Effectiveness of supplement ingredients on infertility treatment in advanced aged women.

AIM: Despite the remarkable progress made in reproductive medical technology in recent years, there has been no improvement in overall pregnancy and birth rates for the rising number of infertile patients. This is thought to be due to the increase in intractable infertility with ovarian dysfunction, as the desired age of pregnancy has increased for women. The aim of this article is to review preclinical studies that used laboratory animals and other tools to examine the effectiveness of diverse supplement ingredients on age-related ovarian dysfunction as well as recent human clinical trials using supplement ingredients. METHOD: We summarized the articles discussing the effectiveness of supplement ingredients on infertility treatment in advanced-aged women by searching PubMed, Cochrane, EMBASE, and Google Scholar databases until December 2022. RESULTS: Supplements are relatively inexpensive and convenient for patients, as they can be purchased at the will of the individual and from among multiple options. Although supplements have been demonstrated to have certain effects in animal studies, evidence of their effectiveness in humans is either lacking or insufficient for reaching a definite conclusion. This may be due to the lack of standardized diagnostic criteria for ovarian dysfunction and poor responders, unclear optimal dosages and duration of supplement intake, and well-designed randomized clinical trials. CONCLUSION: Additional lines of evidence on the effectiveness of supplements in patients with ovarian dysfunction at an older age need to be accumulated in the future.

Improvement of oocyte quality through the SIRT signaling pathway.

Background: Oocyte quality is one of the major deciding factors in female fertility competence. Methods: PubMed database was searched for reviews by using the following keyword "oocyte quality" AND "Sirtuins". The methodological quality of each literature review was assessed using the Preferred Reporting Items for Systematic reviews and Meta-Analyses (PRISMA) 2020 statement. Main Findings: Oxidative stress has been recognized as the mechanism attenuating oocyte quality. Increasing evidence from animal experiments and clinical studies has confirmed the protective roles of the sirtuin family in improving oocyte quality via an antioxidant effect. Conclusion: The protective roles in the oocyte quality of the sirtuin family have been increasingly recognized.

Placensin is a glucogenic hormone secreted by human placenta.

FBN1 encodes asprosin, a glucogenic hormone, following furin cleavage of the C-terminus of profibrillin 1. Based on evolutionary conservation between FBN1 and FBN2, together with conserved furin cleavage sites, we identified a peptide hormone placensin encoded by FBN2 based on its high expression in trophoblasts of human placenta. In primary and immortalized murine hepatocytes, placensin stimulates cAMP production, protein kinase A (PKA) activity, and glucose secretion, accompanied by increased expression of gluconeogenesis enzymes. In situ perfusion of liver and in vivo injection with placensin also stimulate glucose secretion. Placensin is secreted by immortalized human trophoblastic HTR-8/SVneo cells, whereas placensin treatment stimulates cAMP-PKA signaling in these cells, accompanied by increases in MMP9 transcripts and activities, thereby promoting cell invasion. In pregnant women, levels of serum placensin increase in a stage-dependent manner. During third trimester, serum placensin levels of patients with gestational diabetes mellitus are increased to a bigger extent compared to healthy pregnant women. Thus, placensin represents a placenta-derived hormone, capable of stimulating glucose secretion and trophoblast invasion.

Effects of Hochuekkito, a traditional Japanese medicine (Kampo), on reproduction of aging female mice.

PURPOSE: To determine the potentials of Hochuekkito (HET) treatment for aging infertility. METHODS: Mice at 36 weeks of age were fed without (control, n = 40) or with low (100 mg/kg/day, n = 24) and high (1000 mg/kg/day, n = 38) doses of HET for 12 weeks. Aging animals at 48 weeks of age were used for in vitro fertilization-embryo transfer (IVF-ET), and their ovaries were subjected to histological and quantitative inflammation analyses. RESULTS: HET administration decreased transcript levels of ovarian inflammatory markers, interleukin 6 (IL-6), IL-1ß, tumor necrosis factor (TNF)-α, and interferon-gamma (IFN-γ) but suppressed ovulation rates and the number of ovulated oocytes in aging mice. Furthermore, HET treatment decreased the rates of oocytes maturation and fertilization and the cumulus-cell expression of TNF-α-induced protein 6 and epidermal growth factor receptor. After IVF-ET, no improvement of declined live offspring rate by aging was achieved by HET administration, although there were no adverse effects on embryo development and implantation as well as gross morphology and bodyweight of pups. CONCLUSION: Present study indicated HET treatment interfered with ovulation and fertilization in aging mice without affecting ovarian follicle development. No improvement on the age-associated decline of live offspring rate and follicle development was achieved after HET treatment.

Heterosis extends the reproductive ability in aged female mice†.

Heterosis is the beneficial effect of genetical heterogeneity in animals and plants. Although heterosis induces changes in the cells and individual abilities, few reports have described the effect of heterosis on the female reproductive ability during aging. In this study, we investigated the reproductive capability of genetically heterogeneous (HET) mice established by the four-way crossing of C57BL/6N, BALB/c, C3H/He, and DBA/2. We found the HET females naturally and repeatedly produced offspring, even in old age (14-18 months of age). We also found that HET females showed a significantly enlarged body and organ sizes in both youth and old age. In histological analyses, the numbers of primordial follicles, primary follicles, secondary follicles, and corpora lutea were significantly increased in the old ovaries of HET females compared with those in inbred C57BL/6 mice of the same age. In vitro fertilization experiments revealed that aged HET oocytes showed identical rates of fertilization, early development, and birth compared to those of young and old C57BL/6 oocytes. We further found the significantly increased expression of sirtuin genes concomitant with the up-regulation of R-spondin2 in old HET ovaries. These results confirm the novel phenotype, characterized by fertility extension and follicular retention due to heterosis, in old HET females. The HET female will be a valuable model for clarifying the mechanism underlying the effect of heterosis in the field of reproduction.

Preliminary human application of optical coherence tomography for quantification and localization of primordial follicles aimed at effective ovarian tissue transplantation.

PURPOSE: The purpose of this study was to evaluate the possible clinical application of optical coherence tomography for assessing ovarian reserve in individual specimens of human ovarian tissue for fertility preservation. METHODS: Ovarian tissue examination by optical coherence tomography was performed before ovarian tissue cryopreservation. Three of the four subjects had hematological disease or cancer, and they faced a threat to their fertility due to impending chemotherapy. One patient underwent ovarian tissue extraction for in vitro activation of dormant follicles as fertility treatment. RESULTS: The current full-field optical coherence tomography technique can detect primordial follicles in non-fixed and non-embedded human ovarian tissue. These images are well correlated with histological evaluation and the ovarian reserve test, including follicle counts. CONCLUSION: It was demonstrated that optical coherence tomography could assess localization of primordial follicles and ovarian reserve in specimens of non-fixed human ovarian cortex, although optimization for examination of human ovarian tissue is needed for clinical application. Additionally, this technique holds the possibility of assessing the ovarian reserve of patients with unevaluable ovarian reserve. TRIAL REGISTRATION NUMBER: UMIN000023141.

Actin polymerization-enhancing drugs promote ovarian follicle growth mediated by the Hippo signaling effector YAP.

Hippo signaling pathway consists of conserved serine/threonine kinases to maintain optimal organ sizes. Studies have demonstrated that fragmentation of murine ovaries increases actin polymerization and disrupts Hippo signaling, leading to nuclear translocation of Hippo signaling effector Yes-associated protein (YAP) in ovarian follicles and follicle growth. For patients with polycystic ovarian syndrome showing follicle arrest, ovarian wedge resection and laser drilling promote follicle growth. Because these damaging procedures likely involve actin polymerization, we tested whether actin polymerization-promoting drugs could promote YAP translocation and stimulate follicle growth. Treatment of murine ovaries with µM Jasplakinolide (JASP), an actin polymerization-promoting cyclic peptide, or sphingosine-1-phosphate (S1P), a follicular fluid constituent known to promote actin polymerization, increased the conversion of globular actin to the filamentous form, followed by increased nuclear YAP and expression of downstream connective tissue growth factor (CCN2). After short-term treatments with JASP or S1P, in vitro cultured and in vivo grafted ovaries showed follicle growth. Furthermore, induction of constitutively active YAP in ovarian grafts of transgenic mice enhanced follicle development, whereas treatment of human ovarian cortices with JASP or S1P increased CCN2 expression. Thus, JASP and S1P stimulate follicle growth and are potential therapeutic agents for treating polycystic ovarian syndrome and other ovarian disorders.

Hippo signaling disruption and Akt stimulation of ovarian follicles for infertility treatment.

Primary ovarian insufficiency (POI) and polycystic ovarian syndrome are ovarian diseases causing infertility. Although there is no effective treatment for POI, therapies for polycystic ovarian syndrome include ovarian wedge resection or laser drilling to induce follicle growth. Underlying mechanisms for these disruptive procedures are unclear. Here, we explored the role of the conserved Hippo signaling pathway that serves to maintain optimal size across organs and species. We found that fragmentation of murine ovaries promoted actin polymerization and disrupted ovarian Hippo signaling, leading to increased expression of downstream growth factors, promotion of follicle growth, and the generation of mature oocytes. In addition to elucidating mechanisms underlying follicle growth elicited by ovarian damage, we further demonstrated additive follicle growth when ovarian fragmentation was combined with Akt stimulator treatments. We then extended results to treatment of infertility in POI patients via disruption of Hippo signaling by fragmenting ovaries followed by Akt stimulator treatment and autografting. We successfully promoted follicle growth, retrieved mature oocytes, and performed in vitro fertilization. Following embryo transfer, a healthy baby was delivered. The ovarian fragmentation-in vitro activation approach is not only valuable for treating infertility of POI patients but could also be useful for middle-aged infertile women, cancer patients undergoing sterilizing treatments, and other conditions of diminished ovarian reserve.

The role of menstrual cycle phase and AMH levels in breast cancer patients whose ovarian tissue was cryopreserved for oncofertility treatment.

PURPOSE: To determine the factors that affect oocyte extraction efficiency when using the "combined procedure". In the present "combined procedure" ovarian tissue cryopreservation and oocyte extraction from an isolated ovary, later used in In Vitro Maturation (IVM), are performed concurrently. METHODS: Data were analyzed retrospectively and obtained from the clinical records of 27 young breast cancer patients referred for fertility preservation. RESULTS: The patients' mean age was 33.7 (±3.8) years, mean serum anti-Müllerian hormone (AMH) concentration was 3.5 (±2.1) ng/ml, and mean number of extracted oocytes was 8.3 (±6.1). The phase of menstruation (follicular or luteal) did not affect either the number of oocytes extracted (P = 0.99) nor oocyte survival or maturation rates. Likewise, the number of oocytes that could be extracted was not affected by the type of laparoscopic procedure (multiple-port or single-incision laparoscopy; P = 0.94) or the molecular subtype of breast cancer (either Luminal A or B; P = 0.52). Analysis revealed that the number of extracted oocytes was well-correlated with the patient's AMH serum level and age (coefficient of correlation: 0.60 and -0.48, respectively). CONCLUSION: We conclude that the outcome of the "combined procedure" primarily depends upon the patient's serum AMH level and age. Importantly, the "combined procedure" may be used during any phase of the menstrual cycle to preserve the fertility of breast cancer patients.

Successful differentiation to T cells, but unsuccessful B-cell generation, from B-cell-derived induced pluripotent stem cells.

Forced expression of certain transcription factors in somatic cells results in generation of induced pluripotent stem (iPS) cells, which differentiate into various cell types. We investigated T-cell and B-cell lineage differentiation from iPS cells in vitro. To evaluate the impact of iPS cell source, murine splenic B-cell-derived iPS (B-iPS) cells were generated after retroviral transduction of four transcription factors (Oct4, Sox2, Klf4 and c-Myc). B-iPS cells were identical to embryonic stem (ES) cells and mouse embryonic fibroblast (MEF)-derived iPS cells in morphology, ES cell marker expression as well as teratoma and chimera mouse formation. Both B-iPS and MEF-derived iPS cells differentiated into lymphocytes in OP9 co-culture systems. Both efficiently differentiated into T-cell lineage that produced IFN-γ on T-cell receptor stimulation. However, iPS cells including B-iPS cells were relatively resistant to B-cell lineage differentiation. One of the reasons of the failure of B-cell lineage differentiation seemed due to a defect of Pax5 expression in the differentiated cells. Therefore, current in vitro differentiation systems using iPS cells are sufficient for inducing T-cell but not B-cell lineage.

Short-term resveratrol treatment restored the quality of oocytes in aging mice.

The quality of oocytes declines by aging, resulting in their low competences for fertility. Here, resveratrol treatment showed increases in the rates of implantation and live offspring as well as decreases in the abortion rate as short as one week after treatment, although the number of ovulated oocytes and the rates of fertilization and blastocyst formation were not changed following resveratrol treatment. Resveratrol treatment did not cause abnormalities mouse estrous cycles and body weights. No abnormality was detected in both fetuses and placentas after 22 weeks of resveratrol treatment and the fetuses had normal fertility. Positive correlations were found between serum resveratrol levels and pregnancy and live offspring rates as well as ovarian expression levels of Sirt1, Sirt3, Sirt4, Sirt5, and Sirt7. The mitochondrial membrane potential and ATP content but not copy number of mitochondrial DNA in oocytes was increased in aging mice with resveratrol treatment. In conclusion, we demonstrated the restoration of oocyte quality in aging mice in addition to the prevention of their quality decline during aging by restoring mitochondrial functions by resveratrol treatment without adverse effects in the animals and their offspring.

CXCL5-CXCR2 signaling is a senescence-associated secretory phenotype in preimplantation embryos.

Pregnancy rate of women decreases with age due to declining quality of oocytes and embryos. However, there is no established method to improve pregnancy rate in aging women. In this study, we identified a senescence-associated secretory phenotype (SASP) factor partially responsible for the decline in embryo implantation potential. Based on microarray analysis using young and aging human embryos at the same morphological grade, 702 genes showed >fivefold increases in aging human blastocysts. Among these genes, C-X-C motif chemokine 5 (CXCL5) showed 7.7-fold increases in aging human blastocysts. However, no-age-dependent changes in expression of the CXCR2, the cognate receptor for CXCL5, were found. In aging mice, Cxcl5 transcript levels were also increased in oocytes and embryos. Treatment of young mouse embryos with CXCL5 decreased implantation rates, together with increased expression of aging markers (P53, P21, Pai-1, and Il-6). Moreover, CXCL5 treatment suppressed trophoblast outgrowth in young mouse blastocysts. Conversely, suppression of CXCL5-CXCR2 signaling in aging mouse embryos using neutralizing antibodies and a receptor antagonist improved the implantation rate, leading to increases in pregnancy and delivery of normal pups. The gene expression pattern of these embryos was comparable to that in young mouse embryos showing enriched cell proliferation-related pathways. In conclusion, we identified CXCL5 as a SASP factor in human and mouse embryos and suppression of CXCL5-CXCR2 signaling during embryo culture improved pregnancy success in aging mice. Future analysis on CXCL5-CXCR2 signaling suppression in human embryos could be the basis to improve embryo development and pregnancy outcome in middle-aged infertile patients.

Accuracy and safety verification of ovarian reserve assessment technique for ovarian tissue transplantation using optical coherence tomography in mice ovary.

Except for histological study, there are currently no suitable techniques available for the detection and identification of primordial follicles in ovary of primary ovarian insufficiency patients who have undetectable AMH levels. Also, the ability to locate and quantify follicles on ovarian cortex strips, without fixation, is valuable for patients who could undergo subsequent successful ovarian tissue transplantation. Although optical coherence tomography (OCT) is a well-established high resolution imaging technique without fixation commonly applied in biomedicine, few reports are available on ovarian tissue imaging. In present study, we established standard OCT follicle images at each developmental stage, including the primordial follicle, and demonstrated the efficacy of OCT to estimate IVF outcome in transplanted mice ovary like ovarian reserve tests. Unfortunately, the current commercial OCT could not be used to accurate follicle count the number of follicles for whole ovary, because the maximum depth of examination was 100 µm. And we demonstrated the safety of OCT examination, it did not affect IVF outcome and birth defect rate, and reproductive ability. Although there is room for improvement, these findings will be first step to bring OCT examination a step closer to clinical application for measuring true ovarian reserve and localizing follicles.

In Vitro Activation of Follicles and Fresh Tissue Auto-transplantation in Primary Ovarian Insufficiency Patients.

CONTEXT: Recently, two patients with primary ovarian insufficiency (POI) delivered healthy babies after in vitro activation (IVA) treatment followed by auto-transplantation of frozen-thawed ovarian tissues. OBJECTIVE: This study sought to report the first case of live birth after IVA treatment following fresh ovarian tissue grafting in patients with POI, together with monitoring of follicle development and serum hormonal changes. DESIGN: This was a prospective observational cohort study. SETTING: We performed IVA treatment in 14 patients with POI with mean age of 29 years, mean duration since last menses of 3.8 years, and average basal FSH level of 94.5 mIU/mL. INTERVENTIONS: Prior to IVA treatment, all patients received routine hormonal treatments with no follicle development. We removed one ovary from patients with POI and treated them with Akt stimulators. We improved upon early procedures by grafting back fresh tissues using a simplified protocol. MAIN OUTCOME MEASURES: In six of the 14 patients (43%), a total of 15 follicle development waves were detected, and four patients had successful oocyte retrieval to yield six oocytes. For two patients showing no spontaneous follicle growth, human menopausal gonadotropin treatment induced follicle growth at 6-8 months after grafting. After vitro fertilization of oocyte retrieved, four early embryos were derived. Following embryo transfer, one patient became pregnant and delivered a healthy baby boy, with three other embryos under cryopreservation. CONCLUSION: IVA technology can effectively activate residual follicles in some patients with POI and allow them to conceive their own genetic offspring. IVA may also be useful for treating patients with ovarian dysfunction including aging women and cancer survivors.

Development of reconstituted embryos derived from somatic cell nuclei in the rabbit.

Production of cloned laboratory animals is helpful in the establishment of medical models. In this study, we examined to produce reconstituted embryos derived from somatic cell nuclei, and to establish embryonic stem (ES) cell lines from the embryo in rabbits. Metaphase II (M-II) oocytes from superovulated rabbit were used as nuclear recipients. Nuclear donor cells were fibroblasts collected from a Dutch Beleted rabbit. The M-II chromosome and the 1st polar body were aspirated, and a fibroblast was inserted into the perivitelline space of the enucleated oocyte. The pairs were electrofused for cell membrane fusion using a cell fusion apparatus, and reconstituted embryos were produced. The embryos were activated and cultured in modified HTF medium and DMEM. The embryos developed to the blastocyst stage were removed their zona pellucida, and they were cultured on the feeder cell layer. As a result of having observed development of reconstituted embryos, 21.2% of the embryos were developed to the blastocyst stage. In the embryos cultured on the feeder cells, the adhesion on feeder cells was observed. We obtained inner cell mass (ICM) colony derived from reconstituted embryos. At present, we are investigating to establish the ES cell lines derived from the embryos reconstituted by nuclear transfer.

Analysis of late-onset ovarian insufficiency after ovarian surgery: retrospective study with 75 patients of post-surgical ovarian insufficiency.

The primary objectives of the present study are to determine the period of onset of ovarian insufficiency after surgery and to confirm potential risk factors for ovarian insufficiency after surgery for the removal of benign ovarian cysts. Data were obtained from 75 patients who underwent surgery for benign ovarian cysts prior to the onset of ovarian insufficiency. Our analysis included 835 ovarian insufficiency patients who were referred to our institution from July 2003 to July 2013. Several epidemiological parameters of ovarian insufficiency after surgery (age at operation, period of onset of ovarian insufficiency, operation procedure, and pathological diagnosis) were investigated. Of the 835 patients who had ovarian insufficiency, 75 patients (9.0%) underwent ovarian surgery before the onset of ovarian insufficiency. Of those 75 patients, 66 patients (88.0%) underwent cystectomy. For the majority of the 75 patients the surgical indication was the presence of endometriotic cysts (57 patients; 76.0%). Twelve patients (16.0%) underwent multiple surgeries (all bilateral cystectomies). The mean age of the patients at the time of surgery was 27.8±5.5 years-old, and the mean period of onset of ovarian insufficiency was 5.8±3.8 years. In patients with cystectomy, the patient's age at the time of surgery and period of onset of ovarian insufficiency was well-correlated (coefficient of correlation; hemilateral endometriotic cystectomy: -0.64, bilateral endometriotic cystectomy: -0.61, and multiple endimetriotic cystectomy: -0.40). We found that cystectomy of endometriotic cysts is the potential risk factor for ovarian insufficiency after surgery, at times, the onset of ovarian insufficiency long after cystectomy. Therefore, it is important to monitor ovarian reserve for an extended period of time after ovarian surgery. It is particularly important to monitor ovarian reserve long-term for patients who wish to conceive in the future and to suggest a variety of infertility treatments appropriate for their ovarian reserve.

C-type natriuretic peptide stimulates ovarian follicle development.

C-type natriuretic peptide (CNP) encoded by the NPPC (Natriuretic Peptide Precursor C) gene expressed in ovarian granulosa cells inhibits oocyte maturation by activating the natriuretic peptide receptor (NPR)B (NPRB) in cumulus cells. RT-PCR analyses indicated increased NPPC and NPRB expression during ovarian development and follicle growth, associated with increases in ovarian CNP peptides in mice. In cultured somatic cells from infantile ovaries and granulosa cells from prepubertal animals, treatment with CNP stimulated cGMP production. Also, treatment of cultured preantral follicles with CNP stimulated follicle growth whereas treatment of cultured ovarian explants from infantile mice with CNP, similar to FSH, increased ovarian weight gain that was associated with the development of primary and early secondary follicles to the late secondary stage. Of interest, treatment with FSH increased levels of NPPC, but not NPRB, transcripts in ovarian explants. In vivo studies further indicated that daily injections of infantile mice with CNP for 4 d promoted ovarian growth, allowing successful ovulation induction by gonadotropins. In prepubertal mice, CNP treatment alone also promoted early antral follicle growth to the preovulatory stage, leading to efficient ovulation induction by LH/human chorionic gonadotropin. Mature oocytes retrieved after CNP treatment could be fertilized in vitro and developed into blastocysts, allowing the delivery of viable offspring. Thus, CNP secreted by growing follicles is capable of stimulating preantral and antral follicle growth. In place of FSH, CNP treatment could provide an alternative therapy for female infertility.