The pressing problem of female fertility; ovarian follicle pool depletion
Age-related decreased fertility has become an increasing challenge for infertility professionals as the average age of first-time motherhood in industrialized countries has increased dramatically over recent decades. Natural fecundity, as well as the success of any infertility intervention, including ART, decreases dramatically with increasing age due to exhaustion of the pool of resting ovarian primordial follicles (
Broekmans et al., 2009
). Furthermore, approximately 1% of women suffer from premature ovarian insufficiency (POI) characterized by a loss of ovarian activity before 40 years of age.Throughout the female life span under physiological circumstances, the majority of ovarian follicles will not enter the stage of cyclic recruitment but will undergo atresia, until near complete follicular exhaustion coincides with menopause. It is estimated that the onset of natural infertility occurs at a median age of 41 years (
Eijkemans et al., 2014
), while endocrine ovarian activity persists up to menopause at a median age of 51 years. Yet, histological samples reveal that the follicle pool in the ovary is not completely exhausted until early in the eighth decade of life (Gougeon et al., 1994
). The pressing problem for reproductive medicine therefore is how to activate (part of) the remaining ovarian ‘reproductive gold reserve’ in those women with premature cessation of ovarian function or in those who wish to become pregnant at a more advanced age.Regeneration, rejuvenation and activation; many efforts, but so far not a convincing clinical breakthrough
Different approaches to regenerate, rejuvenate or reactivate germ cells in the human ovary have been reported in recent years. These attempts include, amongst many others, the generation of artificial gametes from putative ovarian stem cells (
Woods and Tilly, 2012
; Telfer and Anderson, 2019), the intra-ovarian injection of calcium gluconate-activated autologous platelet-rich plasma (Sills et al., 2018
), the use of autologous mitochondrial transfer into oocytes in vitro (Labarta et al., 2019
), or the administration of androgens to patients with the aim of increasing the sensitivity of the remaining follicles to gonadotrophin stimulation (- Labarta E.
- de Los Santos M.J.
- Herraiz S.
- Escribá M.J.
- Marzal A.
- Buigues A.
- Pellicer A.
Autologous mitochondrial transfer as a complementary technique to intracytoplasmic sperm injection to improve embryo quality in patients undergoing in vitro fertilization-a randomized pilot study.
Fertil. Steril. 2019; 111: 86-96
Nagels et al., 2015
). None of these approaches have entered routine clinical practice, and the evidence available so far is either still pre-clinical or of questionable validity.In 2010, a method of activating dormant follicles in the mammalian ovary was proposed based on the PTEN-PI3K-Akt-Foxo3 pathway (
Li et al., 2010
). These in-vitro observations in mice quickly turned into a clinical trial in the human (Kawamura et al., 2013
; - Kawamura K.
- Cheng Y.
- Suzuki N.
- Deguchi M.
- Sato Y.
- Takae S.
- Ho C.H.
- Kawamura N.
- Tamura M.
- Hashimoto S.
- Sugishita Y.
- Morimoto Y.
- Hosoi Y.
- Yoshioka N.
- Ishizuka B.
- Hsueh A.J.
Hippo signaling disruption and Akt stimulation of ovarian follicles for infertility treatment.
Proc. Natl. Acad. Sci. U. S. A. 2013; 110: 17474-17479
Suzuki et al., 2015
), combining the in-vitro stimulation of the ovarian Akt signaling pathway in ovarian cortex fragments with a method termed ‘hippo-signaling disruption’ (Hsueh et al., 2015
). In short, ovarian cortex fragments from POI patients were vitrified, warmed, fragmented into small pieces (approximately 100 cubes of 1–2 mm2) and incubated with PTEN-inhibiting and PI3K-Akt-stimulating drugs, respectively, for 2 days before laparoscopic auto-transplantation beneath the serosa of the fallopian tubes. To this day, only a small number of pregnancies and births resulting from this method have been reported in the peer-reviewed literature or in the form of an abstract presentation (Fabregues et al., 2018
; Lunding et al., 2018
; Lee and Chang, 2019
).Ovarian in-vitro activation; now one-step and ‘drug-free’
The wide-spread usage of the approach of combining the in-vitro activation by drugs with hippo-signalling disruption has been hampered by three issues: Firstly, the need for two laparoscopic procedures, one for ovarian cortex retrieval, and another for the auto-transplantation procedure after incubation of the cortex fragments with the activating drugs. Secondly, these activating drugs, an inhibitor of the phosphatase PTEN (Phosphatase and Tensin homolog) and an activator of the phosphatidylinositol 3-kinases (PI3K) and serine/threonine-specific protein kinase (Akt), interact with intra-cellular signaling pathways involved in cellular growth, proliferation and differentiation, which may also be involved in carcinogenesis. Uninhibited, the PTEN enzyme drives programmed cell-death and therefore PTEN is thought to act as a tumor suppressor. Accordingly, alterations of the PTEN-PI3K-Akt pathway have been found in numerous cancers (
Luongo et al., 2019
), including, but not limited to, cancers of the ovary and fallopian tube (- Luongo F.
- Colonna F.
- Calapà F.
- Vitale S.
- Fiori M.E.
- De Maria R.
PTEN Tumor-Suppressor: The Dam of Stemness in Cancer.
Cancers (Basel). 2019; 11 (Jul 30pii: E1076)https://doi.org/10.3390/cancers11081076
Dean et al., 2019
; Dvorská et al., 2019
). Given such a safety concern, extensive pre-clinical work appears prudent before large sample-size clinical trial in the human. Thirdly, a 2-day in-vitro culture of ovarian tissue may lead to tissue damage, necrosis and programmed cell-death, which may counteract the motive of ovarian activation.What is ovarian Hippo signaling disruption?
Organ growth and size is controlled by the so-called Hippo signaling pathway, the central enzyme, first identified in Drosophila, being the protein kinase Hpo. In short, the Hippo pathway regulates cell proliferation and apoptosis and thereby organ size by negative growth control (
Pan, 2007
). The name comes from tissue outgrowth (a hippopotamus-like phenotype) observed in the context of mutations in the Hpo gene. Accordingly, disruption of this pathway stimulates growth, cell survival and proliferation, which has also been demonstrated in the mammalian ovary (Kawamura et al., 2013
). It was claimed that Hippo disruption alone can promote growth of dormant primordial follicles (- Kawamura K.
- Cheng Y.
- Suzuki N.
- Deguchi M.
- Sato Y.
- Takae S.
- Ho C.H.
- Kawamura N.
- Tamura M.
- Hashimoto S.
- Sugishita Y.
- Morimoto Y.
- Hosoi Y.
- Yoshioka N.
- Ishizuka B.
- Hsueh A.J.
Hippo signaling disruption and Akt stimulation of ovarian follicles for infertility treatment.
Proc. Natl. Acad. Sci. U. S. A. 2013; 110: 17474-17479
Hsueh et al., 2015
), and a drug-free activation of follicular growth only by mechanical disruption is now being tested in POI patients (Fabregues et al., 2018
), who typically should still have a pool of primordial follicles in addition to follicles up to secondary stage.Can drug-free in-vitro activation resolve infertility in 30-40% of POI patients?
In this issue of RBMO, two uncontrolled, small case series on the empirical use of drug-free in-vitro activation (IVA) in patients with POI involving 11 (
Kawamura et al., 2020
) and 14 (Ferreri et al., 2020
) female patients respectively are reported from two collaborating groups. Briefly, the original IVA protocol (Kawamura et al., 2013
) has now been modified into a drug-free, one-step procedure with laparoscopic retrieval of ovarian fragments, removal of ovarian medulla, dissection of ovarian cortex into small cubes of about 1 mm3 and immediate autologous re-transplantation. Before and after the procedure, endogenous LH levels were kept low, while ovarian stimulation with high doses of FSH commenced shortly after the surgical procedure, eventually ending in oocyte retrieval and IVF. Both groups report not only follicular growth in the transplanted ovarian tissue, but also successful oocyte retrieval, and pregnancies after embryo transfer. Nine out of 25 POI patients (both studies combined) achieved pregnancy (pregnancy rate 36.0%, 95% confidence interval: 20.2 to 55.5).- Kawamura K.
- Cheng Y.
- Suzuki N.
- Deguchi M.
- Sato Y.
- Takae S.
- Ho C.H.
- Kawamura N.
- Tamura M.
- Hashimoto S.
- Sugishita Y.
- Morimoto Y.
- Hosoi Y.
- Yoshioka N.
- Ishizuka B.
- Hsueh A.J.
Hippo signaling disruption and Akt stimulation of ovarian follicles for infertility treatment.
Proc. Natl. Acad. Sci. U. S. A. 2013; 110: 17474-17479
Recently, an uncontrolled trial on 20 women younger than 39 years of age with preserved menstrual cycle but diminished ovarian reserve was reported (
Lunding et al., 2019
). These women underwent laparoscopic biopsy of one ovary and re-transplantation of the fragmented ovarian tissue into a peritoneal pocket, while the contra-lateral ovary served as a control. Endocrine and sonographic follow-up, as well as a trial of IVF did not reveal a higher activity of the fragmented ovarian tissue. An editorial (- Lunding S.A.
- Pors S.E.
- Kristensen S.G.
- Landersoe S.K.
- Jeppesen J.V.
- Flachs E.M.
- Pinborg A.
- Macklon K.T.
- Pedersen A.T.
- Andersen C.Y.
- Andersen A.N.
Biopsying, fragmentation and autotransplantation of fresh ovarian cortical tissue in infertile women with diminished ovarian reserve.
Hum. Reprod. 2019; (Oct 8pii: dez152)https://doi.org/10.1093/humrep/dez152
Steiner, 2019
) therefore demanded the abandonment of this procedure.- Steiner A.Z.
Evidence that biopsying, fragmentation and auto-transplantation of ovarian tissue should be abandoned as a treatment of diminished ovarian reserve.
Hum. Reprod. 2019; (Oct 8. pii: dez158)https://doi.org/10.1093/humrep/dez158
After careful consideration and extensive review, we decided to publish these two uncontrolled small case series concerning POI patients in RBMO, despite the obvious limitations to the external validity of these findings. An efficacious method for ovarian activation is indeed urgently needed and research concerning this topic has recently been identified as a top priority in a comprehensive international stakeholder review (
Duffy, 2019
). Furthermore, by publishing the data in this way both groups comprehensively and openly share their methodology with the scientific community, which should allow other investigators to either replicate the results at a higher level or to refute the paradigm.As editors we also want to express some words caution:
- •An uncontrolled clinical experiment does not allow a reliable estimate of the association of exposure with outcome.
- •A small sample size necessarily results in imprecise effect estimates.
- •Case series have a high risk of selection bias, e.g. only patients likely to respond to the intervention are included and the findings are therefore difficult to cross-compare with other observations, let alone to extrapolate to the heterogeneous group of patients with POI.
- •Although it has been shown that fragmentation of ovarian cortex interacts with the Hippo pathway and induces the production of factors thought to promote primordial up to pre-antral follicular growth, the specific mechanism of action responsible for the activation of dormant follicles has not been elucidated.
- •Little is known about the maternal and neonatal safety of these procedures.
- •Only a sufficiently large, controlled and multi-center clinical trial would allow the establishment of a robust cause-effect relationship and a robust estimate of the magnitude of effect size. This insight is necessary for further clinical judgement, when burden, risks and cost have to be weighed against prospects for a positive outcome.
From experimental to established treatment of ovarian follicle pool activation: where is drug-free IVA now?
The clinical need for effective infertility care in women with POI or diminished ovarian reserve is high, since alternative treatment options beyond oocyte donation do not exist at present. However, in an ideal scenario, the introduction of a new treatment is preceded by pre-clinical research that elucidates putative mechanisms of action. Next, the intervention needs to be sufficiently mature and replicable in practical terms. Obviously, this new treatment should only be offered in a research setting and with approval of a medical-ethics review board. If the biological principle has been understood, and the pre-clinical evidence and the first observed clinical effects are sufficiently promising, further application can be considered – but, again, only in a research setting. The rapidly evolving and competitive field of reproductive medicine often tempts practicing clinicians to jump on the moving bandwagon too early. As editors of RBMO, we encourage a wait-and-see approach for practitioners, and call for more basic science studies into Hippo disruption, as well as a collaborative effort on a controlled trial, before further uncontrolled empirical usage.
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