Ovarian rescue in women with premature ovarian insufficiency: facts and fiction.

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.

Single intraovarian dose of stem cell- and platelet-secreted factors mitigates age-related ovarian infertility in a murine model.

BACKGROUND: Systemic administration of soluble factors from bone marrow-derived stem cells combined with activated platelet-rich plasma (SC-PRP) restored ovarian function, mediated through paracrine signaling, in murine models of chemotherapy-induced ovarian damage and human tissue from poor responder patients. However, the effects against age-related infertility and the efficacy of local administration have not been evaluated yet. OBJECTIVE: This study aimed to assess whether a single intraovarian dose of stem cells combined with activated platelet-rich plasma can recover ovarian function, oocyte quality, and developmental competence in older mice. STUDY DESIGN: The effects of stem cells combined with activated platelet-rich plasma against age-related infertility were assessed following controlled ovarian stimulation in an aging murine model reproducing 3 physiological stages of women's reproductive life, namely young, advanced maternal age, and menopausal (n=12 animals per group). Female mice were randomized to receive a single intraovarian injection (10 µL/ovary) of either saline, activated platelet-rich plasma, or stem cells combined with activated platelet-rich plasma. Seven days later, the mice were stimulated, naturally mated, and sacrificed to harvest their ovaries for histologic assessment and molecular analysis and their oviducts to evaluate oocyte maturation and to assess early embryo development. RESULTS: A single intraovarian injection of stem cells combined with activated platelet-rich plasma promoted follicle activation and development in young, advanced maternal age, and old mice. Furthermore, stem cells combined with activated platelet-rich plasma rescued fertility in older mice by enhancing the quantity and quality of ovulated mature oocytes and supporting early embryo development to the blastocyst stage in all the evaluated ages. These fertility outcomes were positively associated with mitochondrial quality, treatment-increased mitochondrial DNA copy numbers, and reduced oxidative damage and apoptosis. Finally, the effects observed by histologic analysis were supported at the proteomic level. Functional proteomic analyses revealed molecular mechanisms involved in oocyte maturation and quality, mitochondrial function, and recovery of the ovarian stroma. CONCLUSION: Bone marrow-derived stem cells combined with activated platelet-rich plasma is a promising treatment with the potential to improve the reproductive outcomes of women with age-related infertility, exceeding the restorative effects of platelet-rich plasma alone. Although further research in human ovarian samples is still required, the autologous nature of stem cell factors collected by noninvasive mobilization, their combination with platelet-rich plasma, and the local administration route suggest that stem cells combined with activated platelet-rich plasma treatment could be a potentially effective and safe application for future clinical practice.

Transcriptomic landscape of granulosa cells and peripheral blood mononuclear cells in women with PCOS compared to young poor responders and women with normal response.

STUDY QUESTION: Are transcriptomic profiles altered in ovarian granulosa cells (GCs) and peripheral blood mononuclear cells (PBMNCs) of women with polycystic ovary syndrome (PCOS) compared to young poor responders (YPR) and women with normal response to ovarian stimulation? SUMMARY ANSWER: RNA expression profiles in ovarian GCs and PBMNCs were significantly altered in patients with PCOS compared with normoresponder controls (CONT) and YPR. WHAT IS KNOWN ALREADY: PCOS is characterised by a higher number of follicles at all developmental stages. During controlled ovarian hyperstimulation, PCOS women develop a larger number of follicles as a result of an exacerbated response, with an increased risk of ovarian hyperstimulation syndrome. Despite the number of developing follicles, they are often heterogeneous in both size and maturation stage, with compromised quality and retrieval of immature oocytes. Women with PCOS appear to have a longer reproductive lifespan, with a slightly higher menopausal age than the general population, in addition to having a higher antral follicular count. As a result, the ovarian follicular dynamics appear to differ significantly from those observed in women with poor ovarian response (POR) or diminished ovarian reserve. STUDY DESIGN, SIZE, DURATION: Transcriptomic profiling with RNA-sequencing and validation using quantitative reverse transcription PCR (qRT-PCR). Women with PCOS (N = 20), YPR (N = 20) and CONT (N = 20). Five patients for each group were used for sequencing and 15 samples per group were used for validation. PARTICIPANTS/MATERIALS, SETTING, METHODS: PCOS was defined using the revised Rotterdam diagnostic criteria for PCOS. The YPR group included women <35 years old with <4 mature follicles (at least 15 mm) on the day of the trigger. According to internal data, this group represented the bottom 15th percentile of patients' responses in this age group. It was consistent with Patient-Oriented Strategies Encompassing Individualize D Oocyte Number (POSEIDON) criteria for POR (Group 3). The young CONT group included women <35 years without PCOS or anovulation, who developed >14 mature follicles (at least 15 mm on transvaginal ultrasound). According to internal data, a threshold of >14 mature follicles was established to represent the top 25% of patients in this age group in this clinic.Overall, n = 60 GCs and PBMNCs samples were collected and processed for total RNA extraction. To define the transcriptomic cargo of GCs and PBMNCs, RNA-seq libraries were successfully prepared from samples and analysed by RNA-seq analysis. Differential gene expression analysis was used to compare RNA-seq results between different groups of samples. Ingenuity pathway analysis was used to perform Gene Ontology and pathways analyses. MAIN RESULTS AND THE ROLE OF CHANCE: In PBMNCs of PCOS, there were 65 differentially expressed genes (DEGs) compared to CONT, and 16 compared to YPR. In GCs of PCOS, 4 genes showed decreased expression compared to CONT, while 58 genes were differentially expressed compared to YPR. qRT-PCR analysis confirmed the findings of the RNA-seq. The functional enrichment analysis performed revealed that DEGs in GCs of PCOS compared to CONT and YPR were prevalently involved in protein ubiquitination, oxidative phosphorylation, mitochondrial dysfunction and sirtuin signaling pathways. LARGE SCALE DATA: The data used in this study is partially available at Gene Ontology database. LIMITATIONS, REASONS FOR CAUTION: The analysis in PBMNCs could be uninformative due to inter-individual variability among patients in the same study groups. Despite the fact that we considered this was the best approach for our study's novel, exploratory nature. WIDER IMPLICATIONS OF THE FINDINGS: RNA expression profiles in ovarian GCs and PBMNCs were altered in patients with PCOS compared with CONT and YPR. GCs of PCOS patients showed altered expression of several genes involved in oxidative phosphorylation, mitochondrial function and sirtuin signaling pathways. This is the first study to show that the transcriptomic landscape in GCs is altered in PCOS compared to CONT and YPR. STUDY FUNDING/COMPETING INTEREST(S): This study was partially supported by grant PI18/00322 from Instituto de Salud Carlos III, and European Regional Development Fund (FEDER), 'A way to make Europe' awarded to S.H. M.C., S.H., S.T., L.R., M.R., I.R., A.P. and R.C. declare no conflict of interests concerning this research. E.S. is a consultant for and receives research funding from the Foundation for Embryonic Competence. TRIAL REGISTRATION NUMBER: N/A.

Use of dopamine agonists to target angiogenesis in women with endometriosis.

Endometriosis requires medical management during a woman's reproductive years. Most treatments aim to create a hypoestrogenic milieu, but for patients wishing to conceive, drugs that allow normal ovarian function are needed. Targeting angiogenesis, a hallmark of the disease, using dopamine agonists (DAs) is a promising strategy for endometriosis treatment. Herein, we review experimental and clinical data that investigate this concept. In experimental models of endometriosis, DAs (bromocriptine, cabergoline, quinagolide) downregulate proangiogenic and upregulate antiangiogenic pathways in inflammatory, endothelial and endometrial cells, blocking cellular proliferation and reducing lesion size. Impaired secretion of vascular endothelial growth factor (VEGF) and inactivation of its receptor type-2 are key events. VEGF inhibition also reduces nerve fiber density in lesions. In humans, quinagolide shows similar effects on lesions, and DAs reduce pain and endometrioma size. Moreover, a 20-fold downregulation of Serpin-1, the gene that encodes for plasminogen activator inhibitor 1 (PAI-1), has been observed after DAs treatment. Pentoxifylline, a PAI-1, increases pregnancy rates in women with endometriosis. Thus, the data support the use of DAs in the medical management of endometriosis to reduce lesion size and pain while maintaining ovulation. A combined approach of DAs and pentoxifylline is perhaps a smart way of targeting the disease from a completely different angle than current medical treatments.

Stem cell-secreted factor therapy regenerates the ovarian niche and rescues follicles.

BACKGROUND: Ovarian senescence is a normal age-associated phenomenon, but increasingly younger women are affected by diminished ovarian reserves or premature ovarian insufficiency. There is an urgent need for developing therapies to improve ovarian function in these patients. In this context, previous studies suggest that stem cell-secreted factors could have regenerative properties in the ovaries. OBJECTIVE: This study aimed to test the ability of various human plasma sources, enriched in stem cell-secreted factors, and the mechanisms behind their regenerative properties, to repair ovarian damage and to promote follicular development. STUDY DESIGN: In the first phase, the effects of human plasma enriched in bone marrow stem cell soluble factors by granulocyte colony-stimulating factor mobilization, umbilical cord blood plasma, and their activated forms on ovarian niche, follicle development, and breeding performance were assessed in mouse models of chemotherapy-induced ovarian damage (n=7 per group). In addition, the proteomic profile of each plasma was analyzed to find putative proteins and mechanism involved in their regenerative properties in ovarian tissue. In the second phase, the most effective plasma treatment was validated in human ovarian cortex xenografted in immunodeficient mice (n=4 per group). RESULTS: Infusion of human plasma enriched bone marrow stem cell soluble factors by granulocyte colony-stimulating factor mobilization or of umbilical cord blood plasma-induced varying degrees of microvessel formation and cell proliferation and reduced apoptosis in ovarian tissue to rescue follicular development and fertility in mouse models of ovarian damage. Plasma activation enhanced these effects. Activated granulocyte colony-stimulating factor plasma was the most potent inducing ovarian rescue in both mice and human ovaries, and proteomic analysis indicated that its effects may be mediated by soluble factors related to cell cycle and apoptosis, gene expression, signal transduction, cell communication, response to stress, and DNA repair of double-strand breaks, the most common form of age-induced damage in oocytes. CONCLUSION: Our findings suggested that stem cell-secreted factors present in both granulocyte colony-stimulating factor-mobilized and umbilical cord blood plasma could be an effective treatment for increasing the reproductive outcomes in women with impaired ovarian function owing to several causes. The activated granulocyte colony-stimulating factor plasma, which is already enriched in both stem cell-secreted factors and platelet-enclosed growth factors, seems to be the most promising treatment because of its most potent restorative effects on the ovary together with the autologous source.

Emerging follicular activation strategies to treat women with poor ovarian response and primary ovarian insufficiency.

PURPOSE OF THE REVIEW: Female reproductive aging remains one of the key unsolved challenges in the field of reproductive medicine. This article reviews three of the most recent and cutting-edge strategies that are currently being investigated to address the issues of poor ovarian response (POR) and primary ovarian insufficiency (POI). RECENT FINDINGS: Publications revealing the mechanism of mechanical disruption of the Hippo signaling pathway paved the way to studies on its potential application for fertility treatments. This, in combination with Akt stimulation, resulted in live births and ongoing pregnancies in women with POI. Building on previous reports on the effects of bone marrow transplants on fertility after chemotherapy, another approach involved autologous stem cell ovarian transplantation (ASCOT). The method proved effective in achieving live births in women previously diagnosed with POR. A third approach, intraovarian injection of autologous platelet-rich plasma, resulted in live births and ongoing pregnancies both spontaneously and via in vitro fertilization (IVF) in women with POI and POR. SUMMARY: New paths are being charted to address the issues of POI and POR. Although these are preliminary studies that should be interpreted with caution, they represent great promise for the women affected by these conditions and the physicians treating them.

Disease-inducing potential of two leukemic cell lines in a xenografting model.

PURPOSE: Cryopreserved ovarian tissue transplant restores ovarian function in young cancer patients after gonadotoxic treatment. However, leukemia is associated with increased risk of malignant cell transmission. We aimed to assess the tumor-inducing potential of two different leukemic cell lines when xenografted to immunodeficient mice. METHODS: Fifty-four female immunodeficient mice were grafted with either 100, 200, 500, 1000, and 10,000 chronic myeloid leukemia in blast crisis (BV-173) cells or relapsed acute lymphoblastic leukemia (RCH-ACV) cells, embedded inside a fibrin scaffold along with 50,000 human ovarian stromal cells. Two mice per cell line received the fibrin matrix without leukemic cells as negative controls. Clinical signs of disease were monitored for 20 weeks. Grafts, liver tissue, and masses were collected for macroscopic analysis and gene expression of BCR-ABL1 and E2A-PBX fusion transcripts present in BV-173 and RCH-ACV respectively. RESULTS: BV-173 cells: Mice grafted with 100, 200, or 500 cells showed no sign of disease after and were negative for BCR-ABL1 expression. Three of the 5 animals grafted with 1000 cells and all mice with 10,000 cells developed disease and showed BCR-ABL1-positive expression. RCH-ACV cells: Two out of 4 mice grafted with 100 cells developed disease and were E2A-PBX1-positive. All the animals grafted with higher cell doses showed signs of disease and all but one were E2A-PBX1-positive. CONCLUSION: The present work proves that the disease-inducing potential of BV-173 and RCH-ACV leukemic cells xenografted to SCID mouse peritoneum differs between cell lines, depending on cell number, type, status, and cytogenetic disease profile when ovarian tissue is harvested.

Dexamethasone does not prevent malignant cell reintroduction in leukemia patients undergoing ovarian transplant: risk assessment of leukemic cell transmission by a xenograft model.

STUDY QUESTION: Does dexamethasone (DXM) incubation avoid the reintroduction of leukemic malignant cells after ovarian tissue retransplantation in vivo? SUMMARY ANSWER: DXM incubation prior to retransplantation of ovarian tissue does not prevent reintroduction of leukemic cells. WHAT IS KNOWN ALREADY: Retransplantation of cryopreserved ovarian cortex from patients diagnosed with acute lymphoblastic leukemia (ALL) involves a risk of reintroducing malignant cells. DXM treatment is effective at inducing leukemic cell death in vitro. STUDY DESIGN, SIZE, DURATION: This was an experimental study where ovarian cortex fragments from patients with ALL were randomly allocated to incubation with or without DXM (n = 11/group) and grafted to 22 immunodeficient mice for 6 months. In a parallel experiment, 22 immunodeficient mice were injected i.p. with varying amounts of RCH-ACV ALL cells (human leukemia cell line) and maintained for 4 months. PARTICIPANTS/MATERIALS, SETTING, METHODS: Cryopreserved ovarian fragments from patients with ALL were exposed in vitro to 0.4 µM DXM or basal media (control) prior to xenograft into ovariectomized severe combined immunodeficiency (SCID) mice (experiment 1). After 6 months of monitoring, leukemia cell contamination was assessed in ovarian grafts and mouse organs by histology, PCR (presence of mouse mtDNA and absence of p53 were together considered a negative result for the presence of human cells) and detection of immunoglobulin monoclonality and specific ALL markers if present in the patient.In experiment 2, a series of 22 immunodeficient female mice was injected with specific doses of the leukemia cell line RCH-ACV (103 - 5 × 106, n = 4/group) to assess the engraftment competence of the SCID model. MAIN RESULTS AND THE ROLE OF CHANCE: ALL metastatic cells were detected, by PCR, in five DXM-treated and one control human ovarian tissue graft as well as in a control mouse liver, although malignant cell infiltration was not detected by histology in any sample after 6 months. In total, minimal residual disease was present in three DXM-treated and three control mice.RCH-ACV cells were detected in liver and spleen samples after the injection of as little as 103 cells, although only animals receiving 5 × 106 cells developed clinical signs of disease and metastases. LIMITATIONS, REASONS FOR CAUTION: This is an experimental study where the malignant potential of leukemic cells contained in human ovarian tissues has been assessed in immunodeficient mice. WIDER IMPLICATIONS OF THE FINDINGS: These results indicate that DXM incubation prior to retransplantation of ovarian tissue does not prevent reintroduction of leukemic cells. Therefore, caution should be taken in retransplanting ovarian tissue from patients with leukemia until safer systems are developed, as leukemic cells present in ovarian grafts were able to survive, proliferate and migrate after cryopreservation and xenograft. STUDY FUNDING/COMPETING INTEREST(S): Funded by the Regional Valencian Ministry of Education (PROMETEO/2018/137) and by the Spanish Ministry of Economy and Competitiveness (PI16/FIS PI16/01664 and PTQ-16-08222 for S.H. participation). There are no competing interests.

Treatment potential of bone marrow-derived stem cells in women with diminished ovarian reserves and premature ovarian failure.

PURPOSE OF REVIEW: We review the techniques recently tested in both animal models and humans to provide a state-of-the-art on adult stem cell ovarian transplant to achieve ovarian rejuvenation in patients with diminished ovarian reserves. RECENT FINDINGS: As the firsts reports of spontaneous pregnancies achieved after bone marrow transplantation in oncologic women with primary ovarian insufficiency, increasing evidence supports the regenerative effects of stem cell-based therapies in the ovarian niche. Adult stem cells from several origins promote follicular development, increase ovarian local vascularization, increase follicle and stromal cell proliferation and reduce cell apoptosis and follicular atresia, although they do not modify embryo quality. Therefore, residual quiescent follicles of aged or damaged ovaries might produce competent oocytes in an adequate ovarian environment. Nevertheless, further research is needed to properly evaluate underlying mechanisms, identify best cell sources and design less invasive infusion techniques. SUMMARY: Stem cells may be a relevant therapeutic alternative for ovary regeneration and follicular development in patients with impaired ovaries, such as poor ovarian responders or women diagnosed with primary ovarian insufficiency.

Single site laparoscopy for fertility preservation: a cohort study.

STUDY OBJECTIVE: To compare operative and postoperative results of ovarian cortex retrieval by conventional laparoscopy (1cm umbilical site and 3 accessory 5-mm-reusable working ports) (HASS) versus single site laparoscopy (SSL). DESIGN: Prospective cohort study. SETTING: Fertility Preservation Programme at La Fe University Hospital-University of Valencia, Valencia, Spain, 2011 to 2012. Fertility Preservation Programme at La Fe University Hospital of Valencia, Valencia, Spain. PATIENTS: Twenty-one patients with cancer (breast cancer: n = 17; Hodgkin's lymphoma: n = 3; and non-Hodgkin's lymphoma: n = 1). INTERVENTION: Ovarian cortex retrieval either by conventional laparoscopy using an umbilical Hasson port and 3 accessory ports (HASS group: n = 11) or by SSL (SSL group: n = 10). MEASUREMENTS AND MAIN RESULTS: Operative length, blood loss, postoperative pain (visual analog scale for pain at 6, 24, and 48 hours), need of additional analgesia, quality of life (European Quality of Life-5 Dimensions), cosmesis of the scar, and patient's self-perception were assessed at 24 and 48 hours and 3 months after surgery. Baseline characteristics were similar between groups. Estimated blood loss, operative length, and postoperative pain did not differ between groups. The start of chemotherapy was not delayed in either group, and cosmesis and image self-perception were also similar. CONCLUSION: The SSL approach can be considered a safe option compared with the classic multisite approach.

Intraovarian PRP injection improves oocyte quality and early embryo development in mouse models of chemotherapy-induced diminished ovarian reserve.

Intraovarian injection of platelet-rich plasma (PRP) has been recently proposed, with encouraging results to provide an alternative option to patients diagnosed with POR or POI. However, the broad spectrum of PRP effects on the reproductive function and the mechanisms of action in follicular activation, response to stimulation, and embryo quality have not yet been studied. In this study, we first induced poor ovarian reserve (POR) and premature ovarian insufficiency (POI) ovarian phenotypes in CD1 mice undergoing PRP or sham intraovarian injection. PRP administration reduced those alterations induced by chemotherapy in ovarian stroma and follicle morphology in both the POR and POI conditions. After ovarian stimulation, we found that PRP did not modify the MII-oocyte yield. Nevertheless, the amount of obtained 2-cell embryos and fertilization rate were increased, being especially relevant for the POI model. Further in vitro embryo culture led to improved blastocyst formation rates and higher numbers of good quality blastocysts in PRP vs. sham females in both the POR and POI conditions. These positive results of PRP injection were also validated in the C57Bl/6 stain. Altogether, our findings suggest a possible effect on oocyte and embryo quality. This effect is likely due to the increase of local paracrine signaling through the released growth factors in PRP-treated ovaries.

Growth factor-loaded ovarian extracellular matrix hydrogels promote in vivo ovarian niche regeneration and enhance fertility in premature ovarian insufficiency preclinical models.

Premature ovarian insufficiency (POI) means menopause before 40 years of age affecting about 1 % of women. Approaches based on cell therapy and the paracrine effects of stem cells or bioproducts such as platelet-rich plasma have been proposed, but concerns remain about undesired systemic effects, as well as the need to optimize delivery methods through bioengineering methods. This study explores the efficacy of decellularized bovine ovarian cortex extracellular matrix (OvaECM) hydrogels alone and as a growth factor (GF) carrier (OvaECM+GF) in a chemotherapy-induced POI murine model. In vitro assays showed a gradual release of GF from the OvaECM sustained for two weeks. Chemotherapy drastically reduced follicle numbers, but OvaECM+GF treatment restored pre-antral follicle development. Moreover, this treatment notably regenerated the ovarian microenvironment by increasing cell proliferation and microvessel density while reducing chemotherapy-induced apoptosis and fibrosis. Whole-ovary RNA sequencing and gene set enrichment analysis revealed an upregulation of regeneration-related genes and a downregulation of apoptotic pathways. The OvaECM+GF treatment also yielded significantly better outcomes following ovarian stimulation and in vitro fertilization. After two consecutive crossbreeding cycles, OvaECM+GF-treated mice showed normal reproductive function. This research showcases the biocompatibility and efficacy of OvaECM to reverse POI in mice, setting a foundation to explore innovative bioengineering-based POI therapies. STATEMENT OF SIGNIFICANCE: Premature ovarian insufficiency (POI) affects about 1 % of women worldwide, causing early menopause before 40 years old. Current treatments alleviate symptoms but do not restore ovarian function. This study explores an innovative approach using ovarian cortex extracellular matrix hydrogels to deliver growth factors into the murine ovarian niche and reverse POI. In vitro release kinetic assays demonstrated a gradual and sustained release of growth factors. In a POI-induced mouse model, intraovarian injections of the hydrogel encapsulating growth factors restored pre-antral follicle development, increased cell proliferation, reduced apoptosis and fibrosis, and improved ovarian response and in vitro fertilization outcomes. Long-term benefits included larger litter sizes. This innovative technique shows promise in regenerating the ovarian environment and improving reproductive outcomes.

Deciphering reproductive aging in women using a NOD/SCID mouse model for distinct physiological ovarian phenotypes.

Female fertility is negatively correlated with age, with noticeable declines in oocyte quantity and quality until menopause. To understand this physiological process and evaluate human approaches for treating age-related infertility, preclinical studies in appropriate animal models are needed. Thus, we aimed to characterize an immunodeficient physiological aging mouse model displaying ovarian characteristics of different stages during women's reproductive life. NOD/SCID mice of different ages (8-, 28-, and 36-40-week-old) were employed to mimic ovarian phenotypes of young, Advanced Maternal Age (AMA), and old women (~18-20-, ~36-38-, and >45-years-old, respectively). Mice were stimulated, mated, and sacrificed to recover oocytes and embryos. Then, ovarian reserve, follicular growth, ovarian stroma, mitochondrial dysfunction, and proteomic profiles were assessed. Age-matched C57BL/6 mice were employed to cross-validate the reproductive outcomes. The quantity and quality of oocytes were decreased in AMA and Old mice. These age-related effects associated spindle and chromosome abnormalities, along with decreased developmental competence to blastocyst stage. Old mice had less follicles, impaired follicle activation and growth, an ovarian stroma inconducive to growth, and increased mitochondrial dysfunctions. Proteomic analysis corroborated these histological findings. Based on that, NOD/SCID mice can be used to model different ovarian aging phenotypes and potentially test human anti-aging treatments.

Systemic changes induced by autologous stem cell ovarian transplant in plasma proteome of women with impaired ovarian reserves.

Patients with poor ovarian response (POR) and premature ovarian insufficiency (POI) are challenging to treat, with oocyte donation remaining as the only feasible option to achieve pregnancy in some cases. The Autologous stem cell ovarian transplantation (ASCOT) technique allows follicle development, enabling pregnancies and births of healthy babies in these patients. Previous results suggest that growth factors and cytokines secreted by stem cells are partially responsible for their regenerative properties. Indeed, ASCOT beneficial effects associate with the presence of different bone marrow derived stem cell- secreted factors in plasma. Therefore, the aim of this study was to assess whether ASCOT induce any modifications in the plasma proteomic profile of patients with impaired ovarian reserves. Discriminant analysis highlighted clear distinctions between the plasma proteome before (PRE), during stem cell mobilization and collection (APHERESIS) and three months after ASCOT (POST) in patients with POR and POI. Both the stem cell mobilization and ASCOT technique induced statistically significant modifications in the plasma composition, reversing some age-related protein expression changes. In the POR group, functional analysis revealed an enrichment in processes related to the complement cascade, immune system, and platelet degranulation, while in the POI group, enriched processes were also associated with responses to oxygen-containing compounds and growth hormones, and blood vessel maturation. In conclusion, our findings highlight the potential proteins and biological processes that may promote the follicle activation and growth observed after ASCOT. Identifying plasma proteins that regenerate aged or damaged ovaries could lead to more effective, targeted and/or preventive therapies for patients.

Distress response in granulosa cells of women affected by PCOS with or without insulin resistance.

PURPOSE: In this study, we investigated whether metabolic dysfunction in women with Polycystic ovarian syndrome (PCOS) induces granulosa cell (GC) stress and activates in the endoplamatic reticulum and the mitochondria (UPRer and UPRmt, respectively). METHODS: Women who were diagnosed with PCOS (based on the Rotterdam criteria), were divided into two groups, PCOS with insulin resistance (PCOS-IR; n = 20) and PCOS with no insulin resistance (PCOS-nIR; n = 20), and compared to healthy oocyte donors (CONT; n = 20). Insulin resistance (IR) was assessed on the results of homeostasis model assessment (HOMA) that determines IR using the concentration of fasting plasma glucose and fasting insuline. Expression of UPRer genes (i.e., IRE1, ATF4, ATF6, XBP1, BIP, and CHOP), and UPRmt genes (i.e., HSP60, HSP10, CLPP, and HSP40) was assessed in cumulus GCs by qRT-PCR. RESULTS: We found that several genes involved in UPRer and UPRmt were overexpressed in the GCs of PCOS-IR and PCOS-nIR compared to CONT. IRE1, ATF4 and XBP1, that are activated by ER stress, were significantly overexpressed in PCOS-IR compared to CONT. BIP and CHOP were overexpressed in PCOS groups compared to CONT. HSP10 and HSP40 were upregulated in PCOS-IR and PCOS-nIR groups compared to the CONT. HSP60 and CLPP showed no statistical different expression in PCOS-IR and PCOS-nIR compared to CONT group. CONCLUSION: Our findings suggest that the GCs of women with PCOS (with or without IR) are metabolically distressed and upregulate UPRer and UPRmt genes. Our study contributes to the understanding of the molecular mechanisms underlying the pathological changes that occur in the follicular microenvironment of women with PCOS.

Impaired telomere pathway and fertility in Senescence-Accelerated Mice Prone 8 females with reproductive senescence.

Ovarian aging is the main cause of infertility and telomere attrition is common to both aging and fertility disorders. Senescence-Accelerated Mouse Prone 8 (SAMP8) model has shortened lifespan and premature infertility, reflecting signs of reproductive senescence described in middle-aged women. Thus, our objective was to study SAMP8 female fertility and the telomere pathway at the point of reproductive senescence. The lifespan of SAMP8 and control mice was monitored. Telomere length (TL) was measured by in situ hybridization in blood and ovary. Telomerase activity (TA) was analyzed by telomere-repeat amplification protocol, and telomerase expression, by real-time quantitative PCR in ovaries from 7-month-old SAMP8 and controls. Ovarian follicles at different stages of maturation were evaluated by immunohistochemistry. Reproductive outcomes were analyzed after ovarian stimulation. Unpaired t-test or Mann-Whitney test were used to calculate p-values, depending on the variable distribution. Long-rank test was used to compare survival curves and Fisher's exact test was used in contingency tables. Median lifespan of SAMP8 females was reduced compared to SAMP8 males (p = 0.0138) and control females (p < 0.0001). In blood, 7-month-old SAMP8 females presented lower mean TL compared to age-matched controls (p = 0.041). Accordingly, the accumulation of short telomeres was higher in 7-month-old SAMP8 females (p = 0.0202). Ovarian TA was lower in 7-month-old SAMP8 females compared to controls. Similarly, telomerase expression was lower in the ovaries of 7-month-old SAMP8 females (p = 0.04). Globally, mean TL in ovaries and granulosa cells (GCs) were similar. However, the percentage of long telomeres in ovaries (p = 0.004) and GCs (p = 0.004) from 7-month-old SAMP8 females was lower compared to controls. In early-antral and antral follicles, mean TL of SAMP8 GCs was lower than in age-matched controls (p = 0.0156 for early-antral and p = 0.0037 for antral follicles). Middle-aged SAMP8 showed similar numbers of follicles than controls, although recovered oocytes after ovarian stimulation were lower (p = 0.0068). Fertilization rate in oocytes from SAMP8 was not impaired, but SAMP8 mice produced significantly more morphologically abnormal embryos than controls (27.03% in SAMP8 vs. 1.22% in controls; p < 0.001). Our findings suggest telomere dysfunction in SAMP8 females, at the time of reproductive senescence.

Bioengineering trends in female reproduction: a systematic review.

BACKGROUND: To provide the optimal milieu for implantation and fetal development, the female reproductive system must orchestrate uterine dynamics with the appropriate hormones produced by the ovaries. Mature oocytes may be fertilized in the fallopian tubes, and the resulting zygote is transported toward the uterus, where it can implant and continue developing. The cervix acts as a physical barrier to protect the fetus throughout pregnancy, and the vagina acts as a birth canal (involving uterine and cervix mechanisms) and facilitates copulation. Fertility can be compromised by pathologies that affect any of these organs or processes, and therefore, being able to accurately model them or restore their function is of paramount importance in applied and translational research. However, innate differences in human and animal model reproductive tracts, and the static nature of 2D cell/tissue culture techniques, necessitate continued research and development of dynamic and more complex in vitro platforms, ex vivo approaches and in vivo therapies to study and support reproductive biology. To meet this need, bioengineering is propelling the research on female reproduction into a new dimension through a wide range of potential applications and preclinical models, and the burgeoning number and variety of studies makes for a rapidly changing state of the field. OBJECTIVE AND RATIONALE: This review aims to summarize the mounting evidence on bioengineering strategies, platforms and therapies currently available and under development in the context of female reproductive medicine, in order to further understand female reproductive biology and provide new options for fertility restoration. Specifically, techniques used in, or for, the uterus (endometrium and myometrium), ovary, fallopian tubes, cervix and vagina will be discussed. SEARCH METHODS: A systematic search of full-text articles available in PubMed and Embase databases was conducted to identify relevant studies published between January 2000 and September 2021. The search terms included: bioengineering, reproduction, artificial, biomaterial, microfluidic, bioprinting, organoid, hydrogel, scaffold, uterus, endometrium, ovary, fallopian tubes, oviduct, cervix, vagina, endometriosis, adenomyosis, uterine fibroids, chlamydia, Asherman's syndrome, intrauterine adhesions, uterine polyps, polycystic ovary syndrome and primary ovarian insufficiency. Additional studies were identified by manually searching the references of the selected articles and of complementary reviews. Eligibility criteria included original, rigorous and accessible peer-reviewed work, published in English, on female reproductive bioengineering techniques in preclinical (in vitro/in vivo/ex vivo) and/or clinical testing phases. OUTCOMES: Out of the 10 390 records identified, 312 studies were included for systematic review. Owing to inconsistencies in the study measurements and designs, the findings were assessed qualitatively rather than by meta-analysis. Hydrogels and scaffolds were commonly applied in various bioengineering-related studies of the female reproductive tract. Emerging technologies, such as organoids and bioprinting, offered personalized diagnoses and alternative treatment options, respectively. Promising microfluidic systems combining various bioengineering approaches have also shown translational value. WIDER IMPLICATIONS: The complexity of the molecular, endocrine and tissue-level interactions regulating female reproduction present challenges for bioengineering approaches to replace female reproductive organs. However, interdisciplinary work is providing valuable insight into the physicochemical properties necessary for reproductive biological processes to occur. Defining the landscape of reproductive bioengineering technologies currently available and under development for women can provide alternative models for toxicology/drug testing, ex vivo fertility options, clinical therapies and a basis for future organ regeneration studies.

Follicular activation in women previously diagnosed with poor ovarian response: a randomized, controlled trial.

OBJECTIVE: To investigate whether ovarian fragmentation for follicular activation (OFFA) improves ovarian reserve markers and in vitro fertilization (IVF) outcomes in women with poor ovarian response (POR). DESIGN: Randomized, controlled trial, with parallel assignment. SETTING: University hospital. PATIENT(S): Thirty-four women with POR according to the European Society of Human Reproduction and Embryology criteria. INTERVENTION(S): Women with POR were randomly allocated to receive ovarian fragmentation in 1 ovary or to no intervention (control group). Ovarian reserve markers were followed at 2-week intervals for 6 months. In vitro fertilization cycles were initiated when the antral follicle count (AFC) doubled or at the end of follow-up. MAIN OUTCOME MEASURE(S): The primary outcome was the number of metaphase II (MII) oocytes obtained. Antral follicle count, antimüllerian hormone level, and reproductive outcomes were recorded as secondary outcomes. Exploratory outcomes included surgical results and analysis of protein and gene expression. RESULT(S): Ovarian fragmentation for follicular activation resulted in an increase in AFC in the intervention ovary compared with the control ovary and an increase in total AFC in the OFFA group compared with controls. Serum antimüllerian hormone and follicle-stimulating-hormone levels did not improve in the OFFA group throughout the follow-up period. Fifteen patients from each arm underwent IVF. In the control group, 33 MII oocytes were retrieved and 18 embryo transfers were performed, with a 20% pregnancy rate and an 18.7% live birth rate per cycle. In the OFFA group, 23 MII oocytes were retrieved and 11 embryo transfers were performed, with a 13.3% pregnancy rate and a 6.7% live birth rate per cycle. Reproductive outcomes did not significantly differ between the groups. Hippo pathway inhibition was confirmed by an 18.8% reduction in the phospho-YAP/YAP (Yes-associated protein 1) ratio and BIRC and CCN overexpression after fragmentation. CONCLUSION(S): Ovarian fragmentation for follicular activation in women with POR resulted in an increase in AFC but did not modify IVF outcomes when compared with controls. CLINICAL TRIAL REGISTRATION NUMBER: NCT02354963.

Mitochondrial enrichment in infertile patients: a review of different mitochondrial replacement therapies.

Poor ovarian responders exhibit a quantitative reduction in their follicular pool, and most cases are also associated with poor oocyte quality due to patient's age, which leads to impaired in vitro fertilisation outcomes. In particular, poor oocyte quality has been related to mitochondrial dysfunction and/or low mitochondrial count as these organelles are crucial in many essential oocyte processes. Therefore, mitochondrial enrichment has been proposed as a potential therapy option in infertile patients to improve oocyte quality and subsequent in vitro fertilisation outcomes. Nowadays, different options are available for mitochondrial enrichment treatments that are encompassed in two main approaches: heterologous and autologous. In the heterologous approach, mitochondria come from an external source, which is an oocyte donor. These techniques include transferring either a portion of the donor's oocyte cytoplasm to the recipient oocyte or nuclear material from the patient to the donor's oocyte. In any case, this approach entails many ethical and safety concerns that mainly arise from the uncertain degree of mitochondrial heteroplasmy deriving from it. Thus the autologous approach is considered a suitable potential tool to improve oocyte quality by overcoming the heteroplasmy issue. Autologous mitochondrial transfer, however, has not yielded as many beneficial outcomes as initially expected. Proposed mitochondrial autologous sources include immature oocytes, granulosa cells, germline stem cells, and adipose-derived stem cells. Presently, it would seem that these autologous techniques do not improve clinical outcomes in human infertile patients. However, further trials still need to be performed to confirm these results. Besides these two main categories, new strategies have arisen for oocyte rejuvenation by improving patient's own mitochondrial function and avoiding the unknown consequences of third-party genetic material. This is the case of antioxidants, which may enhance mitochondrial activity by counteracting and/or preventing oxidative stress damage. Among others, coenzyme-Q10 and melatonin have shown promising results in low-prognosis infertile patients, although further randomised clinical trials are still necessary.