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In females, oocytes cease their meiotic division at the first meiotic prophase I germinal vesicle stage before birth and stay arrested at this stage for many years. At puberty, upon appropriate stimulation by follicle stimulating hormone (FSH) and luteinizing hormone (LH), the nuclear membrane dissolves, the chromosomes arrange themselves on the first meiotic spindle and meiosis progresses. The completion of the first meiotic division is characterised by the expulsion of the first polar body. The second meiotic division is initiated rapidly after completion of the first meiotic division and prior to ovulation meiosis arrests again at metaphase II until fertilization .
In in-vitro fertilisation cycles (IVF), treatment initially involves the inhibition of the endogenous production of the gonadotropins which are responsible for the growth and maturation of the follicles containing the oocytes. After that, the growth of multiple follicles is induced by controlled exogenous administration of gonadotropins. The oocytes are finally matured by injection of hCG (human chorionic gonadotropin) which acts as a surrogate LH surge. Add/compare significance of LH surge in natural ovulation
In ovarian stimulated cycles hCG is administrated for sufficient time what does this mean?(28-38h) to induce ovulation of mature oocytes. However, the retrieved oocytes could be at varying stages of meiotic maturity. Usually about 8-15% of the retrieved oocytes are immature (at the germinal vesicle or metaphase I stage) ; but sometimes the percentage of immature oocytes may be greater (>25%) than usual and this could lower the likelihood of successful IVF treatment .
A couple was first seen at Homerton Fertility Centre in July 2011. The female patient was 29 years old and had been trying for a pregnancy for the past 2 years. She had regular ovulatory cycles and normal ovaries. Investigations showed normal Anti-Müllerian hormone (AMH) (21.62pmol/L) levels as well as normal LH, FSH and oestradiol levels (6.0u/L, 4.6uL, 57pmol/L respectively). The male patient was 27 years old with no medical or surgical history of importance. His semen analysis was normal. The couple initially had 3 unsuccessful cycles of intrauterine insemination (IUI).
The first in-vitro fertilisation (IVF) cycle started in June 2012. Pituitary suppression was achieved by gonadotropin releasing hormone (GnRH) antagonist (see table 1). Ovarian response was monitored using transvaginal scans. Also the serum oestrogen (E2) levels were measured on Day 6 of the stimulated cycle. The patient showed normal response to medication and 11 follicles of the expected size (~18mm) developed after 11 days of stimulation. Oocyte retrieval was performed 35 hours after the hCG injection. On the day of egg collection 8 oocytes were collected. The semen parameters were normal and conventional IVF was performed. Sperm insemination was performed 40 hours after the trigger time. On the day of fertilisation check (16 hours after insemination) none of the oocytes had fertilised. The oocyte maturity was classified according to their nuclear status: presence (MII) or absence (MI) of the first polar body or possession of an intact germinal vesicle (GV). All the oocytes were defined as immature at the GV stage. The immature oocytes were further cultured until the following day in cleavage medium (Sage, USA) but their maturity status did not change.
The second IVF cycle was performed in September 2012. This time, the patient was put under the GnRH agonist protocol (see table 1 for details on the stimulation protocol). The patient again showed normal response to medication and 12 follicles (~18mm) developed after 11 days of stimulation. On the day of egg collection 12 oocytes were collected. Although the sperm parameters were normal again, due to previous failed fertilization with standard IVF, intra-cytoplasmic sperm injection (ICSI) was planned this time. After denudating the 6 oocytes (2 hours after the egg collection), all were defined as immature at the GV stage. Considering the patient's history, the decision was made not to denudate the rest of the oocytes; and to perform standard IVF for the rest of them. On the day of fertilisation check all the oocytes were still immature at the GV stage, apart from one that was mature but which did not fertilise. The immature oocytes failed to proceed in meiosis even after extended culture in cleavage medium until the following day.
Drugs taken for stimulation
Days of stimulation
Serum E2 levels before the trigger
Dose of hCG for trigger
No of oocytes collected
Short protocol with
GnRH antagonist (Cetrotide, 0.25mg)
- Combined FSH and LH (Menopur, 75 IU)
- Recombinant FSH (Gonal F, 125 IU)
Isn't this a little low and may indicate a problem?
Pregnyl, 5000 IU
Long protocol with GnRH agonist (Supracure, 0.5ml)
- Recombinant FSH (Gonal F, 225 IU for 7 days and 150 IU for 3 days)
Table 1. Gonadotropin stimulation and follicular response for the two separate cycles
Very few studies have been published reporting complete oocyte maturation failure of oocytes arrested at the GV stage after IVF treatment [3-6].
The first cause to be examined is the response of the developing follicles to gonadotropins. FSH is responsible for follicular growth. Insufficient oocyte growth cannot explain the reason for oocyte maturation arrest in our case, since the size of the developed follicles was within the appropriate range (17-20mm). Serum oestrogen levels were also monitored. Apart from the ultrasound scan, oestrogen monitoring acts as an additional witness of follicular growth as it shows the response of follicles to FSH. This is because Granulosa follicular cells, through their FSH receptors, bind the FSH which is then responsible for the aromatisation of androgens to oestrogens . However, the full growth of the follicle is not always accompanied by the maturation of the egg in it. So, sometimes immature oocytes can be aspirated from big follicles. Because of the normal size of the follicles on the aspiration day, the possibility of premature luteinisation can also be excluded. Premature luteinisation results in early oocyte ovulation and thus only small follicles containing immature oocytes are present on the aspiration day .
As hCG (surrogate for LH) is responsible for oocyte maturation in IVF, the presence of only immature eggs could be a result of incomplete or absent LH effect. An incomplete LH surge could happen if the patient did not self-administer the trigger injection on time. This is unlikely to be the case for this patient due to the repeated occurrence of the phenomenon in both attempts and the patient's confirmation that the medication was taken on time. However, serum LH levels should be monitored at the trigger time to fully exclude this possibility. Complete failure of induction of an LH surge could be explained by a problematic batch of inactive hCG hormone. This is not applicable on this occasion as no other patients faced this problem. Also, the same phenomenon occurred in both cycles where different types of hCG were used. Finally, an absent LH effect could be a result of dysfunctional gonadotropin receptor (LHR). Studies in LHR knockout mice showed lack of development of ovulatory follicles . Women with mutations of the LH receptors also showed no preovulatory follicles and no formation of corpora lutea . This could be an explanation because before use more scientific language the LH surge, LH receptors are accumulated by the Granulosa cells and LH is used for the synthesis of progesterone which is essential for the formation of the corpus luteum . So, an investigation to examine a possible dysfunction of the LH receptor would be to test the progesterone levels of the patient after the trigger. However, the best confirmation would be a genetic analysis which would reveal the absence or not of the gene coding for the LH receptor protein.
Hartshorne et al  exposed the immature oocytes in vitro to culture media supplemented with FSH or hCG. Expansion of cumulus was observed but the oocytes still failed to resume meiosis. They showed that although the cumulus cells were expressing gonadotropins receptors and were responding to them, the signal was not able to reach the oocyte. Thus, a defective communication of the oocyte with its surrounding cells should also be considered as a possible explanation of our results. It is known that cAMP (cyclic adenosine monophosphate) is produced by the somatic cells of the follicle and is diffused to the oocyte and causes maturation arrest . The hCG surge, removes the oocyte from its contact with granulosa cells and it acts as a 'switch' which minimises the flow of cAMP inducing germinal vesicle break down and oocyte maturation [10, 11]. Studies in animals showed that dysfunction of the phosphodiesterase type 3 (PDE3) (which is a molecule involved in that signalling pathway of oocyte-cumulus communication) leads to elevated levels of cAMP and thus to a constant arrest at the GV stage . The possibility of abnormal signalling between the cumulus cells and the oocyte cannot be excluded in our case.
Finally, failure of oocyte maturation could be a result of an intrinsic oocyte defect. During oocyte maturation, various signal transduction pathways should be activated in order to promote the activation of the maturation-promoting factor (MPF) via phosphorylation . This factor is then responsible for the remodelling of the nuclear (chromosome condensation) and the cytoplasmic compartments (Golgi, ribosomes and mitochondria) during oocyte maturation. Studies in animals which involved complete inactivation of genes, like Cdc25, responsible for the expression of key proteins of the MPF pathway resulted in oocyte maturation arrest at the GV stage . Abnormalities resulting in abnormal nuclear and oocyte remodelling could be a possible explanation for our results.
Extending the period of stimulation until the follicles reach 22-23mm, extending the HCG to retrieval interval and increasing the hCG dose have been some of the suggestions for dealing with the problem of repeated retrieval of immature oocytes (reviewed in ). Additionally, it has been suggested that immature retrieved oocytes can be matured in vitro after culture in favourable conditions (in-vitro maturation, IVM) . However the success rates of this technique are still unclear. Finally, intracytoplasmic injection of donor cytoplasm or maturation factors, or the transfer of the GV from a patient's oocyte into a donor's enucleated oocyte could be theoretical additional treatment options . However, these techniques are at an experimental stage and still a lot of more research is required until their approval and their clinical application. Egg donation seems to be the most viable option in these cases.
In our the Homerton case, the patient is planning to come for another treatment cycle at Homerton Hospital. Since at Homerton IVM is not performed, the doctors have decided to modify her the stimulation treatment plan in the next cycle. This will include a long protocol with a combined dose of FSH and LH for stimulation as these have been shown to improve the maturity of the retrieved oocytes [16, 17]. Also a double dose of hCG will be used as trigger. Serum LH levels will also be monitored to confirm that the trigger was taken on time.
The mechanisms that lead to complete oocyte maturation failure are not completely understood as the process of oocyte maturation is complex. Further studies and research is needed to solve the problem and improve the outcome of IVF.