia which has a great effect on this LH-mediated steroidogenic activity).
• Both LH and FSH signal though cAMP.

Question 23

LH and FSH have same 2nd messenger.

How does the cell distinguish between them?

Answer 23

• LH and FSH have same 2nd messenger = cAMP
• FSH produces low cAMP levels, LH produces high cAMP levels
• Difference in density of FSHr & LHr (LHr>FSHr or LHr more effectively coupled to cAMP generation)
• LH and FSH are thought to produce different cAMP levels. This is linked to the difference in density between the LHr and the FSHr; LHr is thought to be more prevalent than the FSHr once induced or it could be that the LHr is more effectively coupled to generate high cAMP levels.
• cAMP is not only important as a signalling molecule; other effects are highlighted on slide = activates pKa for conversion of cholesterol and conversion of progesterone.
• provides energy for biosynthetic activity
• mediates effects of FSH and LH on protein production eg. aromatase, SCC, LHr, proteolytic enzymes

Question 24

What is needed to support follicle growth in folliculogenesis?

Answer 24

• FSH
• LH
• Androgens
• Ovarian angiogenesis

Question 25

Why is angiogenesis needed and what are the important angiogenic factors?

Answer 25

• Constant re-modelling to allow for growth of follicle (2-20mm) through the ovarian tissue, angiogenesis of CL when it forms, tissue repair of ovulated surface epithelium etc.
• Angiogenic factors stimulated primarily by androgens but also oestrogens = theca, gc, stroma all involved. Oestrogens also have angiogenic effects, so all of the cells are involved in this process.
• Basic fibroblast growth factor (bFGF) = endothelial cell mitogen, most potent angiogenic factor. bFGF is a mitogen for the endothelial cells which form the walls of the blood vessels (very potent)
• Vascular endothelial growth factor (VEGF) = endothelial cell mitogen, enhances vascular permeability. VEGF is also a potent mitogen for the endothelial cells (not as potent as bFGF)
• Ovarian lymphatic vessels recruited to theca and stroma layers around growing follicle, under control of VEGF-R3. Theca and stroma layers around the follicle have ovarian lymphatic vessels which are under the control of VEGF-R3.

Question 26

How do androgens act on the endothelial cells?

Answer 26

• liganded AR induces HIF-1 expression which is transcription factor for VEGF
  Lebbe & Woodruff (2013) Mol. Human Reprod. 19:828-837
• The current working model is that when androgen binds to its receptor (the androgen receptor is a nuclear receptor), the complex (the liganded AR) is translocated to the nucleus. Binds to androgen response elements on the HIF-1 gene and induces HIF-1 expression. The HIF-1 that is produced will bind to HIF response elements (HRE) on the VEGF gene and induce production of VEGF. VEGF binds to its receptor on endothelial cells and induces endothelial cell proliferation..

Question 27

How is AFC and ovarian reserve calculated to determine fertility?

Answer 27

• Has implications for fertility and conditions such as premature ovarian insufficiency.
• Some of the problems brought about by diminishing ovarian reserve can be counteracted by growing follicles in vitro (in the laboratory).
• When small antral follicles are at about 4mm, the GC are producing the maximum amount of AMH. As the follicles get bigger and progress through other stages, AMH levels decrease and become undetectable.
• This AMH secreted by the GC enters the bloodstream and can be measured as serum AMH.
• Hence, serum AMH measured will reflect the small, growing antral follicle pool.
• The AF are the ones that have been recruited to enter into the menstrual cycle and continue their growth; AFC in the early follicular phase correlates with numbers of growing follicles only
  Those small AF are also visible under ultrasound (have fluid-filled spaces); ultrasound can easily detect follicles larger than 2mm. Can correlate with AMH serum levels from a blood test. Use ultrasound to count number of 2-8mm follicles at start of menstrual cycle (early follicular phase) & correlate ≈ AMH serum levels (from a blood test)
• A woman having trouble conceiving can be scanned to carry out an AFC and AMH serum marker (one of the tests they can do). Once the number of AF have been correlated with serum AMH, they may conclude that fertility problems are due to a low number of growing follicles for her age (correlated more to a woman who is approaching perimenopause in her early 40’s, for example; gives an indication that there is a problem)
• Low numbers of antral follicles are a sign of ovarian ageing
• This change in follicles and AMH is observable earlier than a rise in FSH serum levels (traditionally used as a marker for perimenopause). FSH levels are controlled by feedback from oestrogen produced by the antral follicles. As women age reproductively and the number of follicles decrease (in the perimenopausal period), oestradiol levels decrease. Negative feedback is released and FSH levels rise. Therefore, FSH is traditionally used as a marker for women approaching menopause. With the development of good ultrasound techniques and the discovery of AMH, AFC and AMH are now used as an indication of a woman’s potential fertility.
• Intriguingly, right ovary has been shown to be larger and have higher AFC than left ovary → thought to be due to larger PF pool in right ovary formed in fetal life (Korsholm AS et al (2016) Gyne. Endocrinol). It is thought that there is a larger pool of primordial follicles formed in foetal life in the right ovary.
• Have to be quite cautious regarding AFC as a fertility marker. This is because women with PCOS have very high levels of AMH, which is a reflection of the high number of arrested follicles but also each follicle is producing more AMH than normal. In addition, it is always better to also look at several (not just AMH and AFC). Have to also take inhibin and FSH into account.
• To summarise, So before selection (early part of menstrual cycle) will have a cohort (~10 is normal) of follicles about 2-10mm diameter visible on ultrasound. So if scan ovary in early folliclular phase can count them – known as AFC and it has been shown that this correlated well with what is known as “functional” ovarian reserve. Preantral follicles and those <2mm diameter cannot be visualised by current u/s technology. All serum markers are the function of the GC of growing follicles and NONE are products of the primordial follicle. Hence only reflect number and size distribution of AFs.
  AMH production is highest in AF≤4mm & AF of 5-8mm contribute most to serum AMH levels.

Question 28

What markers are used in combination to determine functional ovarian reserve?

Answer 28

• AFC & serum markers FSH, AMH, E2 and Inhibin B used to determine “functional ovarian reserve”, but does not indicate true PF reserve ie what you are born with
• By looking at all of these markers together, the functional ovarian reserve can be determined, i.e. what is going on with the growing antral follicles. This does NOT indicate the true primordial follicle reserve (what you are born with) – impossible to know this in any individual woman, because we don’t have any biomarkers or secreted substances from the primordial follicle pool (which is in the resting state).
• It is known that women’s fertility does decline gradually and then appears to fall drastically once she passes the age of around 35. This is seen in numerous studies (can look at pregnancy rates and live birth rates?????)
• Other studies show that there is a reduction in the number of follicles retrieved per patient depending on their age group. This occurs across all stages and classes of follicles. The error bars are very large and overlap between younger women (meaning there are women in the <30 group who would have a follicle reserve equivalent to women in their 30’s and women can have a better follicle reserve than other women in the same age group etc.)! The women in 31-39 age group seem to be more consistent, but this is just one snapshot (more numbers would need to be considered to get a meaningful picture).

Question 29

What is Premature Ovarian Failure/Primary Ovarian Insufficiency (POI)?

Answer 29

• POI is clinical term for premature ovarian failure. POI has replaced the term premature menopause
• Affects 1% of women worldwide
• Defined as ovarian dysfunction <40yrs → oligomenorrhoea or amenorrhoea (irregular/arrested cycles). Main feature is infertility due to something increasing loss of follicles or born with less follicles than normal.
• Overarching feature is infertility resulting from accelerated depletion or reduced follicle reserve.
• Many reasons have been given for this; aetiology is poorly understood.
  1) Among them, it is known that pool of follicles in the ovary is affected in women who undergo chemo/radiotherapy (agents are toxic to the body and induce DNA damage) = Environmental genotoxins induce DNA damage eg chemo/radio-therapy for cancer treatment
  2) Mutations in genes, e.g. BRCA1 and BRCA2, that repair DNA double-stand breaks, resulting in diminished ovarian reserve. This is thought to do with setting up ovarian reserve in the foetus (DNA double-stranded breaks that occur during mitosis and meiosis in the embryo need to be repaired). If they can’t be repaired, there will be a large number of oocytes/follicles being eliminated as they are being formed.
  3) Altered hormonal signalling increases depletion from pool at all stages
  4) Chromosomal defects, e.g. Turner’s syndrome (XO) → associated with streak ovaries (have no/few follicles)
  5) Autoimmune diseases, including thyroiditis & Addison disease, are also linked with diminished ovarian reserve and POI.
• Treatment options are limited, short of freezing ovaries or freezing biopsies. Can get donation of eggs. Will come back to ovarian cryopreservation. Another aspect that could be considered is growing follicles in vitro.

Question 30

How are artificial ovaries made?

Answer 30

• 3D-printer makes ovary from microporous hydrogel scaffolds
• Follicles seeded through out to create mouse bio-prosthetic ovary
• Scaffold provide 3D support to follicles – allowing for vascularisation and ovulation
• Ovarian function restored when implanted in surgically sterilized mice
• Pups born through natural mating! Laronda MM et al (2017) Nature Communications
• Following on from this, 3D printers have been used to make artificial ovaries that act as scaffolding using hydrogel materials. Has to be tested in animals first; the scaffolds are made of hydrogels, follicles are seeded through, lie within the scaffold and start to grow. The scaffolding allows blood vessels to grow etc. They supported growth of follicles really well and the whole thing could be implanted into mice with removed ovaries. They were able to form embryos through natural mating and give birth to pups. The follicles which were seeded onto there (into the artificial ovary) and implanted back were then able to produce live pups.
• The field is always changing and expanding; hope to see much more progress as we go forward, but it is a challenge with human follicles and they do not behave in the same way as mice follicles (but we remain ever hopeful).

Question 31

How can follicles be grown in vitro to treat POI?

Answer 31

• Another aspect that could be considered is growing follicles in vitro. Attempts have invariably failed in the past and the follicles did not progress very far. It was then discovered that follicles survive much better if they have a three dimensional matrix around them to support them. This increases communication and contact between GC and oocyte.
• Development of 3D culture systems
• Hydrogel matrix to support follicle so that contact maintained between oocyte and gc
• Also to be permeable to media
• Modifiable in terms of rigidity
• 2 types:
  1) Collagen
  2) Alginate (product of seaweed)
• Alginate gels were able to produce antral follicles from small 2° preantral follicles in both monkey & human
  (reviewed in Smitz et al (2010) Human Reprod Update vol.16)
• Various hydrogel matrices were developed to support the follicle (had really good success). The problem with developing these hydrogel matrices is that they have to be permeable, because media has to get through, but they also have to be rigid enough to support the growing follicles and modifiable so the follicle has space to grow in size.
• There are two types that have been found to be particularly useful and successful.
• The progress is ongoing and they are working on different ways of doing this, but it hasn’t gone as fast as we could have hoped. As seen in previous lectures, mouse follicles can been grown up completely from small preantral stages to mature oocyte without any problem in vitro.