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.

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.

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.

Pathways and factors regulated by bone marrow-derived stem cells in human ovarian tissue.

OBJECTIVE: To describe molecular and paracrine signaling changes produced by human bone marrow-derived stem cells (BMDSC) in human ovarian cortex. DESIGN: Experimental study. SETTING: University hospital research laboratories. PATIENT(S): Ovarian cortex from poor responder women (n = 7). ANIMALS: Immunodeficient NOD/SCID female mice (n = 18). INTERVENTION(S): Human ovarian cortex strips were xenografted into ovariectomized NOD/SCID female mice. A week later, mice were infused with phosphate-buffered saline, 1 × 106 BMDSC, or 3 × 105 CD133+ cells via tail vein. Gene expression changes and enriched pathways were assessed by RT2 Profiler Arrays. Several upregulated genes were validated in individual samples by real-time quantitative PCR, and transcriptomic results were reinforced by a proteomic assessment. MAIN OUTCOME MEASURE(S): Gene expression changes, enriched Kyoto Encyclopedia of Genes and Genomes pathways, and paracrine factors. RESULT(S): Seventy-four Kyoto Encyclopedia of Genes and Genomes pathways were upregulated, with the PI3K-Akt signaling pathway the most enriched after BMDSC and CD133 treatments. The greatest transcriptomic changes were seen on day 14 in the BMDSC group, affecting the regulation of paracrine factors such as KITLG, THBS1, SERPINF1, and TIMP2. Proteomics data verified changes in FoxO signaling, actin cytoskeleton remodeling, and apoptosis by BMDSC. CONCLUSION(S): We identified paracrine factors and pathways regulated by BMDSC that may be future targets of treatment for the increasing number of poor responder women. Our findings suggest that BMDSC upregulated soluble factors such as KITLG, THBS1, SERPINF1, and TIMP2 as well as PI3K-Akt signaling and regulation of actin cytoskeleton pathways. The identification of these putative underlying mechanisms informs future experiments aiming to optimizing clinical application of BMDSC.

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.

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.

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.

Diminished Ovarian Reserve Chemotherapy-Induced Mouse Model: A Tool for the Preclinical Assessment of New Therapies for Ovarian Damage.

Diminished ovarian reserve (DOR) and primary ovarian insufficiency (POI) are primary factors leading to infertility. However, there is a lack of appropriate animal models of DOR usable for assessing new therapeutic strategies. In this study, we aimed to evaluate whether chemotherapy treatment in mice could reproduce features similar of that observed in women with DOR. Twenty-one Nonobese diabetic/severe combined immunodeficiency (NOD/SCID) female mice were allocated to 3 groups (n = 7/group): control, single dose of vehicle (Dimethyl Sulfoxide [DMSO]); DOR, single reduced chemotherapy dose; and POI, single standard chemotherapy dose. After 21 days, mice underwent ovarian hyperstimulation and mating. Part of the animals were harvested to analyze ovarian reserve, ovulation and fertilization rates, and morphology, apoptosis, and vascularization of the ovarian stroma. The remaining mice underwent multiple matings to assess pregnancy rates and litter sizes. The DOR and POI mice showed an impaired estrous cyclicity and a decrease in ovarian mass, number of follicles, Metaphase II (MII) oocytes, and embryos as well as in ovarian stroma vascularization. Mice in both models showed also an increase in the percentage of morphologically abnormal follicles, stromal degeneration, and apoptosis. Similar to that observed in DOR and POI patients, these impairments were less severe in DOR than in POI mice. None of the POI females were able to achieve a pregnancy. Meanwhile, DOR females achieved several consecutive pregnancies, although litter size was decreased when compared to controls. In conclusion, a mouse model which displayed most of the ovarian characteristics and fertility outcomes of women with DOR has been established using a single dose of chemotherapy.

Optimizing ovarian tissue quality before cryopreservation: comparing outcomes of three decortication methods on stromal and follicular viability.

OBJECTIVE: To evaluate whether specific ovarian decortication techniques vary in promoting ovarian cortex cryopreservation and transplant outcomes. DESIGN: Experimental design. SETTING: University hospital. ANIMAL(S): Nonobese diabetic (NOD)/severe combined immunodeficiency (SCID) female mice. INTERVENTION(S): Human ovarian biopsy samples allocated to one of the following decortication procedures: scratching with scalpel blade (B), cutting with microsurgical scissors (M), separation with slicer (S), or no-separation (control, C). Parallel, in vivo experiment: decortication techniques combined with slow freezing (SF) and vitrification (VT) before xenograft into immunodeficient mice. MAIN OUTCOME MEASURE(S): Follicular counts, apoptosis, shear stress, Hippo pathway and inflammation. In vivo: recovered grafts analyzed for follicular counts, angiogenesis, proliferation, and fibrosis. RESULT(S): There were no differences in follicular density or number of damaged follicles between the decortication techniques in the in vitro study. Nevertheless, the M samples showed statistically significantly increased stromal damage compared with the controls and S samples, and up-regulation of Hsp60 shear stress gene expression. Decortication by both M and S inhibited the Hippo pathway, promoting gene expression changes. In the 21-day xenograft, total follicular density statistically significantly decreased compared with the nongrafted controls in all groups. Nevertheless, no differences were observed between the decortication techniques. Ovarian stroma vascularization was increased in the vitrified samples, but among the slow-freezing samples, the B samples had the lowest microvessel density. The M decorticated xenografts had increased fibrosis. CONCLUSION(S): Decortication with a slicer causes less damage to ovarian tissue than other commonly used methods although microsurgical scissors seem to preserve slightly increased follicular numbers. Nevertheless, blade decortication seems to be a reliable technique for maintaining acceptable follicular conditions without inducing serious stromal impairment.

Stem Cell Paracrine Signaling for Treatment of Premature Ovarian Insufficiency.

Premature ovarian insufficiency is a common disorder affecting young women and represents the worst-case ovarian scenario due to the substantial impact on the reproductive lifespan of these patients. Due to the complexity of this condition, which is not fully understood, non-effective treatments have yet been established for these patients. Different experimental approaches are being explored and strategies based on stem cells deserve special attention. The regenerative and immunomodulatory properties of stem cells have been successfully tested in different tissues, including ovary. Numerous works point out to the efficacy of stem cells in POI treatment, and a wide range of clinical trials have been developed in order to prove safety and effectiveness of stem cells therapy-in diminished ovarian reserve and POI women. The main purpose of this review is to describe the state of the art of the treatment of POI involving stem cells, especially those that use mobilization of stem cells or paracrine signaling.

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.

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.

OBJECTIVE: To study if autologous mitochondrial transfer (AUGMENT) improves outcome in patients with previously failed in vitro fertilization (IVF). DESIGN: Randomized, controlled, triple-blind, experimental study. SETTING: Private infertility center, Valencian Institute of Infertility (IVI-RMA), Valencia, Spain. PATIENT(S): Infertile women ≤42 years of age, body mass index <30 kg/m2, antimüllerian hormone ≥4 pmol/L, >5 million/mL motile sperm, at least one previous IVF with at least five metaphase oocytes (MIIs) collected, and low embryo quality. INTERVENTIONS(S): An ovarian cortex biopsy was performed to isolate egg precursor cells to obtain their mitochondria. Sibling MIIs were randomly allocated to AUGMENT (experimental) or intracytoplasmic sperm injection (Control). In AUGMENT, mitochondrial suspension was injected along with the sperm. Viable blastocysts from both groups were biopsied for preimplantation genetic testing for aneuploidy. MAIN OUTCOME MEASURE(S): Pregnancy, embryo quality. RESULT(S): An interim analysis was conducted. The patients' mean age was 36.3 ± 3.6 years, and they had an average of 2.5 ± 1.5 previous IVF cycles. Two of the 59 enrolled patients spontaneously conceived (one miscarried). Fifty-seven patients had ovarian biopsies and underwent stimulation. Oocyte retrieval was performed in 56 patients (premature ovulation; n = 1). A total of 253 MIIs were inseminated in AUGMENT and 250 in Control; fertilization rates were 62.7 ± 30.0% and 68.7 ± 29.1%, respectively. Statistical differences were observed in day 5 blastocyst formation rates (23.3 ± 32.0% vs. 41.1 ± 36.9%). Neither the euploid rate per biopsied blastocyst (43.8 ± 41.7% vs. 63.8 ± 44.1%) nor the euploid rate per MII (9.8 ± 20.5% vs. 11.9 ± 16.1%) between AUGMENT and Control achieved statistical significance. Moreover, no differences were seen regarding mitochondrial DNA content and relevant morphokinetic variables. Thirty patients were able to undergo embryo transfer. Cumulative live birth rates per transferred embryo were 41.6% in AUGMENT and 41.2% in Control. CONCLUSION(S): AUGMENT does not seem to improve prognosis in this population. Therefore, the study has been discontinued. CLINICAL TRIAL REGISTRATION NUMBER: NCT02586298.

Autologous stem cell ovarian transplantation to increase reproductive potential in patients who are poor responders.

OBJECTIVE: To evaluate effects of autologous stem cell ovarian transplant (ASCOT) on ovarian reserve and IVF outcomes of women who are poor responders with very poor prognosis. DESIGN: Prospective observational pilot study. SETTING: University hospital. PATIENT(S): Seventeen women who are poor responders. INTERVENTION(S): Ovarian infusion of bone marrow-derived stem cells. MAIN OUTCOME MEASURE(S): Serum antimüllerian hormone levels and antral follicular count (AFC), punctured follicles, and oocytes retrieved after stimulation (controlled ovarian stimulation) were measred. Apheresis was analyzed for growth factor concentrations. RESULT(S): The ASCOT resulted in a significant improvement in AFC 2 weeks after treatment. With an increase in AFC of three or more follicles and/or two consecutive increases in antimüllerian hormone levels as success criteria, ovarian function improved in 81.3% of women. These positive effects were associated with the presence of fibroblast growth factor-2 and thrombospondin. During controlled ovarian stimulation, ASCOT increased the number of stimulable antral follicles and oocytes, but the embryo euploidy rate was low (16.1%). Five pregnancies were achieved: two after ET, three by natural conception. CONCLUSION(S): Our results suggest that ASCOT optimized the mobilization and growth of existing follicles, possibly related to fibroblast growth factor-2 and thrombospondin-1 within apheresis. The ASCOT improved follicle and oocyte quantity enabling pregnancy in women who are poor responders previously limited to oocyte donation. CLINICAL TRIAL REGISTRATION NUMBER: NCT02240342.

Fertility rescue and ovarian follicle growth promotion by bone marrow stem cell infusion.

OBJECTIVE: To assess if infusion of human bone marrow-derived stem cells (BMDSCs) could promote follicle development in patients with impaired ovarian functions. DESIGN: Experimental design. SETTING: University research laboratories. ANIMAL(S): Immunodeficient NOD/SCID female mice. INTERVENTION(S): Human BMDSCs were injected into mice with chemotherapy-induced ovarian damage and into immunodeficient mice xenografted with human cortex from poor-responder patients (PRs). MAIN OUTCOME MEASURE(S): Follicle development, ovulation, and offspring. Apoptosis, proliferation, and vascularization were evaluated in mouse and human ovarian stroma. RESULT(S): Fertility rescue and spontaneous pregnancies were achieved in mice ovaries mimicking PRs and ovarian insufficiency, induced by chemotherapy, after BMDSC infusion. Furthermore, BMDSC treatment resulted in production of higher numbers of preovulatory follicles, metaphase II oocytes, 2-cell embryos, and healthy pups. Stem cells promoted ovarian vascularization and cell proliferation, along with reduced apoptosis. In xenografted human ovarian tissues from PRs, infusion of BMDSCs and their CD133+ fraction led to their engraftment close to follicles, resulting in promotion of follicular growth, increases in E2 secretion, and enhanced local vascularization. CONCLUSION(S): Our results raised the possibility that promoting ovarian angiogenesis by BMDSC infusion could be an alternative approach to improve follicular development in women with impaired ovarian function. CLINICAL TRIAL REGISTRATION NUMBER: NCT02240342.

3D NIR-II Molecular Imaging Distinguishes Targeted Organs with High-Performance NIR-II Bioconjugates.

Greatly reduced scattering in the second near-infrared (NIR-II) region (1000-1700 nm) opens up many new exciting avenues of bioimaging research, yet NIR-II fluorescence imaging is mostly implemented by using nontargeted fluorophores or wide-field imaging setups, limiting the signal-to-background ratio and imaging penetration depth due to poor specific binding and out-of-focus signals. A newly developed high-performance NIR-II bioconjugate enables targeted imaging of a specific organ in the living body with high quality. Combined with a home-built NIR-II confocal set-up, the enhanced imaging technique allows 900 µm-deep 3D organ imaging without tissue clearing techniques. Bioconjugation of two hormones to nonoverlapping NIR-II fluorophores facilitates two-color imaging of different receptors, demonstrating unprecedented multicolor live molecular imaging across the NIR-II window. This deep tissue imaging of specific receptors in live animals allows development of noninvasive molecular imaging of multifarious models of normal and neoplastic organs in vivo, beyond the traditional visible to NIR-I range. The developed NIR-II fluorescence microscopy will become a powerful imaging technique for deep tissue imaging without any physical sectioning or clearing treatment of the tissue.

Oocyte vitrification versus ovarian cortex transplantation in fertility preservation for adult women undergoing gonadotoxic treatments: a prospective cohort study.

OBJECTIVE: To compare the efficacy of oocyte vitrification (OV) with that of ovarian cortex cryopreservation and transplantation (OCT) in women undergoing gonadotoxic treatments. DESIGN: Prospective observational cohort study. SETTING: Not applicable. PATIENT(S): Candidates for chemo-/radiotherapy who joined our fertility preservation (FP) program were included in this study between 2005 and 2015. One cohort included 1,024 patients undergoing OV; the other cohort included 800 patients undergoing OCT. INTERVENTION(S): OV using the cryotop device and OCT using a slow freezing protocol. MAIN OUTCOME MEASURE(S): Live-birth rate (LBR) and clinical pregnancy rate (CPR). RESULT(S): Basal antimüllerian hormone levels of the patients revealed no differences in ovarian reserve before FP (OV, 11.6 pM [5.4-24.7]; OCT, 11.8 pM [6.4-21.9]). In the OV cohort, 49 patients used the vitrified oocytes after a mean storage time of 3.9 years. In the OCT cohort, 44 sought pregnancy after a mean storage time of 5.5 years. A trend toward higher CPR and LBR (per patient) was observed in the OV group (risk ratio [RRCPR], 1.31 [95% confidence interval, 0.90-1.92]; RRLBR 1.39 [95% confidence interval, 0.95-2.03]), although differences were not statistically significant. In the OCT group, 46.7% of pregnancies occurred spontaneously and no pregnancy was achieved when the tissue was harvested beyond the age of 36 years. All patients except three undergoing OCT resumed or improved endocrine ovarian function. CONCLUSION(S): Although we observed a trend toward higher LBR after OV, OCT is a very effective method to preserve fertility, allows for natural pregnancy, and restores ovarian function. In clinical scenarios where OV is not feasible, OCT remains the FP technique of choice and should no longer be considered experimental.