Fertility Preservation in Patients with Severe Ovarian Dysfunction

Ovarian function progressively declines during aging and in some pathophysiological conditions including karyotype abnormality, autoimmune diseases, ...

International University of Health and Welfare School of Medicine

Is ovarian tissue cryopreservation using vitrification followed by in vitro activation (IVA) of dormant follicles a potential approach for infertility treatment of patients with primary ovarian insufficiency (POI)?

Successful fertility preservation following ovarian tissue vitrification in patients with primary ovarian insufficiency.

Ovary transplantation: to activate or not to activate.

The mammalian ovary is an organ that controls female germ cell development, storing them and releasing mature oocytes for transporting to the oviduct. During the fetal stage, female germ cells change from a proliferative state to meiosis before forming follicles with the potential for the growth of surrounding somatic cells. Understanding of molecular and physiological bases of germ cell development in the fetal ovary contributed not only to the elucidation of genetic disorders in primary ovarian insufficiency (POI), but also to the advancement of novel treatments for patients with POI. Accumulating evidence indicates that mutations in ,  and genes are associated with POI. In addition, cell biology studies revealed the important roles of these genes as essential translational factors for germ cell development. Recent insights into the role of the PI3K (phosphatidylinositol 3-kinase)-Akt signaling pathway in primordial follicle activation allowed the development of a new infertility treatment, IVA ( activation), leading to successful pregnancy/delivery in POI patients. Furthermore, elucidation of genetic dynamics underlying female germ cell development could allow regeneration of oocytes from ES (embryonic stem)/iPS (induced pluripotent stem) cells in mammals. The purpose of this review is to summarize basic findings related to female germ cell development and potential clinical implications, especially focusing on POI etiologies. We also summarize evolving new POI therapies based on IVA as well as oocyte regeneration.

Disorganization of the germ cell pool leads to primary ovarian insufficiency.

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.

Heterosis extends the reproductive ability in aged female mice†.

Do gut microbiota associate with the ovulatory cycle in women showing normogonadotrophic anovulation? In humans, the gut microbiota affects diverse physiological functions and dysbiosis (microbial imbalance) may lead to pathological syndromes. However, there is comparatively little information on the relevance of gut microbiota to reproductive functions in women. Here, a group of women with idiopathic chronic anovulation were examined, who do not exhibit any apparent endocrinological disorder, as they are suitable for investigating the relationship between intestinal bacteria and ovulatory disorders.

Distinctive subpopulations of the intestinal microbiota are present in women with unexplained chronic anovulation.

The recently developed in-vitro activation (IVA) approach provides a promising infertility treatment for patients with premature ovarian insufficiency. The IVA method promotes growth of residual ovarian follicles following ovarian tissue fragmentation leading to Hippo signalling disruption, together with in-vitro incubation with Akt stimulators. As poor ovarian response (POR) patients with decreased ovarian reserve (DOR) have multiple secondary follicles, this study tested whether Hippo signalling disruption alone using in-vitro ovarian cortical fragmentation, followed by autologous grafting, was sufficient to promote follicle growth.

Drug-free in-vitro activation of follicles for infertility treatment in poor ovarian response patients with decreased ovarian reserve.

The ovarian follicle pool size is limited; it decreases with age and following germ cell-damaging chemo- or radiation therapies. Due to a trend of delaying child-bearing age in the modern society, it is important to investigate the possibility to maintain the follicle reserve for middle-aged women and cancer-bearing patients subject to therapies. Earlier studies demonstrated the important role of the mammalian targets of the rapamycin (MTOR) signaling pathway in the activation of primordial follicles and suggested that treatment with the MTOR inhibitor rapamycin could maintain the follicle pool in rodents. Here, we confirmed the ability of rapamycin treatment for 3 weeks to suppress primordial follicle development and to maintain follicle pool size in mice. We further demonstrated that the developmental potential of oocytes was not affected by rapamycin treatment and the effect of rapamycin to decrease initial follicle recruitment is reversible. Using human ovarian cortical fragments grafted into immune-deficient mice, we demonstrated the ability of rapamycin to suppress follicle growth from the primordial stage. Our studies provide the basis for further studies on the possibility of using MTOR inhibitors to maintain follicle reserve in middle-aged women and cancer patients before/during germ cell-damaging therapies.

Rapamycin treatment maintains developmental potential of oocytes in mice and follicle reserve in human cortical fragments grafted into immune-deficient mice.

The Hippo signaling pathway, which is important in organ size regulation, is present in organisms from the fly to mammals. Disruption of the Hippo signaling pathway leads to increased nuclear translocation of the effector Yes-associated protein (YAP), resulting in the expression of cystein-rich 61, connective tissue growth factor, and nephroblastoma overexpressed (CCN) growth factors and baculoviral inhibitors of apoptosis repeat containing (BIRC) apoptosis inhibitors to increase organ sizes. Furthermore, genome-wide knockdown of genes in insect cells demonstrated that actin polymerization promoted nuclear translocation of YAP. In the mammalian ovary, we demonstrated the expression of Hippo signaling pathway genes and showed that ovarian fragmentation increased actin polymerization, leading to YAP nuclear translocation and increased expression of cystein-rich 61, CCN growth factors and BIRC apoptosis inhibitors, followed by enhanced follicle growth. Here we summarize evidence suggesting the role of mechanical stress on follicle growth in the ovary and describe recent use of ovary-damaging procedures to treat ovarian infertility. Ovarian fragmentation, together with in vitro incubation with Akt-stimulating drugs, formed the basis of an in vitro activation (IVA) therapy to treat patients with premature ovarian insufficiency, whereas ovarian fragmentation alone (drug-free IVA) was successful in treating patients with premature ovarian insufficiency with recent menses cessation. For middle-aged women with poor ovarian responses and diminished ovarian reserve, drug-free IVA was also effective in promoting follicle growth for infertility treatment. In addition, an in vivo follicle activation approach based on laparoscopic ovarian incision showed promise for patients with resistant ovary syndrome. With initial success using mechanical disruption approaches, future investigation could evaluate possibilities to refine mechanical methods and to locally administer actin polymerization-enhancing drugs for ovarian infertility treatment.

Hippo signaling disruption and ovarian follicle activation in infertile patients.

To demonstrate our procedures of drug-free in vitro activation (IVA) for treating patients with premature ovarian insufficiency (POI) or diminished ovarian reserve (DOR), as well as the laparoscopic ovarian incision (LOI) procedure for treating patients with resistant ovary syndrome (ROS).

Surgical approaches of drug-free in vitro activation and laparoscopic ovarian incision to treat patients with ovarian infertility.

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.

Kazuhiro Kawamura

Advanced Reproduction Medicine Research Center,
Department of Obstetrics and Gynecology,
Advanced Reproduction Medicine Research Center, Department of Obstetrics and Gynecology

International University of Health and Welfare School of Medicine

Fertility Preservation in Patients with Severe Ovarian Dysfunction

Kazuhiro Kawamura

.css-o250i3{font-size:18px;font-family:Open Sans,sans-serif;line-height:21.6px;}Advanced Reproduction Medicine Research Center,Department of Obstetrics and Gynecology,Advanced Reproduction Medicine Research Center, Department of Obstetrics and Gynecology

International University of Health and Welfare School of Medicine

Fertility Preservation in Patients with Severe Ovarian Dysfunction

Advanced Reproduction Medicine Research Center,
Department of Obstetrics and Gynecology,
Advanced Reproduction Medicine Research Center, Department of Obstetrics and Gynecology