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The ovary has a comparatively short functional lifespan compared with other organs, and genetic and pathological injuries can further shorten its functional life. Thus, preserving ovarian function should be considered in the context of women with threats to ovarian reserve, such as ageing, premature ovarian insufficiency (POI) and diminished ovarian reserve (DOR). Indeed, one-third of women with POI retain resting follicles that can be reactivated to produce competent oocytes, as proved by the in-vitro activation of dormant follicles. This paper discusses mechanisms and clinical data relating to new therapeutic strategies using ovarian fragmentation, stem cells or platelet-rich plasma to regain ovarian function in women of older age (>38 years) or with POI or DOR. Follicle reactivation techniques show promising experimental outcomes and have been successful in some cases, when POI is established or DOR diagnosed; however, there is scarce clinical evidence to warrant their widespread clinical use. Beyond these contexts, also discussed is how new insights into the biological mechanisms governing follicular dynamics and oocyte competence may play a role in reversing ovarian damage, as no technique modifies oocyte quality. Additional studies should focus on increasing follicle number and quality. Finally, there is a small but important subgroup of women lacking residual follicles and requiring oocyte generation from stem cells.
Health is one of the fundamental principles and rights of human beings. While the last century was characterized by population growth, the 21st century is predicted to be one of population ageing. By 2050, an estimated 30% of the population will be over the age of 65 (
) and the knowledge that overall functional decline is slow but steady. Yet, the ovary is unique in that this organ has an abrupt reduction in function that reaches extinction within roughly 10 years, while other organs remain fully functional. The ovarian ageing phenomenon could be caused by the high demand of the physiological requirements needed for function, leading to diminished ovarian reserve (DOR) and increased oocyte/embryo aneuploidy (
), common features of physiological ageing found in young women with premature ovarian insufficiency (POI).
POI is a heterogeneous condition affecting 1% of women of reproductive age and is defined by elevated gonadotrophins, including FSH, and irregular or absent menstruation before the age of 40 years (
). DOR or poor ovarian response to stimulation is also heterogeneous and is frequently encountered during fertility treatment in regularly menstruating women (
Diminished ovarian reserve in the United States assisted reproductive technology population: diagnostic trends among 181,536 cycles from the Society for Assisted Reproductive Technology Clinic Outcomes Reporting System.
). Many – but not all – cases of DOR observed in clinical practice are physiological and represent an age-dependent decline in ovarian reserve. However, a common feature of all these clinical conditions is the reduced reproductive potential to achieve motherhood with the woman's own eggs.
The possibility of activating dormant follicles within the ovarian cortex piqued interest after their reactivation to grow and produce healthy offspring was shown by Li and colleagues using an in-vitro activation (IVA) protocol for dormant follicles involving PTEN (phosphatase and tensin homolog) inhibitors and PI3K (phosphatidylinositol 3 kinase) stimulators (Akt [serine/threonine protein kinase 1] stimulation) (
). This finding can be considered as the stepping stone to ovarian reactivation techniques, together with the demonstration in ovarian biopsies from women with POI that almost 40% retained some (30%) or many (9%) follicles in the cortex (
) have found that the ovary can spontaneously re-start functioning and even achieve naturally conceived pregnancies. In particular, there seems to be a huge difference in prognosis among individuals with high FSH concentrations and long periods of amenorrhoea, where naturally conceived pregnancies have been described in 4.4% of cases, and those with fluctuating POI (slightly elevated serum FSH, sporadic spotting), who have a 17.5% naturally conceived pregnancy rate (
). Thus, although the best clinical model to test alternatives to activate resting follicles is POI, the important number of patients with DOR and their demands to avoid the use of third-party reproduction has forced researchers and clinicians to apply the same principles of follicular activation in women with POI to those with DOR in an attempt to circumvent their damaged follicular pool. Because of the different aetiologies, POI and DOR are not comparable and perhaps experience will bring an understanding of which method of activating follicles works in which condition.
This review discusses the techniques available, the potential indications and the outcomes of studies aimed at follicular rescue and ovarian reactivation via prolonging reproductive function. It also discusses their advantages and limitations for future use in the clinical setting and new innovative scientific developments that promise major changes in the field.
Search methods
PubMed was searched for peer-reviewed original English-language manuscripts published in the last 10 years to identify studies on established and experimental techniques proposed to restore ovarian reproductive function. Conditions of interest were POI, DOR, poor ovarian response (POR) and older age. The following keywords were used: oocyte ageing, fertility preservation, ovarian reserve, anti-Müllerian hormone (AMH), antral follicle count (AFC), resting/dormant follicles, follicular rescue, IVA, ovarian fragmentation, stem cells, bone marrow-derived stem cells (BMDSC), platelet-rich plasma (PRP) and autologous stem cell ovarian transplantation (ASCOT), in various combinations. The study also included relevant papers cited within these primary references. The literature was screened to identify experimental data regarding ovarian function and oocyte quality with potential translational applications to expand ovarian lifespan. Keywords such as spindle transfer, soluble growth factors, DNA damage and repair, double-strand breaks, apoptosis, telomeres, oocyte generation, stem cells and ovarian ageing were searched alone and in different combinations.
Full articles, including clinical trials (randomized or non-randomized), case reports and case series, were retrieved and reviewed. In addition, registered clinical trials (from www.clinicaltrials.gov) were reviewed to explore indications and alternative methods for expanding the function of human ovaries after injury or at a patient's request. A total of 207 original references were included in this review.
Pathways to induce follicular activation and ovarian rescue
Folliculogenesis in mammals starts with the activation of a few dormant primordial follicles (around 1000 /month) to initiate growth (
) after menarche. The transition from primordial to primary stage is characterized by both oocyte growth and the differentiation of flattened pregranulosa cells into proliferative cuboidal granulosa cells. Follicles then progress through different developmental stages to finally undergo ovulation or atresia. Although a developmental programme intrinsic to the oocyte is crucial for orchestrating follicular growth rate, whether activation of the oocyte and/or pregranulosa cells is coordinated remains unclear (
Several signalling pathways (Supplementary Table) are involved in follicle activation guidance through the control of oocyte growth initiation and maintenance, and mediating the primordial or primary transition, such as PTEN deletion on chromosome 10, PI3K, FOXO3 (the transcription factor forkhead box O3) and mTORC1 (the mammalian target of rapamycin complex 1). Indeed, knockout mouse models with an oocyte-specific deletion of Pten or Foxo3 showed a reduced reproductive lifespan due to the activation of all dormant follicles (
) and is considered the primary regulator of activation. PTEN inhibition promotes the activation of PI3K to phosphorylate intermediate effectors including Akt to finally induce the nuclear export of downstream FoxO3 proteins, leading to primordial follicle activation (
). This maintains phosphorylation of the transcription factor YAP (Yes1 associated transcriptional regulator), preventing it from moving from the cytoplasm to the nucleus. This is known as the Salvador–Hippo–Warts (SHW) pathway (
). When the rigidity of the extracellular matrix decreases (for example, after ovarian tissue fragmentation), actin polymerization increases in the granulosa cells and Rho and ROCK activity decreases, leading to a lower degree of YAP phosphorylation and resulting in an interruption of the intracellular SHW signalling (
). Migration of phosphorylated YAP to the nucleus promotes the expression of CCNs (growth promoters, namely CYR61 [cysteine-rich angiogenic protein 61 or CCN1], CTGF [connective tissue growth factor or CCN2] and NOV [nephroblastoma overexpressed or CCN3]) and BIRC (baculoviral inhibitors of apoptosis repeat containing, which are apoptosis inhibitors), and subsequently promotes small secondary follicle growth and granulosa cell formation in addition to primordial activation (
Techniques to induce ovarian rescue in humans: basic experimental studies and clinical data in DOR, POI and older women
IVA and ovarian fragmentation
The findings described above prompted a proposed protocol for the IVA of dormant follicles using PTEN inhibitors and PI3K stimulators (Akt stimulation) over 48 h in mice and human ovaries (
). This can be considered as the proof of concept for oocyte/ovarian reactivation because it demonstrates that residual dormant follicles, remaining in the ovaries when ovarian function is lacking, can be rescued to extend the reproductive lifespan of diseased ovaries.
Kawamura and colleagues improved the former IVA technique by including inhibition of the Hippo pathway to promote follicle activation, bringing about a synergic effect in different follicle populations, and increasing development to antral stages to produce mature oocytes and embryos to transfer (
). These characteristics can be found in both physiological ageing and in young women with POI. Since the disruption of the extracellular matrix results in growth factors being released after Hippo inhibition, it has been postulated that the disruption of the extracellular matrix can also stimulate somatic cell proliferation and therefore contribute to improving the ovarian niche to support follicle development (
). This theory is further supported by the fact that in animal models, the use of antifibrotic drugs for pulmonary fibrosis allowed a reversal of age-associated ovarian fibrosis while improving ovulation and extending fertility (
Since the first manuscript describing the technique for women with POI that consists of mechanical removal of the ovarian cortex via laparoscopy and subsequent fragmentation into 1 mm3 cubes to inhibit the Hippo pathway followed by IVA over 48 h with Akt stimulators before tissue autografting into the patient via laparoscopy (Figure 1), many case series and cohort studies using the same approach have reported varying degrees of success in women with DOR for POI (
). Based on the authors’ experience with fertility preservation for oncological patients, for which the surgical procedures are similar to those of IVA, except for the culture with Akt stimulators, it was learned that oncological patients in whom the ovarian cortex was fragmented into small pieces and subsequently reimplanted performed much better in terms of revascularization, return to cycling and pregnancy outcomes compared with women whose entire cortex was first removed and subsequently reimplanted as a bigger fragment (
Oocyte vitrification versus ovarian cortex transplantation in fertility preservation for adult women undergoing gonadotoxic treatments: a prospective cohort study.
With this background, the authors modified the IVA technique to what was referred to as ovarian fragmentation for follicular activation (OFFA), which was subsequently also employed as ‘drug-free IVA’ (
Drug-free in-vitro activation of follicles and fresh tissue autotransplantation as a therapeutic option in patients with primary ovarian insufficiency.
) (Figure 1A). This new approach reduces the number of operations required while avoiding ovarian tissue cryopreservation and associated follicular loss. This drug-free approach is more readily applicable to clinical practice due to the potential carcinogenic effects of PTEN inhibition (
Inhibition of phosphatase and tensin homologue (PTEN) in human ovary in vitro results in increased activation of primordial follicles but compromises development of growing follicles.
The overall cumulative pregnancy rate ranged from a success rate as high as 30% in a well-characterized population with POI to 60% in women with DOR whose inclusion criteria were solely based on repeated serum AMH concentrations of ≤5 pmol/l (0.7 ng/ml) (
). The heterogeneity in reported results could be in part due to differences in the definition of the primary end-points between studies: while some used the number of follicles in response to stimulation (
), others did not use predefined outcomes to assess the success of the technique. The protocols used to activate follicles also varied between experimental settings (Table 1). Other limitations of the above-described studies include the low number of participants included or the lack of a control group.
Table 1Studies reporting the effect of ovarian fragmentation, in POR, POI, premenopausal, and postmenopausal patients.
Intervention
Diagnosis and age of patients
Study design
Outcomes overall
Unilateral or bilateral ovarian fragmentation, ovarian tissue vitrification, warming + incubation with bpV(HOpic) + 740-YP and reimplantation of the tissue during the same surgical procedure to the serosa of both Fallopian tubes (
9/37 developed follicles 3/37 achieved a biochemical pregnancy 2/37 had a live birth
Unilateral ovarian fragmentation + incubation with bpV(HOpic) + 740-YP and reimplantation of the tissue during the same surgical procedure to the serosa of both Fallopian tubes (
POR and infertility 37 years old (30–39) Preserved menstrual cycles AMH ≤5 pmol/l
Prospective cohort study (n = 20)
7/20 experienced elevation of AMH 12/20 resulted in a clinical pregnancy without ART 16/20 started at least one IVF cycle (38 cycles performed in total) 7/20 had a live birth after IVF (18.4% cumulative LBR per IVF cycle) 1/20 had a live birth after IUI (+1 termination) 3/20 had a live birth after natural conception
Unilateral ovarian fragmentation (without incubation) and reimplantation to ovary (
9/11 increased AMH levels (baseline prior to the procedure) 3/11 resulted in a clinical pregnancy after IVF (1 miscarriage, 1 ongoing, 1 live birth) 1/11 resulted in a natural conception (ongoing)
Unilateral ovarian fragmentation (without incubation) and reimplantation to the contralateral ovary and peritoneal pocket (
Drug-free in-vitro activation of follicles and fresh tissue autotransplantation as a therapeutic option in patients with primary ovarian insufficiency.
POR and infertility Previous failed IVF 36 years (34–39) AMH 0-0.6
g/ml All intended to undergo IVF
Randomized controlled trial (n = 34) Control arm: no intervention
Higher AFC after intervention in the operated ovary (difference 2 antral follicles, P = 0.021) Clinical pregnancy rate after IVF 2/15 with intervention versus 3/16 controls Live birth rate after IVF 1/15 with intervention versus 3/16 controls
Recently, the current authors’ group published the results of a randomized controlled trial in which the efficacy of OFFA to increase the number of antral follicles was assessed in women with DOR (
). Although OFFA resulted in an inhibition of the Hippo pathway, confirmed by an 18.8% reduction in the phospho-YAP/YAP ratio and BIRC and CCN overexpression after fragmentation, and an increase in AFC (P = 0.021), serum AMH or FSH concentrations did not improve. Indeed, reproductive and IVF cycle outcomes were not statistically different between the groups.
More research is needed in this field, but given evidence of a molecular and biological effect, patients for whom egg donation is not an alternative treatment and in whom follicles are very unlikely to develop (individuals with POI) may have follicular activation through fragmentation, which could still play a role in treatment. Indeed, clinicaltrials.gov reveals several ongoing studies evaluating this technique, and new modifications of the procedure, for both DOR and POI patients (NCT04608695, NCT04390308, NCT03670407, NCT03298750 and NCT02322060).
The use of stem cells
Up to 20 different types of adult stem cell are found in human tissues with the capacity to differentiate into the cells of the tissue in which they were originally located (
). Among them, both haemopoietic and mesenchymal cells have become increasingly important in regenerative medicine, with mesenchymal stem cells (MSC) popularly tested due to a higher replication potential and the capability to differentiate into distinct cells (
Human endometrial mesenchymal stem cells restore ovarian function through improving the renewal of germline stem cells in a mouse model of premature ovarian failure.
Bone marrow transplantation generates immature oocytes and rescues long-term fertility in a preclinical mouse model of chemotherapy-induced premature ovarian failure.
Effects of single and multiple transplantations of human umbilical cord mesenchymal stem cells on the recovery of ovarian function in the treatment of premature ovarian failure in mice.
Different therapeutic effects of cells derived from human amniotic membrane on premature ovarian aging depend on distinct cellular biological characteristics.
CD44+/CD105+ human amniotic fluid mesenchymal stem cells survive and proliferate in the ovary long-term in a mouse model of chemotherapy-induced premature ovarian failure.
) also demonstrate the ability to recover hormone synthesis, follicle development and fertility in rodent models, suggesting their role as follicle reactivating agents (
). However, not all MSC types may have an autologous source: for instance, cells from the umbilical cord or amniotic fluid will probably have an allogenic origin (
). BMDSC are mononuclear cells, including MSC, haemopoietic stem cells and endothelial progenitors, with low immunogenicity that can self-renew and differentiate after tissue damage (cytokine liberation) (
). Moreover, the proliferation and mobilization of clinically relevant cell numbers into the peripheral blood is achieved by employing pharmacological treatments such as granulocyte colony-stimulating factor (G-CSF), and BMDSC can easily be collected by apheresis, avoiding invasive techniques.
As adult stem cells, they can secrete soluble factors, such as cytokines and growth factors, to stimulate neighbouring cells (Figure 2A) (
Human endometrial mesenchymal stem cells restore ovarian function through improving the renewal of germline stem cells in a mouse model of premature ovarian failure.
Cytokines produced by bone marrow cells can contribute to functional improvement of the infarcted heart by protecting cardiomyocytes from ischemic injury.
Am. J. Physiol. Heart. Circ. Physiol.2006; 291: H886-H893
). This paracrine hypothesis has been supported by other studies using different sources of stem cells and their secretomes, including MSC-derived microvesicles and exosomes, to restore ovarian function in animal models (
Human Umbilical Cord Mesenchymal Stem Cell-Derived Exosomes Improve Ovarian Function and Proliferation of Premature Ovarian Insufficiency by Regulating the Hippo Signaling Pathway.
Human amnion-derived mesenchymal stem cell (hAD-MSC) transplantation improves ovarian function in rats with premature ovarian insufficiency (POI) at least partly through a paracrine mechanism.
Al-Hendy A. Human BM-MSC secretome enhances human granulosa cell proliferation and steroidogenesis and restores ovarian function in primary ovarian insufficiency mouse model.
Figure 2Mechanisms of ovarian ovarian reactivation techniques (ovarian fragmentation for follicle activation). (A) Autologous ovarian stem cell transplantation (ASCOT) of bone marrow-derived stem cells (BMDSC) and infusion of secreted soluble factors. (B) Ovarian injection of platelet-rich plasma (PRP). Created with BioRender.com.
The current group recently reported that the infusion of human BMDSC promotes human and mouse follicular growth by increasing ovarian vascularization and stromal cell proliferation, and reduces cell death in immunosuppressed animals in which conditions of POR and POI were mimicked, allowing fertility restoration resulting in natural conception (
). In both cases, injected cells arrived from the animal's tail vein to the ovarian tissue by a process known as ‘homing’, where the damaged ovarian niche attracts undifferentiated stem cells, specifically from bone marrow, to maintain homeostasis (
This ovarian rescue appears to be partially mediated by a paracrine mechanism (Figure 2A), as a characterization of the BMDSC secretome revealed soluble factors involved in the cell cycle, gene expression, pathways related to RhoGTPases, and Hippo among others (
), and key regulators of the neoangiogenic process and vessel stability. Because follicular growth also depends on cyclic microvascular remodelling throughout the menstrual cycle (
Quantification of ovarian stromal Doppler signals in poor responders undergoing in vitro fertilization with three-dimensional power Doppler ultrasonography.
Clinically, follicular reactivation and niche regeneration could also explain several reports of fertility recovery and natural conception in infertile women after cancer treatment and bone marrow transplant (
Recovery of female fertility after chemotherapy, irradiation, and bone marrow allograft: further evidence against massive oocyte regeneration by bone marrow-derived germline stem cells.
Recovery of female fertility after chemotherapy, irradiation, and bone marrow allograft: further evidence against massive oocyte regeneration by bone marrow-derived germline stem cells.
); interestingly, analysis of the baby's, the mother's and the bone marrow donor's DNA revealed a genetic compatibility between the mother and the child. This finding dismissed a genetic relationship between the donor and the baby, so the probability that stem cells replenish oocytes directly is unlikely; instead, stem cells may promote regeneration of the ovarian niche, allowing the growth and rescue of existing residual follicles, as supported by several experimental studies (
Within the clinical set-up of MSC, umbilical cord-derived MSC (UCMSC) have been shown to increase the ovarian function in 61 young patients (mean age 31 years, range 39-34 years) with POI, after transvaginal intra-ovarian allogenic stem cell infusion of UCMS isolated from donor healthy full-term human placental samples (
). This methodology resulted in an increased AFC, initiation of ovarian stimulation and overall pregnancy rate of 6.6% (4/61; three IVF and one natural conception) with no safety issues reported even when using an allogenic cell source. Positive results were increased in women with a shorter duration of amenorrhoea. Authors reported mature follicles only in ovaries receiving UCMSC. However, even within the same clinical trial, there were differences in the procedure, as some women received one transplant while others received two or three.
Most clinical investigations of MSC have published the use of bone marrow-derived mesenchymal cells (BM-MSC) in humans after iliac crest puncture and posterior in-vitro culture (Table 2). However, the objective is to achieve the same results while adopting less invasive methods. For instance, BM-MSC were employed in 30 women with POI who were aged 40 years or less, with one group receiving a direct laparoscopic infusion into the ovarian stroma, and the other group undergoing unilateral catheterization of the ovarian artery (
). Overall, hormone improvement was observed in 86.7% of participants from both groups, while 60% showed ovulation at some point during follow-up. Only 10% of women underwent IVF cycles, and one natural conception was achieved. Comparable results were obtained in 10 women (26–33 years old) with POI after BM-MSC injection into both ovaries via laparoscopy (
Role of Autologous Bone Marrow-Derived Stem Cell Therapy for Follicular Recruitment in Premature Ovarian Insufficiency: Review of Literature and a Case Report of World's First Baby with Ovarian Autologous Stem Cell Therapy in a Perimenopausal Woman of Age.
Intraovarian injection of autologous human mesenchymal stem cells increases estrogen production and reduces menopausal symptoms in women with premature ovarian failure: Two case reports and a review of the literature.
Increase of oestrogen levels 6–9 months after treatment (first case from <10.9 to 26 pg/ml; second case some controls missing) Progressive decrease in FSH levels over the 6–9-month follow-up Recovery of menstruation 7 months after treatment
Prospective observational pilot study (n = 20; ongoing at time of publication: report on 17 patients)
Improvement of ovarian reserve biomarkers in 13 women 2 weeks after treatment (AFC and AMH) 28 IVF cycles initiated (78 days after ASCOT) Oocyte retrieval in 24/28 of the cycles, and embryos in 19/28 of the cycles 5 pregnancies, 2 miscarriages and 3 live births
Bone marrow derived stem cells restore ovarian function and fertility in women with POI: Interim report of a randomized trial comparing mobilization versus ovarian injection.
in: 36th Annual Meeting of European Society Reproduction and Embryology (ESHRE). 2020
POI (still ongoing): report on 10 patients <38 years old
Randomized prospective pilot study (n = 20)
Follicular growth 90–140 days after treatment Positive response to BMDSC (AMH + FC): 66.6% in the ASCOT group, 50% in the mobilization group 6 women started IVF, with 16 cycles initiated 4 MII oocytes and 2 embryos obtained 1 ongoing pregnancy, with 1 live birth
). This innovative treatment received the name of ‘autologous stem cell ovarian transplantation’ (ASCOT). A total of 17 women with DOR according to the ESHRE criteria (
) (≤40 years, 3–5 years of infertility) were included in the study. Initially, BMDSC were mobilized and collected from peripheral blood by apheresis after a 5-day pharmacological treatment (G-CSF). The cells were then infused by intra-arterial catheterization into one ovarian artery, leaving the other ovary as a control. During the 6-month follow-up, ovarian reserve biomarkers (AFC and AMH) were enhanced in 81.3% of the participants, but importantly, there was a significant positive association with the presence of the BMDSC-secreted growth factors fibroblast growth factor 2 (FGF-2), involved in follicle growth (
), and thrombospondin-1 (key regulator of neoangiogenesis, associated with AMH increase) present in the plasma from apheresis.
These women had a total of 24 previous failed IVF cycles, in which no pregnancies were achieved. After ASCOT, 88.2% of women started IVF cycles at some point during the follow-up, retrieving a total of 51 metaphase II (MII) oocytes and obtaining 36 embryos after intracytoplasmic sperm injection (ICSI). In total, the pregnancy rate after ASCOT was 33.3%, with three naturally conceived pregnancies. However, the euploidy rate was only 16%, due to the advanced maternal age of the participants.
Different conclusions were drawn after this pilot study. First, the high pregnancy rate suggests that invasive cell collection followed by cell culture is not mandatory to obtain good reproductive results. Second, AFC increased in both the treated and the control ovary, suggesting that circulating growth factors and BMDSC have beneficial effects on the non-infused ovary, and bringing into question the requirement for direct arterial cell infusion. Furthermore, ovarian reserve biomarker improvement was observed 4 weeks after ASCOT treatment, supporting the theory that secondary and pre-antral follicles, already present at the time of the treatment, benefit from the stem cells’ properties and growth factors to regenerate the ovarian niche and stimulate the growth of the granulosa while preventing atresia. Although some antral and pre-antral follicles may be present in women with DOR, there is have a damaged ovarian niche that makes it impossible to maintain physiological requirements and follicular growth (
; Richardson et al., 2014). Finally, BMDSC may increase ovarian reserve but have no effect on oocyte quality, which is directly related to age and exponentially decreases from 38 years onwards.
A second prospective pilot study of ASCOT involving 20 women with POI (NCT03535480) according to the ESHRE POI criteria was carried out (
Bone marrow derived stem cells restore ovarian function and fertility in women with POI: Interim report of a randomized trial comparing mobilization versus ovarian injection.
in: 36th Annual Meeting of European Society Reproduction and Embryology (ESHRE). 2020
). Participants were randomized into two study arms: the first group received 5 days of G-CSF treatment, with cells remaining in circulation in the peripheral blood to test whether cells and growth factors would arrive at the wounded ovaries; the second group were treated identically to the first ASCOT study (with blood apheresis, and cell infusion into one ovarian artery). Preliminary interim analysis in 10 participants revealed follicular development in both arms when compared with basal concentrations; these follicular growth waves were detected 90–140 days after treatments. Six women started IVF cycles after treatment in both arms (60%), 4 MII oocytes were retrieved and two vitrified embryos were successfully transferred, one in each study arm. The pregnancy rate was 10%, with one healthy live birth at 38 weeks of pregnancy. Half of the participants reported an improvement of vasomotor symptoms at some point during follow-up, although a significant decrease of FSH concentrations was not recorded.
Stem cell studies in patients with POI addressed amelioration of ovarian reserve after more than 3 months of follow-up (
Role of Autologous Bone Marrow-Derived Stem Cell Therapy for Follicular Recruitment in Premature Ovarian Insufficiency: Review of Literature and a Case Report of World's First Baby with Ovarian Autologous Stem Cell Therapy in a Perimenopausal Woman of Age.
Intraovarian injection of autologous human mesenchymal stem cells increases estrogen production and reduces menopausal symptoms in women with premature ovarian failure: Two case reports and a review of the literature.
Bone marrow derived stem cells restore ovarian function and fertility in women with POI: Interim report of a randomized trial comparing mobilization versus ovarian injection.
in: 36th Annual Meeting of European Society Reproduction and Embryology (ESHRE). 2020
). These patients have no stimulable antral follicles, so researchers have turned their attention to the dormant primordial follicles. They postulate that stem cells could restore the ovarian niche, allowing the residual follicle pool to successfully grow and develop (
). This could explain why ovarian reserve biomarkers are successfully improved from 3 to 5 months after the intervention, as human primordial follicles take up to 5 months to reach the antral stage (
Likewise, the objective should be the identification of less invasive treatments in women with DOR and POI. Indeed, at the time of this publication, several studies are using different stem cell sources being developed for these women (NCT04009473, NCT03877471, NCT03069209, NCT02043743, NCT01853501, NCT01742533 and NCT02603744).
Platelet-rich plasma
Paracrine actions and regenerative cell-independent mechanisms in ovarian tissue activate pre-existing quiescent follicles through a variety of soluble growth factors involved in follicular growth, angiogenesis, viability and ovarian response to ovarian stimulation (
). In fact, a paracrine signalling mechanism is also the basis of the use of PRP, defined as the plasma fraction with the highest platelet concentration, at least 1 million/μl obtained by autologous blood centrifugation (
PRP is enriched in terms of both platelets and growth factors, including transforming growth factor-β, vascular endothelial growth factor (VEGF), platelet-derived growth factor (PDGF), insulin-like growth factor-1, FGF-ß (
). Platelets mediate haemostasis following trauma or local ischaemia by mechanisms such as adhesion, aggregation, thrombin generation and fibrin formation (
). In addition, they play an important role in modulating cellular migration, extracellular matrix remodulation, cell proliferation, angiogenesis, cell differentiation and apoptosis (
). Platelet alpha-granules discharge a range of molecules, such as hepatocyte growth factor, stromal cell derived factor-1, adenosine diphosphate (ADP), serotonin and sphingosine-1-phosphate, among others (
Autologous activated platelet-rich plasma injection into adult human ovary tissue: Molecular mechanism, analysis, and discussion of reproductive response.
). These substances promote tissue repair, incite neoangiogenesis, have pro-inflammatory effects (Figure 2B) and activate the clearance of dead cells and the eradication of detritus (
Autologous activated platelet-rich plasma injection into adult human ovary tissue: Molecular mechanism, analysis, and discussion of reproductive response.
). PRP is applied in multiple fields, including periodontal and oral surgery, orthopaedics, aesthetics, burns treatment, ophthalmology and spinal, cardiac, and trauma surgery (
In the context of ovarian tissue, PRP could play an important role in folliculogenesis and ovulation by providing an appropriate environment to support follicle selection and growth (
) in the same way that platelets are involved in wound repair and tissue regeneration after cell deterioration. Indeed, cyclic injury in the ovary following ovulation could be repeatedly healed by platelets (
). PRP cytokines have proangiogenic properties; for example, VEGF and PDGF are involved in the increased vascularization observed during follicular maturation until final ovulation (
The vascular endothelial growth factor (VEGF)/VEGF receptor 2 pathway is critical for blood vessel survival in corpora lutea of pregnancy in the rodent.
), which show a significant increase in peritoneal VEGF. In contrast, antagonism of VEGF and PDGF in animals induces reduced vascularization and increased apoptosis, leading to impaired folliculogenesis and atresia (
Inhibition of platelet-derived growth factor (PDGF) receptor affects follicular development and ovarian proliferation, apoptosis and angiogenesis in prepubertal eCG-treated rats.
Autologous activated platelet-rich plasma injection into adult human ovary tissue: Molecular mechanism, analysis, and discussion of reproductive response.
An advantage of PRP is that it can be easily obtained by minimal intervention. Autologous peripheral blood is isolated from the patient, and centrifuged to separate PRP from platelet-poor plasma, ensuring a platelet concentration up to 10 times greater than the circulating concentration (
). Various commercial PRP kits are available, providing predetermined platelet concentrations. However, the use of different PRP concentrations can lead to protocol deviations and heterogeneity in results.
‘Platelet activation’ is proposed as an important step to promote platelet growth factor release (
Autologous activated platelet-rich plasma injection into adult human ovary tissue: Molecular mechanism, analysis, and discussion of reproductive response.
). Although the role of platelet activation has not been fully established, platelet activation yields significant platelet degranulation with growth factor discharge and pro-inflammatory cytokine release (
). Several techniques exist to activate platelets, including ADP, thrombin, collagen, calcium chloride, calcium gluconate or a combination of these substances (
Autologous activated platelet-rich plasma injection into adult human ovary tissue: Molecular mechanism, analysis, and discussion of reproductive response.
). In clinical practice, fine ultrasound-guided needles help to introduce PRP into the human ovary. PRP is ideally injected directly into the ovarian cortex, to favour penetration due to its large molecular weight (
), allowing an interaction with receptors and ligands, activating inter- and intracellular signalling pathways, and consequently enhancing cell and follicular differentiation (
Although mechanistic experimental studies are limited, preclinical trials have been accomplished. Hosseini and colleagues evaluated the effect of PRP on primordial follicles, isolated from previously healthy deceased woman and embedded in a PRP-enriched 3D matrix (previously activated with thrombin), detecting follicle growth after 10 days in culture (
). For a better understanding of the mechanism of action of PRP in ovarian repair and to clearly distinguish its benefits in different pathologies, the latest studies with separation of the pathologies are discussed here. However, not all investigations operate with the same PRP protocol, as different volumes are applied and different activation techniques are used, and some investigations do not use PRP activation. This could explain the heterogeneity of results even when the studied populations have similar characteristics.
The first clinical data on PRP were reported as a pilot study in four women with more than 1 year of infertility, with at least one failed or cancelled assisted reproductive technology (ART) treatment, or amenorrhoea lasting for at least 3 months (
First data on in vitro fertilization and blastocyst formation after intraovarian injection of calcium gluconate-activated autologous platelet rich plasma.
). A significant reduction in FSH was observed with MII oocytes retrieved from all participants, which finally lead to one blastocyst per patient. One woman achieved pregnancy (still ongoing at the time of publication) and the other three women decided to freeze their embryos for future frozen embryo transfers.
Another investigation reported a 15.8% pregnancy rate (three clinical pregnancies including two natural conceptions) after bilateral PRP infusion in 19 patients with DOR (
). The results showed a significant increase in the number of oocytes retrieved in ART treatments when compared with cycles before the intervention, with natural conceptions occurring 2–4 months after the PRP intervention. Similar results were obtained in a retrospective study with the same PRP protocol in 96 women with POR according to the Patient-Oriented Strategies Encompassing IndividualizeD Oocyte Number (POSEIDON) criteria for POR (
Evaluation of intra-ovarian platelet-rich plasma administration on oocytes-dependent variables in patients with poor ovarian response: A retrospective study according to the POSEIDON criteria.
). Although there were no significant differences in hormone concentrations after treatment, there was a significant increase in the number of oocytes retrieved and an increase in MII oocytes in all POSEIDON groups. Although the overall pregnancy rate was 14.6%, young participants within group 1 benefited most from the intervention (71.4% pregnancy rate) (
Evaluation of intra-ovarian platelet-rich plasma administration on oocytes-dependent variables in patients with poor ovarian response: A retrospective study according to the POSEIDON criteria.
A large non-randomized clinical trial of 83 women with low ovarian reserve allocated patients into two different groups, with 46 receiving activated PRP injections for three consecutive cycles, and the rest receiving no intervention. After a 3-month follow-up, participants receiving PRP showed a significant decrease in FSH and a significant increase in AMH and AFC (compared with the control group). The biochemical and clinical pregnancy rate in the PRP group was statistically significantly higher than in the control group (26.1% versus 5.4%, P < 0.02; and 23.9% versus 5.4%, P < 0.03, respectively), as was the number of live births (5 versus 1) with no statistical differences for miscarriage rate. The researchers concluded that PRP improved the number and quality of the oocytes retrieved, which will lead to a superior quality embryo cohort (
The use of autologous platelet-rich plasma (PRP) versus no intervention in women with low ovarian reserve undergoing fertility treatment: a non-randomized interventional study.
Preliminary report of intraovarian injections of autologous platelet-rich plasma (PRP) in extremely poor prognosis patients with only oocyte donation as alternative: a prospective cohort study.
The effects of intra-ovarian autologous platelet rich plasma injection on IVF outcomes of poor responder women and women with premature ovarian insufficiency.
Preliminary report of intraovarian injections of autologous platelet-rich plasma (PRP) in extremely poor prognosis patients with only oocyte donation as alternative: a prospective cohort study.
Ovarian reserve parameters and IVF outcomes in 510 women with poor ovarian response (POR) treated with intraovarian injection of autologous platelet rich plasma (PRP).
). Ovarian reserve parameters showed a statistically significant increase in AFC, a rise in serum AMH and a decrease in serum FSH values after PRP; the improvement of ovarian reserve markers was positively correlated with the likelihood of producing at least one viable embryo. The natural conception rate was 4.3% (22/510), while the pregnancy rate after IVF was 26.6% (83/312) among women with embryo formation. Overall, PRP injection resulted in a 20.6% pregnancy rate in this cohort (105/510), with a total of 66 live births, 12 from natural conceptions and 54 after IVF. Interestingly, this study established that women aged 38 years or less benefited the most from PRP, as 40 years of age (sensitivity 48.3, specificity 70.9; area under the curve [AUC] 0.612) was the cut-off for the those who would not benefit due to an absence of ovarian response, and age over 38 years increased the risk of miscarriage (sensitivity 61.5, specificity 73.8; AUC 0.705).
Navali and co-workers also conducted a clinical study in 35 participants with POR (
Intraovarian Injection of Autologous Platelet-Rich Plasma Improves Therapeutic Approaches in The Patients with Poor Ovarian Response: A Before-After Study.
). After a 2-month follow up, they identified a significant increase in the number of oocytes and embryos obtained after IVF. Moreover, there was a significant increase in serum oestradiol concentrations after PRP treatment, while FSH and AMH concentrations presented no significant surge after a single injection of PRP. Three women reported a natural conception 4 months after the PRP treatment.
A recent investigation suggests that PRP infusion did not increase ovarian reserve but instead had a positive effect on embryo euploidy rate (
). This study included women with at least one previous failed IVF cycle and/or who produced blastocysts both before and after intra-ovarian PRP treatment. Although neither FSH, AMH nor AFC significantly improved after PRP, the euploidy rate increased from only 8% to 42.8% after PRP infusion. Similarly, a normalization of the embryonic chromosome content after PRP treatment was reported in a case study of a 42-year-old patient with six IVF previous attempts and a total of 20 aneuploid embryos (100% aneuploidy rate), who achieved an euploid embryo after PRP ovarian infusion followed 5 months later by an injection of isolated PDGF (
Normalized Ploidy Following 20 Consecutive Blastocysts with Chromosomal Error: Healthy 46, XY Pregnancy with IVF after Intraovarian Injection of Autologous Enriched Platelet-derived Growth Factors.
A new approach of using platelet-rich autologous plasma to increase the ovarian reservoir in a Syrian patient with ovarian insufficiency: A case report.
) reported increased AMH concentrations (0.39 ng/ml versus 0.94 ng/ml) 15 days after PRP injections into different points of the reproductive system including the ovaries, cervix and uterus, and intramuscularly (the thighs).
PRP treatment in women with POI is different because this group have amenorrhoea or oligomenorrhoea, high FSH concentrations and no antral follicles to be stimulated with exogenous gonadotrophins. A recent pilot study was performed in 311 women with POI who were less than 40 years age (
Effects of intraovarian injection of autologous platelet rich plasma on ovarian reserve and IVF outcome parameters in women with primary ovarian insufficiency.
) and in whom intra-ovarian PRP infusion into at least one ovary was performed. This investigation showed promising results, with a total pregnancy rate of 11.6%: 23 pregnancies were achieved naturally, while 13 were achieved after IVF (pregnancy rate of 22.8% per transfer, 4% of the total). Altogether, 8% accomplished a live birth, while another 8% were cryopreserving embryos at the time of publication. Interestingly, 201 participants showed follicular growth (64.6%) and were able to undergo ART. Additional case reports in POI also describe pregnancies (
Live Birth in Woman With Premature Ovarian Insufficiency Receiving Ovarian Administration of Platelet-Rich Plasma (PRP) in Combination With Gonadotropin: A Case Report.
), in eight perimenopausal women of advanced maternal age, showed a restoration of menstruation in all women 1–3 months after treatment, with oocyte retrieval and vitrification of embryos. Three years later, the same group described similar results in three women: one postmenopausal patient and two women with POI (
A Case Series on Natural Conceptions Resulting in Ongoing Pregnancies in Menopausal and Prematurely Menopausal Women Following Platelet-Rich Plasma Treatment.
Reactivating ovarian function through autologous platelet-rich plasma intraovarian infusion: Pilot data on premature ovarian insufficiency, perimenopausal, menopausal, and poor responder women.
) showed that PRP infused on day 3 of the menstrual cycle, except in amenorrhoeic women, who received it on a random day, improved ovarian reserve biomarkers and the number of oocytes retrieved in women with DOR. In the first group, ovarian reserve biomarkers improved, especially in the first cycle after the intervention, and ICSI performance was enhanced after PRP treatment, with a 46.6% pregnancy rate (compared with no pregnancies before the intervention). In participants with POI, 60% of the women recruited had a restoration of their menstrual cycle, in addition to an improvement in hormone concentrations (AMH and FSH) and AFC. The pregnancy rate was 10%, and all were natural conceptions. In perimenopausal patients recovery of menses occurred in 80%, along with a reduction in FSH. The natural conception rate was 13.3%, followed by three live births. Restoration of menses was observed in 43.3% of the postmenopausal group, with hormone improvement and a 3.3% pregnancy rate with one live birth. The authors concluded that results were more optimistic in women with DOR and perimenopause than with POI and menopause.
In summary, findings in patients with DOR show a restoration of ovarian function in the first 2–3 months after a PRP intervention (
Ovarian reserve parameters and IVF outcomes in 510 women with poor ovarian response (POR) treated with intraovarian injection of autologous platelet rich plasma (PRP).
The use of autologous platelet-rich plasma (PRP) versus no intervention in women with low ovarian reserve undergoing fertility treatment: a non-randomized interventional study.
A new approach of using platelet-rich autologous plasma to increase the ovarian reservoir in a Syrian patient with ovarian insufficiency: A case report.
First data on in vitro fertilization and blastocyst formation after intraovarian injection of calcium gluconate-activated autologous platelet rich plasma.
The effects of intra-ovarian autologous platelet rich plasma injection on IVF outcomes of poor responder women and women with premature ovarian insufficiency.
), similar to that described after stem cell treatment. This is likely to be due to the rescue of secondary and pre-antral follicles, because the PRP stimulus prevents follicle atresia under normal conditions in poor-responder patients (
The use of autologous platelet-rich plasma (PRP) versus no intervention in women with low ovarian reserve undergoing fertility treatment: a non-randomized interventional study.
Evaluation of the ovarian reserve in young low responders with normal basal levels of follicle-stimulating hormone using three-dimensional ultrasonography.
). Follicle growth seen in both POI and perimenopausal or menopausal women is linked to primordial follicle stimulation. However, hormone improvement is not evident in all patients, and some still presented with amenorrha during the follow-up (
Reactivating ovarian function through autologous platelet-rich plasma intraovarian infusion: Pilot data on premature ovarian insufficiency, perimenopausal, menopausal, and poor responder women.
Recent investigations have shown promising results (Table 3). However, the studies use different PRP volumes and activation protocols, and the population characteristics are heterogeneous, which is likely to lead to a variation in outcomes. This makes it difficult to obtain standardized protocols for future studies. Moreover, many of the investigations described are case reports with low-quality data, and controlled clinical trials are missing. Furthermore, as current clinical trials also lack a control group, some outcomes, such as hormone improvement or pregnancy rate after ART, may be inconclusive because some individuals conceive naturally while others opt to store cryopreserved embryos.
Table 3Investigations assessing the effect of intra-ovarian PRP injection for follicular activation, according to the diagnoses of POR and POI, and in premenopausal and post-menopausal patients
First data on in vitro fertilization and blastocyst formation after intraovarian injection of calcium gluconate-activated autologous platelet rich plasma.
Non-significant increase in serum AMH (P = 0.17) and decrease in FSH (P < 0.01) in all cases MII oocyte retrieval (5.3 ± 1.3 MII oocytes) At least one blastocyst per patient 1 pregnancy (9 weeks gestational age)
The use of autologous platelet-rich plasma (PRP) versus no intervention in women with low ovarian reserve undergoing fertility treatment: a non-randomized interventional study.
Prospective controlled, non-randomized comparative study (n = 83) 1. 46 patients with PRP infusion 2. 37 patients with no intervention
200 μl of activated PRP per ovary
0.1 ml of 10% calcium chloride
46/83 patients received the PRP infusion Significant FSH decrease (33%) and increase in AMH (63%) and AFC (75%) in the PRP group 1.5 × more oocytes retrieved after IVF in the PRP group Higher rate of medium- and top-quality embryos in the PRP group (100% versus 55%) 13 pregnancies in the treatment group versus 2 in the control group 5 live births in PRP group, 2 natural conceptions, versus 1 in the control group after IVF
Reactivating ovarian function through autologous platelet-rich plasma intraovarian infusion: Pilot data on premature ovarian insufficiency, perimenopausal, menopausal, and poor responder women.
Improvement of AMH and AFC on first menstrual cycle after PRP ICSI in 96.6% of patients 101 oocytes retrieved, producing 58 embryos 14 pregnancies with 2 miscarriages and 12 live births
Evaluation of intra-ovarian platelet-rich plasma administration on oocytes-dependent variables in patients with poor ovarian response: A retrospective study according to the POSEIDON criteria.
No statistical differences in hormone levels after treatment Increase in MII oocytes retrieved in POSEIDON groups 1, 3 and 4 14 clinical pregnancies, higher pregnancy rate in POSEIDON group 1
Bilateral PRP intra-ovarian infusion in 18 patients. In 2 patients, infusion during a laparoscopic intervention (
AMH and AFC increased in the first and second menstrual cycles after intervention IVF cancellation rate was decreased from 40% before to 15% after PRP Increase in the number of oocytes and embryos obtained after PRP versus previous IVF cycles 2 pregnancies with 2 live births, 1 pregnancy being a natural conception
Ovarian reserve parameters and IVF outcomes in 510 women with poor ovarian response (POR) treated with intraovarian injection of autologous platelet rich plasma (PRP).
POR according to POSEIDON criteria Mean age 40.3 years (30–45)
Prospective observational study (n = 510)
PRP injected in both ovaries (dose not reported)
No
Statistically significant increase in AFC (4.2 ± 2.4 versus 2.6 ± 1.3) 105 pregnancies: 22 natural conceptions and 87 pregnancies after IVF 66 live births, 54 after IVF and 12 from natural conception
Preliminary report of intraovarian injections of autologous platelet-rich plasma (PRP) in extremely poor prognosis patients with only oocyte donation as alternative: a prospective cohort study.
Patients with poor prognosis after failed IVF, 54 with regular menses, 26 with oligomenorrhoeaMean age 44.2 ± 5.4 years
Prospective cohort study (n = 80)
1.5 ml of PRP per ovary
No
No amelioration of hormone concentrations (oestradiol, FSH or AMH) after PRP treatment 6 clinical pregnancies after IVF, with 2 ongoing pregnancies and 2 live births
Intraovarian Injection of Autologous Platelet-Rich Plasma Improves Therapeutic Approaches in The Patients with Poor Ovarian Response: A Before-After Study.
After a 2-month follow-up, patients displayed a significant elevation in the number of oocytes (3.68 ± 0.24 versus 2.22 ± 0.13) and embryos obtained (3.17 ± 0.14 versus 1.41 ± 0.13) compared with previous levels Significant increase in oestradiol levels (404.1 ± 16.76 versus 237.7 ± 13.14 pg/ml) 3 spontaneous pregnancies (4 months after PRP treatment)
Bilateral PRP intra-ovarian infusion (Tülek et al., 2022)
POR Mean age 38.1 ± 4.4 years
Retrospective analysis (n = 50)
2 ml of PRP per ovary
No
Significant increase in number of oocytes and embryos obtained following IVF Cancellation rate lower following PRP 7 clinical pregnancies and 4 live births
Women with at least one failed IVF treatment Mean age 40.08 ± 1.46 years
Prospective pilot study (n = 12)
4 ml of PRP per ovary
No
No significant changes in FSH and AMH levels, or AFC value after PRP Before PRP 3/37 embryos analysed were euploid, after PRP 12/28 embryos analysed were euploid
Bilateral PRP intra-ovarian infusion – 5 months later followed by an injection of isolated platelet-derived growth factors prepared as an enriched, cell-free substrate (Sills et al, 2019)
Advanced age – low ovarian reserve (42 years)
Case report (n = 1)
Not reported
No
1 euploid embryo after treatment 20 aneuploid embryos prior to treatment
PRP injection into different points of the reproductive organs via laparoscopy (
A new approach of using platelet-rich autologous plasma to increase the ovarian reservoir in a Syrian patient with ovarian insufficiency: A case report.
Menstrual cycle 6 weeks after PRP, with: 1. AMH improvement (from 0.02 to 0.08 ng/ml) 2. FSH decrease (from 149 to 27 mIU/ml) 1 pregnancy after a natural cycle, with spontaneous miscarriage in week 5
Bilateral PRP intra-ovarian infusion, combined with recombinant 150 IU of FSH + 75 IU LH (Pergoveris) (
Live Birth in Woman With Premature Ovarian Insufficiency Receiving Ovarian Administration of Platelet-Rich Plasma (PRP) in Combination With Gonadotropin: A Case Report.
A Case Series on Natural Conceptions Resulting in Ongoing Pregnancies in Menopausal and Prematurely Menopausal Women Following Platelet-Rich Plasma Treatment.
Restoration of menses FSH decrease: First patient: from 65 to 10 mIU/ml in the first menstrual cycle, 2 months following PRP Second patient: from 46.5 to 15.0 mIU/ml in the 5th month AMH increase 2 naturally conceived ongoing pregnancies
Bilateral or unilateral PRP intra-ovarian infusion (
Effects of intraovarian injection of autologous platelet rich plasma on ovarian reserve and IVF outcome parameters in women with primary ovarian insufficiency.
201 patients attempted IVF, and oocyte retrieval was performed in 130 82 women obtained at least one cleavage-stage embryo 57 patients underwent ET (for the remaining 25 patients embryos were vitrified) 36 pregnancies, 23 natural conceptions and 13 conceptions after IVF. 9 live births at the time of publication
Reactivating ovarian function through autologous platelet-rich plasma intraovarian infusion: Pilot data on premature ovarian insufficiency, perimenopausal, menopausal, and poor responder women.
Restoration of menstrual cycles in 18 of the 30 POI patients (success group): this subgroup presented a statistically significant improvement in hormone levels (AMH and FSH) and AFC 3 natural conceptions with 3 live births
Bilateral PRP intra-ovarian infusion (Tülek et al.,2022)
POI Mean age 37.9 ± 1.9 years
Retrospective analysis (n = 21)
2 ml of PRP per ovary
Not reported
Menses restored in 10 patients 8 embryos obtained and transferred, but no pregnancy
Reactivating ovarian function through autologous platelet-rich plasma intraovarian infusion: Pilot data on premature ovarian insufficiency, perimenopausal, menopausal, and poor responder women.
A Case Series on Natural Conceptions Resulting in Ongoing Pregnancies in Menopausal and Prematurely Menopausal Women Following Platelet-Rich Plasma Treatment.
Restoration of regular menses since the first month after treatment FSH decrease (from 119 to 27 mIU/ml in the fifth month after PRP) AMH and AFC increased 1 naturally conceived ongoing pregnancy
Reactivating ovarian function through autologous platelet-rich plasma intraovarian infusion: Pilot data on premature ovarian insufficiency, perimenopausal, menopausal, and poor responder women.
Restoration of menses was observed in 13 of 30 patients (success subgroup): in these patients there was statistical significant hormone improvement (AMH, FSH, LH) 1 natural conception with 1 live birth
However, the conclusion that PRP treatment could be an alternative treatment for women with DOR is viable based on the increased pregnancy rates reported in the literature even though randomized controlled trials are lacking. Whereas the results are more encouraging in women with patients, optimistic outcomes were also obtained in those with POI and established menopause, whose reproductive prognosis is worse than that of DOR patients. In the future, PRP as an alternative therapy should be further investigated. There are several ongoing trials involving PRP injection to treat impaired ovaries (NCT04797377, NCT04275700, NCT04149028, NCT03542708 and NCT02992756), some of which already include a proper control group (NCT03937661, NCT05181748, NCT04922398 and NCT04381299), while others aim to compare PRP with PPP (NCT04278313) and another is combining PRP with stem cells (NCT04444245).
Future insights into the mechanisms governing follicular dynamics and oocyte ageing
Ovarian reactivation techniques focus largely on strengthening the process of follicle activation and growth. However, none has shown a relevant effect on increasing oocyte quality, and they rather assess aneuploidy rate by preimplantation genetic testing for aneuploidies, showing values according to patient age (
Ovarian reserve parameters and IVF outcomes in 510 women with poor ovarian response (POR) treated with intraovarian injection of autologous platelet rich plasma (PRP).
). Increasing follicle growth and final oocyte yield increases the possibility of having a good-quality oocyte to produce a euploid and transferable healthy embryo (
). However, because the developmental potential of early embryos relies on oocyte quality, strategies to modify oocyte and embryo quality are increasingly requested as both parameters significantly decline with age (
The nature of aneuploidy with increasing age of the female partner: a review of 15,169 consecutive trophectoderm biopsies evaluated with comprehensive chromosomal screening.
Chromosomal and cytoplasmic context determines predisposition to maternal age-related aneuploidy: brief overview and update on MCAK in mammalian oocytes.
) because mitochondria provide the energy needed for these processes. However, developing a healthy embryo also requires input from other organelles and molecules within the ooplasm machinery that is impaired as age advances (
To overcome this, maternal spindle transfer (MST) was developed in a preclinical study and set up in human oocytes to provide the appropriate cytoplasmic and mitochondrial machinery from a donor to properly develop a euploid embryo using oocytes from different sources with minimal heteroplasmy (
Spindle transfer can enhance the potential of developmentally compromised human oocytes to reach the blastocyst stage: proof of concept with donor oocytes. 34rd Annual Meeting of the European Society of Human Reproduction and Embryology.
First registered pilot trial to validate the safety and effectiveness of maternal spindle transfer to overcome infertility associated with poor oocyte quality.
) in a trial with human oocytes from 25 donors with previous massive embryo arrest, leading to increased fertilization rates after ICSI and to the production of good-quality blastocysts (60.5%) with an overall 50% euploidy rate and less than 1% mitochondrial DNA carryover in the offspring. MST produced at least one good-quality blastocyst in 65% of women, allowing nine embryo transfers and resulting in six clinical pregnancies leading to two live births (and three ongoing pregnancies) in a very poor-prognosis population due to embryo arrest (
First registered pilot trial to validate the safety and effectiveness of maternal spindle transfer to overcome infertility associated with poor oocyte quality.
Recent reports have highlighted the involvement of DNA damage response and repair, metabolism and apoptosis-regulating pathways and genes in the control of ovarian ageing (
), supporting the fact that a variation in cell death after DNA damage response is a crucial mechanism in this process (Figure 3). The competence of follicular cells and oocytes to respond to DNA insults is critical to ensure their viability. In fact, prophase-arrested oocytes, especially those enclosed in dormant primordial follicles, retain the ability to repair exogenous damage through homologous recombination to ensure the transmission of an intact genome if apoptosis activation allows it (
Figure 3Molecular pathways related to oocyte quality with translational potential to extend the ovarian lifespan. Oocyte quality is affected by several factors such as age, disease, impaired bioenergetics, oxidative and metabolic stress, and exogenous agents. Different approaches have been proposed to recover oocyte quality: restoring telomere length and telomerase activity with pharmacological treatments; balancing metabolic and oxidative stress in early-stage oocytes through nicotinamide adenine dinucleotide (NAD+) repletion; improving mitochondrial function and supplementing the oocyte with autologous good-quality mitochondria; maternal spindle transfer to provide the oocyte with an adequate cytoplasmic and mitochondrial machinery from a donor to properly develop a euploid embryo; and priming the DNA damage and repair mechanisms to avoid follicle atresia induced by double-strand breaks by lowering CHEK2 activity to increase the time frame for DNA repair by homologous recombination when possible. Created with BioRender.com.
This pathway involves a conserved DNA damage response checkpoint kinase ATM–CHEK2 (ataxia-telangiectasia mutated–checkpoint serine-threonine kinase 2). The role of CHEK2 is to integrate and determine repair and cellular responses from several DNA repair pathways and to promote apoptosis in response to double-strand breaks (
). CHEK2 reduces the threshold of apoptosis activation in damaged primordial oocytes, allowing DNA repair and therefore reducing follicular depletion. Indeed, carriers of CHEK2 loss-of-function variants showed natural menopause 3.5 years later than non-carriers (
), supporting the fact that reduced CHEK2 activity allows oocyte restoration and increases reproductive lifespan. Aged Chek2–/– knockout mice showed increased ovarian reserve, response to ovarian stimulation and reproductive potential even with a normal follicular reserve at birth (
). This finding might be related to reduced follicular death and atresia as a consequence of lowered CHEK2 levels to allow the rescue of a small number of primordial follicles, suggesting a new strategy for future therapies.
Oocyte mitochondrial bioenergetics
Double-strand breaks are caused by exogenous genotoxic agents as well as by physiological oxidative and metabolic stress due to age (
The nature of aneuploidy with increasing age of the female partner: a review of 15,169 consecutive trophectoderm biopsies evaluated with comprehensive chromosomal screening.
). Indeed, early-stage oocytes and granulosa cells from aged non-human primate ovaries and aged human granulosa cells showed a down-regulation of antioxidant genes such as IDH1, PRDX4 and NDUFB10, increased oxidative damage and apoptosis (
). The observation that impaired bioenergetics and oxidative stress appeared in early-stage oocytes might explain the ineffectiveness of treatments intended to restore mitochondrial bioenergetic balance in mature oocytes during ICSI through the transfer of autologous mitochondria, as these treatments were applied when the damage has already been established in early development (
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.
To amend treatment timing, a pharmacological intervention to restore metabolic and oxidative stress balance in early-stage oocytes was proposed based on the repletion of the redox cofactor nicotinamide adenine dinucleotide (NAD+/NADH), whose deficiency is related to several age-associated pathologies (
). In the context of ovarian tissue, the age-associated decline in NAD+ is more prone to affect the oocyte rather than other ovarian cells. Oral administration of NMN for 4 weeks, a sufficient period for early-stage mouse follicles to activate, grow and mature (
), improved final oocyte yield after ovarian stimulation, spindle assembly, blastocyst formation and euploidy rates in aged mice due to NAD+ repletion in oocytes (
). These encouraging results were confirmed in breeding trials showing improved pregnancy rates of healthy offspring, highlighting the absence of deleterious effects associated with NMN supplementation. Moreover, the authors evaluated the role of NMN supplementation for in-vitro maturation and embryo culture and observed a restoration of oocyte quality.
These findings, together with other reports supplementing several NAD precursors (
), suggest a new approach to restoring the metabolic and oxidative balance of primordial follicles to obtain competent oocytes to produce implantable embryos for aged and young women with poor-quality embryos (Figure 3). Indeed, the ability of primordial follicles to remain dormant but viable and metabolically active for decades relies on their competence to limit reactive oxygen species production through the inhibition of the NADH dehydrogenase (complex I) of the respiratory chain (
Among the factors determining IVF success in ovarian and age-related infertility, telomeres are an attractive target. Telomeres are specialized structures at the ends of chromosomes whose main function is to preserve genome integrity and avoid chromosome fusions (
). Interestingly, the oocyte and surrounding granulosa cells have shown differences in telomere length and telomerase activity, which is especially relevant in granulosa cells due to their impressive proliferation rate. Indeed, a compromised telomere pathway has been found in granulosa (
Impaired telomere length and telomerase activity in peripheral blood leukocytes and granulosa cells in patients with biochemical primary ovarian insufficiency.
Impaired telomere length and telomerase activity in peripheral blood leukocytes and granulosa cells in patients with biochemical primary ovarian insufficiency.
) from young women with impaired ovarian function.
Telomere-related parameters at the time of oocyte retrieval are predictive variables of IVF outcomes and aneuploidy. A higher relative telomere length has been detected in the surrounding cumulus cells of oocytes that fertilized and developed into a good-quality day 3 embryo (
), in which breakpoints that resulted in telomere shortening in mature oocytes led to failed fertilization or the failed development of an implantable healthy embryo (
). Telomere-lengthening capability relies on the oocyte, as demonstrated by the telomere elongation observed in parthenogenetically activated oocytes (
). Thus, follicular cells can be considered as a potential target for this type of therapy, which promotes telomere lengthening in order to improve the outcomes of impaired and aged oocytes (Figure 3).
Plasma- and growth factor-based therapies to restore folliculogenesis and oocyte quality
The human ovary shows senescence and ageing signs early in life compared with other organs (Richardson et al., 2014;
), with a sudden decrease of ovarian reserve after the mid-30s. Although systemic ageing effects seem to manifest around a woman's 60s, a recent characterization of the human plasma proteome identified three different waves of ageing defined by significant changes in protein composition at 34, 60 and 78 years of age (
). Proteomic changes observed around 34 years of age involved a higher number of regulated proteins when compared with those occurring at 60 years and therefore could be involved, or at least be connected, with the early onset of ovarian ageing. Thus, the use of ‘omics’ analyses in ageing studies provides new and useful insights for anti-ageing therapies.
Induction of tissue repair by introducing young growth factor-enriched plasma into damaged and aged organisms (
). The current authors’ recent work seeking to evaluate the potential of paracrine signalling of different stem cell sources in combination with additional platelet-enclosed factors showed that this can be a feasible and less invasive option to reactivate ovarian function (
). Although plasma treatments enriched in BMDSC or umbilical cord stem cells reactivated follicle growth in POI mouse models, BMSDC had a more potent effect in recovering oocyte quality and short- and long-term fertility.
Moreover, the activation process to release additional factors enclosed within platelets increased the effects, which were especially relevant in BMDSC-rich plasma. This difference might be due to a variation in the platelet secretome according to platelet origin (
). These results were also validated in human ovarian biopsies of women with POR xenografted into immunodeficient mice, which is currently being evaluated in an ongoing randomized clinical trial of women with POI that includes a non-treated control group (NCT04475744). The proteomic analysis of this plasma revealed the presence of soluble factors related to cell cycle/apoptosis, gene expression, signal transduction, cell communication, response to stress, signalling by RhoGTPases, nuclear receptors, NOTCH (notch receptor), PDGF and WNT (Wnt family member), and DNA repair of DBS (
). These findings may be relevant to age-induced ovarian damage in addition to chemotherapy insults as a link between reduced oxidant stress response, cell communication, metabolism of proteins and ovarian ageing was recently described (
While these approaches are based on the rescue, repair and reactivation of existing follicles in damaged or senescent ovaries following senotherapeutic interventions (
), there are cases where access to germ cells is not an option and therefore a completely different approach should be taken.
De-novo generation of oocytes
In recent decades, differentiation of stem cell types into oocytes has been a challenge. Nevertheless, a series of cutting-edge studies to differentiate mouse embryonic and induced pluripotent stem cells into fertilizable oocytes (
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