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26. Activation of Ovarian Cortex
Keywords
Premature ovarian insufficiencyOvarian cortical tissue freezingOvarian cortex activationOvarian cortical autotransplantationOogonial stem cellsBone marrow mesenchymal stem cells and ovaryDiminished ovarian reserve26.1 Ovarian Autotransplantation
Ovarian cortical tissue freezing before life-saving treatment in young females has found worldwide acceptance as an important fertility preservation option and currently; it is the sole option for prepubertal female children. The main intention of ovarian cortical tissue freezing is to perform the autotransplantation of such tissues for induction of puberty and to restore fertility [1–4].
Accidental autografting of ovarian tissue to the laparoscopic umbilical incision site was reported after removing the ovarian endometrioma specimen out of the pelvis by using the umbilical port site [5]. Ovarian tissue could regain its function with neovascularization without requiring a vascular pedicle. Although this was reported to be the first case and quoted by many articles concerning ovarian autotransplantation, the history of ovarian autotransplantation itself goes back to the early twentieth century.
The autotransplantation procedure used to be performed during hysterectomy and bilateral salpingo-oopherectomy. Transplantation of parts of one or both ovaries was performed into the abdominal wall. The postmenopausal (as described as “ablation”) symptoms since the performed operation were observed to be substantially relieved during the period that the grafts showed activity in three women. Ovarian autotransplantation was also performed into the wall of the uterus if hysterectomy was not performed, to preserve menstruation [6]. It was eloquently described that the ovaries were completely removed and wrapped in gauze and placed in a vessel containing normal salt solution at about 100 °F. Then a peritoneal pocket was created by blunt dissection between the peritoneum and the undersurface of the rectus muscle. The visually healthy looking ovarian tissue was used for grafting. The ovaries were cut into 2 by 2.5 cm discoid pieces, and two to three pieces were transplanted into those peritoneal pockets without requiring any sutures to keep the grafts in their locations. The authors believed that grafting ovarian tissue in multiple small tissue pieces provides better results since a larger surface area would assure a better blood supply [6]. It was reported that the average age of such women undergoing ovarian tissue autografting was 30.5 years, the youngest being 20 years and the oldest being 41 years. Graft functioning was observed anywhere from 5 weeks to 6 months after the surgery. Indeed, there is a lot to learn from the history of medicine, as now older publications can become reachable electronically. Let us now review the present state of ovarian autotransplantation with the intention to activate ovarian cortex in women with premature ovarian insufficiency.
26.1.1 Applications of Lessons from History
In its landmark article, Pincus pointed out his observations in rabbits. He reported that removal of oocytes from the follicle itself resulted in maturation regardless of the presence or absence of pituitary hormones or of thyroxin in the culture medium. Therefore the maturation of the oocyte could be achieved simply by isolating it from its normal follicular environment which also leads to normal fertilization when exposed to sperm [7]. As mentioned above, it has already been shown that the ovarian tissue when autotransplanted in small pieces within a peritoneal pocket gets activated with follicle development, leading to diminished symptoms of menopause. In oncofertility studies, the activity of the frozen ovarian tissue could be restored more than 93% of the time after thawing and autotransplantation in small fragments and by repeating the autotransplantation procedure ovarian activity could be extended for over 11 years [8, 9].
Silber observed that even the vitrified ovarian tissue autotransplantation was performed in large cortical strips, extended graft survival can be achieved. Contrary to the common belief and to the older models of ovarian aging, increased primordial follicle loss with follicle growth leads to lower oocyte reserve but with eventual slower gradual decline of the ovarian reserve which may lead to extended functioning of the autotransplanted thin but unfragmented strip of the ovarian cortex. This may well be related to the relief of ovarian stromal and medullary pressure to the ovarian cortex, which is achieved through autotransplantation. Hence, such an extended graft survival can even be helpful to physiologically postpone the menopause in females [10, 11].
26.1.2 Relevance to Diminished Ovarian Reserve and/or Premature Ovarian Insufficiency-Associated Gonadotropin Increase
Some clinical data suggest that DOR is not only associated with disturbed ovarian – pituitary and hypothalamic axis – but also with increased gonadotropin levels, which results in some changes in the ovary itself resulting in a forward feedback loop of further deterioration of ovarian function. Hypergonadotropic levels of FSH, mostly above 20 IU/L, may be associated with abnormal follicle growth dynamics, while desensitization of the G protein-coupled receptors may also ensue. Hence, the prolonged suppression of FSH may decrease the forced early follicle selection and may restore the responsiveness of follicles to FSH when needed, perhaps through mitigation of desensitization [12, 13]. Therefore, suppression of FSH in the luteal phase before ovarian stimulation and following ovarian autotransplantation while waiting for ovarian function return was attempted with exogenous estrogen [14]. However, now the effects of chronically elevated LH levels on the ovarian stroma are also under scrutiny.
Ovarian stromal fibrosis can be seen in women with advanced reproductive age (ARA) with increased basal LH levels [15]. Increased risk of ovarian cancer was also associated with increased LH levels in women with polycystic ovary syndrome [16]. Increased stromal androgens with LH increase may result in ovarian stromal hyperplasia and fibrosis [17, 18]. In mice, unexpected accumulation of cells in the ovarian stroma with increased expressions of CYP17A1 (17OHlase/17–20 desmolase for glucocorticoid, androgen, and eventual estrogen production), CYP19A1 (aromatase for estrogen production from androgens), and LH/HCG receptors was noted to be due to increased LH levels [19, 20]. The ovarian stromal fibrosis in mice induced by both elevated LH and androgens was shown to be reversed by prolonged GnRH antagonist treatment [20]. It was also suggested that ovarian stromal fibrosis may impede secondary follicle or pre-antral follicle development, and this can be mitigated by prolonged GnRH antagonist treatment [20]. This aspect is important since there are proposed noninvasive methods to assess ovarian tissue rigidity associated with ovarian fibrosis [21] and treatment protocols can be refined accordingly. As reviewed in mild stimulation section, our prolonged GnRH agonist suppression protocol with estradiol priming until antral follicles are observed before mild stimulation as recommended in patients with profound DOR showing increased gonadotropins may reflect the mitigating factors achieved by prolonged suppression of FSH and LH.
Therefore, actually relieving the mechanical stress within the ovary via medical or surgical means may result in secondary follicle growth possibly through inhibition of the Hippo pathway via decreased phosphorylation of Yes-associated protein (YAP). This can be achieved surgically by cutting the ovary into fragments in vitro or in vivo via autotransplantation of the ovarian cortex [10, 11, 22].
26.1.3 How to Fit In Vitro Activation into This Paradigm
Hippo(potamus) pathway, although not well defined, controls the organ size by regulating cell proliferation, apoptosis, and stem cell self-renewal [23, 24]. It is involved in cell contact inhibition regulated by cytoskeleton and protein ubiquitination. It has been observed that protein kinase Hippo mutations, namely, MST-1/2, lead to organ overgrowth. Furthermore, dysregulation of Hippo pathway may result in cancer development. Hence Hippo signaling is considered as a tumor suppressor cascade [25]. Actually, some members of Hippo pathway like YAP (Yes-associated protein tyrosine kinase)/TAZ are considered as oncogenes, since they play roles in the programming of cancer stem cells with increased proliferation and inhibition of apoptosis. YAP/TAZ (transcriptional co-activator with PDZ binding motif) phosphorylation results in their inactivation by keeping them in the cytoplasm. Disrupted Hippo pathway leads to YAP/TAZ de-phosphorylation leading to their nuclear entry, acting on organ growth, cell proliferation, inhibition of apoptosis, and oncogenesis.
Mechanical stress created in the ovarian cortex may suppress follicle growth by inhibition of YAP/TAZ as part of the mechano-induction system in granulosa cells. With the same rationale, cutting ovarian cortex in pieces in vitro disrupts Hippo signaling. Polymerization of globular actin to filamentous actin was shown to result in Hippo signaling disruption. Fragmentation of ovarian tissue induces actin polymerization, which in turn is associated with nuclear localization of YAP in granulosa cells of primary and secondary follicles in mice [22]. As phosphorylated YAP decreases, nuclear levels for YAP increase. This leads to their interaction with transcription factors TAED, which leads to extracellular matrix enhancement of CCN growth factors that include cysteine-rich angiogenic protein 6 CCN1, connective tissue growth factor CCN2, nephroblastoma overexpressed gene CCN3, and 3 Wnt-induced secreted proteins. CCN growth factors stimulate cell growth and proliferation and inhibit apoptosis [26]. Therefore, the stimulation of secondary follicle growth in the ovary may be achieved through these mechanisms following the fragmentation of ovarian cortex or with decreased pressure to the ovarian cortex from stromal components.
26.2 Ovarian Cortical In Vitro Activation and Autotransplantation
It was observed that anovulation associated with polycystic ovary syndrome (PCOS) responds to surgically induced controlled ovarian injury via wedge resection or ovarian drilling [27, 28]. Although PCOS is associated with high rather than diminished ovarian reserve (DOR), the FSH sensitivity is restored in antral follicles leading to follicle growth and eventual ovulation after such ovarian tissue disruptive procedures.
It is widely believed that primary germ cell proliferation stops at around mid-gestation in the fetus and then germ cells arrested at prophase I of meiosis are surrounded by a layer of somatic granulosa cells to form primordial follicles. The number of primordial follicles may be between 1.5 and 2 million at birth but with follicle atresia decreases to about 400,000 at puberty, and there may be about 1000 primordial follicles still remaining at menopause [29]. In diminished ovarian reserve cases, primordial follicle pool is decreased, and more profound decrease is expected in the presence of premature ovarian insufficiency (POI). In POI cases, primordial follicles still exist as evidenced by the observation that 50% of POI cases may show intermittent ovarian function and 5–10% may conceive [30]. The women with POI may show a resumption of ovulatory function mostly within the first year of diagnosis. The offspring conceived from women who later develop POI or who were diagnosed with POI are reported to be healthy. Even, it was reported that the reproductive capacity of young women with POI (early 20s to early 30s) might be comparable to the women of the same age range [31, 32]. Therefore, primordial follicle can be activated to support follicle growth and ovulation leading to competent oocytes and to healthy offspring.
The stimulation of primordial follicle may be through another oncogenic pathway. It has been shown in mice that oocyte-specific deletion of phosphatase and tensin homolog deleted from chromosome 10 (PTEN) causes premature activation of primordial follicles leading to depletion of ovarian reserve and to an eventual premature ovarian failure [33]. PTEN is a major suppressor of phosphatidylinositol 3-kinase (PI3K) and PI3K induces eventual activation of Akt (acute transforming retrovirus thymoma protein kinase), a serine/threonine kinase also known as protein kinase B. Akt signaling, in general was found to be important in the growth of primordial follicles. PI3K activation in granulosa cells of mice by selective PTEN disruption was also shown to increase ovulated follicles while increasing the life span of corpora lutea [34]. Akt signaling stimulation has been shown to stimulate secondary follicle growth as well [22].
In mice, PTEN inhibitor bpV(HOPIC) was used transiently to activate primordial follicles to generate mature oocytes which were then subjected to IVF to achieve progeny mice [35]. Therefore, from mouse studies, it was suggested that ovarian cortical activation can be achieved in women with POI. Then the technique was translated into human ovaries.
Human ovarian tissue was obtained from women with POI via laparoscopy for in vitro activation based on Hippo signaling pathway disruption and Akt signaling stimulation, aided by autotransplantation followed by in vitro fertilization (IVF) [22]. Ovaries were removed by laparoscopy and then ovarian cortical tissue strips, which were 1–2 mm thick and measuring 1×1 cm, were frozen with vitrification. Then ovarian cortical strips were thawed and further fragmented into smaller sized (1–2 mm) pieces. Then these tissues were cultured with reversible inhibitor of PTEN bisperoxovanadium or bpV (dipotassium bisperoxo meaning 5-hydroxypyridine-2-carboxyl, oxovanadate, HOPIC) and Akt stimulator 740YP (Tocris) for 24 hours and then only with 740YP for another 24 hours followed by a final wash. Then 40 to 80 such pieces were autotransplanted beneath the fallopian tube serosa into a created peritoneal pouch via laparoscopy, and sutures were used to keep these fragments in place. Patients were followed by weekly or biweekly transvaginal ultrasound and serum estradiol and FSH levels. Out of 27 patients, 13 patients showed histological evidence of residual follicles, and 8 patients showed follicular activity. All eight patients showed histological presence of residual follicles in their tissue samples before autografting. All such women developed pre-ovulatory follicles (of about 20 mm in size) in less than 6 months of autotransplantation, and even some patients developed pre-ovulatory follicles in 3 weeks. Hence, secondary follicle development may be the reason for shorter follicle growth times since per common knowledge primordial follicle to pre-ovulatory follicle development phase may require at least 6 months [36]. When follicles reached >5 mm in diameters, FSH treatment was started and HCG trigger was used with the follicular size >16 mm. Oocyte retrieval was scheduled 36 hours after the HCG administration. Mature oocytes could be obtained from 5 women, which were fertilized with intracytoplasmic sperm injection. Three patients underwent embryo transfers. One did not get pregnant, the other one showed only elevated HCG level, and the last one, who was a 29-year-old patient at the time of ovarian removal with POI diagnosed at the age of 25, got pregnant with a singleton pregnancy and delivered a healthy baby at 37 weeks and 2 days with a birth weight of 3254 grams [22].
The same group had a follow-up report about the outcome of in vitro activation and autotransplantation [14]. They described 37 women with POI who underwent the process, 54% (n: 20) of which showed histological evidence of residual follicles before autotransplantation. Then among these 20 women with evidence of residual follicles, 9 showed follicle growth in autografts (45%). Oocyte retrieval was possible in 6 women with a total of 24 oocytes obtained. IVF with embryo transfer was possible in four women. Three had a positive pregnancy test but two had live birth. Hence, the live birth rate among women with histological evidence of residual follicles was 10% (2/20). Much lower rates would be calculated if the denominator were 37 women. The authors initially included the women with POI with a history of amenorrhea for more than 1 year and elevated serum FSH levels of >40 mIU/mL (n = 31). Then the last 6 women were included if they had a history of amenorrhea >4 months, and serum FSH levels of >35 mIU/mL (n = 6) to include POI patients with a shorter history of amenorrhea. Although more studies are required, the reported rates of ovarian activity and pregnancy rates are reminiscent of those reported in POI patients in general as discussed above (50% of POI cases may show intermittent ovarian function and 5–10% may conceive naturally).
26.2.1 Safety Concerns for In Vitro Activation
Since the report of live births with this technique, some arguments followed. It is not clear if the sole fragmentation of the ovarian tissue followed by autotransplantation can be adequate to activate the follicle growth across the board. It seems that Hippo signaling disruption is more relevant to activation of secondary to antral follicles as discussed above. For primordial follicle activation, the in vitro activation focused also on blocking PTEN action while enhancing Akt signaling, and it is not clear whether this step is really necessary due to the concerns about application of potentially oncogenic chemicals.
The data from ovarian cortical tissue autotransplantation after life-saving treatments suggest the primordial follicle growth follows autografting without needing in vitro activation [37, 38]. The main criticism about in vitro activation studies is that they did not include a control group solely with autotransplantation without in vitro activation [39].
There are also some concerns that in vitro activation before autotransplantation may actually decrease the graft longevity since generalized activation of resting follicle pool may result in follicle burnout. Another concern is about keeping the size of the autografted fragments very small in in vitro activation studies. It was suggested that the strips of thawed ovarian cortex resumes function better than the small fragments injected into the ovary [40]. As discussed above, it was observed that vitrification, thawing, and autotransplantation of long strips of ovarian cortical tissue resulted in extended graft survival and function [10, 11, 41]. Extended endocrine function of the graft is also important to mitigate health issues and consequences of early menopause. Naturally, the recruitment rate of follicles may be reduced when the ovarian reserve is decreased. However, with in vitro activation, follicle burnout seems to be accelerated. Hence, the patients undergoing in vitro activation before ovarian cortical tissue autotransplantation should look into immediate, short-term outcomes for follicle activation and pregnancy [39].
The researchers working on in vitro activation before autotransplantation in patients with POI also acknowledge that ovarian cortical tissue cryopreservation for fertility preservation is different than in vitro activation which is in fact for infertility treatment. They even recommend in vitro activation of the fresh ovarian cortex without vitrification and thawing, but just autotransplanting the ovarian cortical tissue fragments after 2 days of treatment [42]. Certainly, this approach may raise some other safety concerns that the growing follicles already exposed to these potentially toxic chemicals may result in a pregnancy. Then the authors stated that in vitro drug treatment could also be omitted and immediate autotransplantation could be undertaken if the tissues still show residual secondary and early antral follicles. Hence, the terms cryopreservation-free in vitro activation and drug- and cryopreservation-free in vitro activation were introduced quoting an ongoing pregnancy in China with the former. The latter method involving just ovarian cortical tissue procurement and autografting back in fragments was performed in women with poor ovarian response (POR) per Bologna criteria with the observation of an increased number of oocytes retrieved [42].
In terms of chemicals used for in vitro activation, the authors defend that ovarian cubes exposed to these chemicals for 2 days are rinsed extensively before grafting and no abnormal growth in the graft sites was observed under ultrasound. In addition, the patients getting their repeat autografting did not show any abnormalities in the past autografting sites as observed by laparoscopy [42]. Certainly, any long-term observational data is lacking to address such safety concerns at this time. However, there is a tendency to promote such approaches for women with DOR and POR and to those who underwent cryopreserved ovarian cortical tissue before their life-saving treatment [43].
On the other hand, human ovarian tissue fragments cultured with 1 μM of bpV (HOPIC) or control medium for 24 hours followed by culture only in control medium for both groups for another 5 days demonstrated that the follicle activation occurred in both treatment groups but with significantly more secondary follicles developed in the group treated with HOPIC. This increased activation with HOPIC was associated with increased Akt phosphorylation and increased nuclear localization of forkhead box O3 (FOXO3) as expected from PTEN inhibition. Nevertheless, isolated and cultured such follicles showed restricted growth and decreased survival if the ovarian tissue was exposed to HOPIC as compared to control tissues not exposed to the chemical. Therefore, PTEN inhibition promotes follicle activation toward the secondary stage but then severely compromises the survival of secondary follicles [44].
In summary, time will show if these surgical approaches with or without chemical treatment will, in fact, become widely accepted approaches for women with POI or profound DOR.
26.3 Platelet-Rich Plasma to Activate Ovarian Cortex
Platelet-rich plasma (PRP) concentrates have found some new uses in regenerative medicine. Platelets, in addition to playing role in hemostasis, secrete various products playing role in cell migration, proliferation, and differentiation and in angiogenesis and tissue repair [45, 46]. Therefore, PRP has been used to support tissue repair and regeneration in various clinical scenarios such as androgenic alopecia [47]. One such attempt was made on four DOR patients older than 35 years of age, with infertility and at least one failed or canceled IVF cycle or amenorrhea for at least 3 months. The authors prepared the PRP from 8 mL of blood collected from patients through a regular venipuncture followed by centrifugation for platelet separation; platelets were then activated by using calcium gluconate. Then PRP was injected into the ovary by using a 17 G, 35 cm single lumen needle while paying attention that about 1 ml of PRP sample was deposited just under the ovarian capsule. The procedure was applied to both ovaries. The patients did not require any sedation or anesthesia. All patients were then started with monitoring every 2 weeks. Especially cycle day 2 or 3, FSH and E2 testing were obtained after the procedure. When increased AMH and/or decreased FSH is noted, the patients were subjected to IVF. All four patients showed decreased FSH levels as compared to the levels before PRP injection. Also, three out of four patients showed an AMH level increase shortly after PRP injection. IVF resulted in at least one day five blastocysts in all four patients which were frozen for banking purposes in three patients. One patient underwent embryo transfer and had an ongoing pregnancy [48].
Since the anti-aging industry has thrived with similar statements, some of the fertility practices, worldwide, advertise for “ovarian rejuvenation” directed toward women with POI and those with DOR. The PRP applications along with ovarian autotransplantation methods as discussed above and stem cell approaches are involved with such promotions. We will then briefly mention about stem cells in relevance to the ovary.
26.4 Activation of Ovaries with Stem Cells and Other Proposed Stem Cell Involved Treatments
The presence of ovarian stem cells postulated by Jonathan Tilly’s group was briefly introduced at the beginning of this book. Oogonial stem cells (OSCs) have been defined in mice as mitotically active cells leading to oocyte renewal, which are also putatively present in the bone marrow and the peripheral blood of adult mice [49, 50]. Although its relevance to humans has been debated, OSCs were isolated from both murine and human ovarian cortex. The current lack of gene promoter exclusively expressed in OSCs but not in differentiating pre-meiotic germ cells or oocytes has been found concerning to definitely prove the presence of OSCs. It seems that OSCs in the ovarian cortex are found in very small numbers. It was estimated that OSCs can be 0.014% of all cells in mouse ovaries, and their numbers decline with chronologic aging. In addition, the introduction of the human OSCs, engineered to express green fluorescent protein (GFP), into human ovarian cortical tissue leads to the formation of follicles containing GFP-positive oocytes about 2 weeks after xenotransplantation into immune-deficient female mice [51].
In order to isolate OSCs from neonatal mice, two-step enzymatic digestion of ovarian tissue was followed by immunomagnetic separation based on DEAD box polypeptide 4 protein (DDX4/VAS, MVH in mice) expression as the membrane marker with cytoplasmic tail common to cells of germ cell lineage in both sexes since it is also expressed in spermatogonia. These cells, also called female germline stem cells, could be cultured more than 15 months with more than 68 passages, unexpected from any somatic cell. The similar isolation could also be made from adult mice. After transfection to express GFP, injection of these cells into the ovaries of mice sterilized with chemotherapy, resulted in GFP and DDX4 expressing oocytes [52]. The use of a membrane marker with cytoplasmic tail produced some concerns. DDX4 is expressed in the cytoplasm of oocytes but it has been claimed that DDX4 has a transmembrane component in OSCs which can be used for cell sorting [53]. In addition, it was shown that GFP by itself causes mitosis since, without GFP, mitosis of putative OSCs did not occur. Some groups developed a different fluorescent mouse model for tracking of DDX4 expressing OSCs and could not detect fluorescence in the ovaries with a conclusion that OSCs do not enter mitosis and hence do not contribute to egg renewal [54].
Small OSC like cells producing oocyte-like cells in culture were also isolated from human ovarian cortical cell cultures by using a strict membrane marker, stage-specific embryonic antigen-4 (SSEA-4) [55, 56]. Some other researchers used other stem cell markers to detect and define OSCs, but in one study, only Fragilis worked [57]. It is agreed that the cells with characteristics of OSCs exist in the ovary of many species including humans. Literature is still undecided about their best method of detection. It is also not clear if these cells contribute to ovarian follicle development in women [58].
One of the proposed applications of OSCs in humans has been some sort of autologous cytoplasmic transfer to improve oocyte quality in women undergoing treatment with assisted reproductive technologies (ART). This technique is called autologous germline mitochondrial energy transfer (AUGMENT) which still requires more studies [59, 60]. One recent prospective randomized trial though did not show any clinically significant benefit of AUGMENT in women with a history of poor embryo quality [61].
Most proposed ovarian stem cell-based therapies have been directed to women with POI or profound DOR. These approaches are usually explored under the basic title of ovarian rejuvenation. One of such protocols is based on the bone marrow-derived stem cell therapy. This protocol is entitled as rejuvenation of premature ovarian failure with stem cells (ROSE-1) (ClinicalTrials.gov identifier: NCT02696889). The study is based on bone marrow aspiration with separation of bone marrow mesenchymal stem cells (MSCs). Then autologous MSCs are injected into the biopsied right ovary. The first abstract on this technique was presented in March of 2018. The authors presented two patients. Human autologous MSCs were isolated from the posterior iliac crest and injected into the ovary through a laparoscopic approach. The patients followed for up to 1 year after the procedure. Both patients resumed menses and had relief from their postmenopausal symptoms with increased estrogen levels. At the time of this review, the study was still open to enroll more subjects [62].
Autologous stem cell ovarian transplantation (ASCOT) was attempted in women with poor ovarian response. Bone marrow-derived stem cells were obtained from peripheral blood after mobilization of such cells with a 5-day course of granulocyte colony-stimulating factor. Then CD34+ cells were collected with apheresis. Then the samples were subjected to the separation of CD133+ cells, which were then infused into one of the ovaries through ovarian artery catheterization. Seventeen patients underwent the procedure. Within 2 weeks of infusion, antral follicle count increased in patients especially in the treated ovary. Some patients showed increased AMH level. The treatment did not increase the embryo euploidy rate but resulted in three natural conceptions and two conceptions with IVF [63].
26.5 Conclusion
In women with POI and profound DOR, ovarian cortical activation has been attempted via ovarian autotransplantation with or without conditioned culture systems or with an ovarian injection of platelet-rich plasma. In addition, several stem cell-based therapies were proposed for a similar population of those with poor ovarian response or poor embryo quality with ART. In that respect, OSC mitochondria have been injected into the retrieved oocytes with no proven improvement in pregnancy rates as of yet. Bone marrow MSCs have been used in a limited number of women with POI with some endocrinologic response. Bone marrow-derived CD-133+ stem cell infusion with ovarian artery catheterization has also been reported in women with a poor ovarian response with some encouraging results. All these interventions are still considered experimental but should be seen as milestones of progress in the management of women with POI, DOR, or POR.
