Research progress of in vitro activation mechanism and clinical application of female ovarian tissue

The activation and development of primordial follicles is the key to the maturation of female gametes.Premature ovarian insufficiency(POI)patients are unable to complete the primordial follicle activation and development due to follicular dormancy and unbalanced developmental regulation in the body,leading to female infertility.Ovarian tissue in vitro activation(IVA)technology has become a new way to solve the problem of patients who cannot auto-activate primordial follicles to obtain their own mature oocytes.In IVA research,signaling pathways such as PI3K/PTEN/Akt and Hippo have become the focus of current research.This review will describe the relevant research progress and clinical application of the IVA mechanism,and provide a reference for clinical research on ovarian tissue culture and activation in vitro.

Detection of HBsAg, HBcAg, and HBV DNA in ovarian tissues from patients with HBV infection

AIM: To investigate the presence of HBsAg, HBcAg, and HBV DNA in ovarian tissues from patients with HBV infection.METHODS: HBsAg and HBcAg were examined in ovarian biopsy tissues from 26 patients with HBV infection by immunocytochemistry, and HBV DNA was detected in ovarian tissues by PCR.RESULTS: HBsAg and HBcAg were present with the same positive rate of 34.6% (9/26). The total positive rate was 46.2% (12/26). HBsAg and HBcAg were positive in 6 (23.1%) of the 26 patients. Brown positive particles were diffusely distributed in ovarian cells. The positive rate of HBV DNA was 58.3% (7/12).CONCLUSION: HBsAg, HBcAg, and HBV DNA can be detected in ovarian tissues from patients with HBV infection. The presence of HBsAg and HBcAg in ovarian tissues does not correlate with the HBV markers in serum.

Prevention and treatment of iatrogenic premature ovarian insufficiency:r interpretation of the first Chinese guideline on ovarian tissue cryopreservation and transplantation

In recent years,with the rapid development of medical research,cancer diagnosis and treatment technology have significantly improved young cancer patient’s survival rate.Anticancer therapy such as chemotherapy,radiotherapy,or hematopoietic stem cell transplantation can lead to premature ovarian insufficiency.The endocrine and reproductive function of the ovary is critical to women’s physical and mental health.Ovarian tissue cryopreservation and transplantation can protect not only female fertility but also preserve ovarian endocrine function.This paper interprets the guidelines for ovarian tissue cryopreservation and transplantation issued by the Chinese Society of Gynecological Endocrinology affiliated to the International Society of Gynecological Endocrinology.The purpose of this guideline’s interpretation is to promote more medical workers to understand the technology of ovarian tissue cryopreservation and transplantation,which can provide patients with more choices of fertility protection methods and improve their quality of life.

Morphological and ultrastructural changes in rat ovarian tissues after cryopreservation

Objective: The aim of the study was to observe and compare the effects of cryopreservation and thawing meth- ods on rat ovarian tissues. Methods: Twenty 5-6 weeks old SPF-SD female rats were randomly divided into two groups, with ten rats in each group. Freshly isolated ovaries saved as a control (group 1: fresh ovaries) in formalin-fixed or vitrified immediately after dissection (group 2: vitrified ovaries). Ovaries in vitrified group were processed into thin slices then cryo- preserved, stored in liquid nitrogen for 21 days, rapidly thawed and grossly examined. All of the collected ovaries underwent hematoxylin and eosin-stained paraffin serial sections and observed the microscopic evaluation in vitrified ovaries. Results: Grossly the vitrified ovaries turned pale color and the size was same as before freeze. The vitrified ovarian tissue had normal anatomical structures of cortex and medulla under the microscope and had no difference with the fresh control ovarian tis- sue. The number and distribution of the follicles were similar with the fresh ovarian tissue, but had smaller size and the gap between oocyte and the surrounding granulosa cells was increased. Few ooctyes were in irregular appearance however the morphology of follicular cells did not give a different appearance as compared to the fresh control ovarian tissue. Conclusion: Cryopreservation of ovarian tissues by vitrification method has some detrimental effect on the morphology of follicles but does not induce negative impact on the number, density and survival of the primordial ovarian follicles. However the whole follicle anatomical structures also had no significant changes.

Morphology and survival of cryopreserved-thawed ovarian tissues after heterotopic autotransplantation

Objective： The aim of our study was to observe the survival and morphological changes of thawed ovarian tis- sues after heterotopic transplantation. Methods： Twenty SPF-SD female rats （5-6 weeks old） were equally randomized into the control group and experimental group. In control group, the freshly isolated ovaries were fixed in formalin. In experimental group, the freshly isolated ovaries were vitrified immediately and cut into thin slices. After stored in liquid nitrogen for 21 days, the tissues of experimental group were rapidly thawed and transplanted into back muscles of rats for 2 or 4 weeks, respectively. After that, all rats in experimental group were sacrificed and the ovarian tissues were collected and fixed in 4% formaldehyde solution. Then the ovarian tissues were stained with HE and observed under the light confocal microscope. Re- suits： With the naked eyes, there was no specific alteration except the size reduction with color changing. Under microscopy, we found normal cortex and medulla in the ovary, and the primordial follicles and follicles in various stages were observed in the cortex. The normal oocytes in ovarian tissues of experimental group were significant decreased than in the control group. Conclusion： The ovarian tissues survive well in experimental group and there is no significant difference in the proportion of follicles between different times （2 and 4 weeks） after grafting. Our results suggest that thawed ovarian tissues could survive after heterotopic transplantation into back muscles of rat models and maintain their morphology and function.

Effects of heterotopic autotransplantation of cryopreserved-thawed ovarian tissues on sex hormone level

Objective: The aim of our study was to measure and compare the serum hormone level of transplant group with blank control and castrated control groups after heterotopic autotransplantation of cryopreserved-thawed ovarian tissues into back muscles. Methods: A total of 40 SPF-SD female rats(5–6 week-old) were randomly divided into three groups: blank control group(group A), castration control group(group B) and transplant group(group C). Ovaries were removed by surgical procedure, then after cryopreservation and thawing procedures the ovarian tissues were implanted into the back muscles of mice in group C. After 4 weeks of ovarian tissues transplantation, all rats blood sampling were measured for E2, LH and FSH hormone levels by ELISA. Results: E2 level was significantly higher in group C and group A than group B [(38.98 ± 5.66) pg/mL,(8.14 ± 3.24) pg/mL; P < 0.05) and [(36.30 ± 6.90) pg/mL,(8.14 ± 3.24) pg/mL; P < 0.05)]. However, E2 level in group C and group A had no significant difference. FSH level in group B, group A and group C was(18.87 ± 2.54) nmol/L,(7.77 ± 0.87) nmol/L and(9.39 ± 2.12) nmol/L respectively. FSH level increased significantly in group B compared with group A, and the difference had statistical significance(P < 0.05). FSH level was slightly increased in group C compared with group A, and the difference was not statistically significant(P > 0.05), but compared with group B, FSH level was significantly reduced and being statistically significant(P < 0.05). Conclusion: Autotransplantation of cryopreserved-thawed ovarian tissue into back muscles can sustain follicular development and re-establish endogenous hormone production by restoring the factors such as angiogenesis and innervations at the graft site.

Study on the Estrous Cycle Regularity of Cryopreserved Rat Ovarian Tissues after Heterotopic Transplantation

Objective: To evaluate the re-initiation of ovarian function in cryopreserved ovarian grafts by means of vaginal smear of transplant rats. Methods: A total of 40 SPF-SD female rats (5 - 6 week-old) were randomly divided into three groups (blank control, castration control and transplant group). Ovaries were removed by surgical procedure then after cryopreservation and thawing procedures the ovarian tissues pushed inside the back muscles gap in transplant group. On the first PO day, vaginal smear collection was daily initiated. After 30 days, the PO day when the estrous cycle was re-initiated was considered for analysis as well as the estrous days and the number of estrous cycles. Results: Normal control group had a regular estrous cycle, while the transplant group had an estrous cycle disorder within first 2 weeks and later 2 weeks after the transplantation while the duration of diestrus cycle lasted longer. At the same time, the castration group had lost the normal estrous cycle, keeping continue in the stage of diestrus. Conclusion: In transplanted animals the re-initiated ovarian function can be predicted with alteration between estrous and diestrus phases with predominant estrous irregularity. Moreover, short autotransplanted graft duration needs time to perfuse by new blood vessels and hormone secretions, so could not directly affect its target organs to function properly.

Modified vitrification method for cryopreservation of human ovarian tissues

Background Vitrification is a prospective technology in ovarian tissue cryopreservation, but it is still in an initial stage. This study was conducted to investigate a modified vitrification protocol for human ovarian tissue, which can be used as an alternative to preserve fertility for young women with cancer who have to undergo cytotoxic therapy and sterilization. Methods Ovarian tissue samples were collected from 15 patients and randomly allocated to groups of fresh, vitrification, and conventional slow freezing. A modified carrierless vitrification method was applied. The proportion of morphologically intact follicles in fresh ovarian tissues was compared with that in warmed/thawed tissues. The initial growth of the follicles and the concentrations of estradiol and progesterone were detected to determine the viability and endocrine function of the cryopreserved tissues. Results The proportion of morphologically intact primordial follicles in the fresh group （97.6%） was significantly higher than that in the other two groups （vitrification group 80.3% and slow-freezing group 72.6%, P〈0.001）. In both the vitrification and slow-freezing groups, estradiol and progesterone were secreted continuously during 2-week culture in vitro, the proportion of primary follicles were both significantly increased compared to the fresh group. No statistically significant differences existed between the two groups after cryopreservation in the proportion of both primordial and primary follicles, and the concentrations of estradiol and progesterone （P〉0.05）.Conclusion The modified vitrification method for cryopreservation of human ovarian tissues is effective, simple, and inexpensive.

Experimental study on the vitrification and xenotransplantation of human ovarian tissue

Background:Cryopreservation of ovarian tissue is a promising method for preserving fertility.Transmission electron microscopy(TEM)is an evaluation system for cryo-injury during the cooling and warming process which is very laborious and needs to be optimized.Objective:In this study,we evaluated that serum 17β-oestradiol(E2)may be used as an indicator of vitrified ovarian tissue.Methods:Immunodeficient nude mice were used as hosts for xenografting of vitrified-warmed human ovarian tissues.A total of 54 mice were divided into two group:vitrified ovarian xenotransplant(VOX)group(n?45)and non-transplant control group(n?9).The transplanted mice were grouped into vitrified/warmed grafted-4 weeks(VOX-4w,n?15),vitrified/warmed grafted-6weeks(VOX-6w n?15)and vitrified/warmed grafted-12 weeks(VOX-12w n?15)according to the time after transplantation.The viable and functional recovery of grafted ovarian tissue was assessed by light microscopy,transmission electron microscopy,and hormone(E2)assays.Results:Serum E2 concentration was significantly higher in VOX-6w(group(21.07 pg/ml)than that of VOX-12w group(15.59 pg/ml).VOX-12w group showed a lower value(12.61 pg/ml)for E2 concentration.The trend for E2 concentration was consistent with the morphological identification of the grafts.Conclusion:In vivo serum hormone E2 released by cortical biopsies can be used as a functional marker for xenotransplanted vitrified-warmed human ovarian tissue reserve.

Offspring from oocytes grown in frozen-thawed ovarian tissues transplanted to male and female bodies

Objective: There are few reports of live births from heterotopic transplantation of frozen-thawed ovarian tissue. The purpose of this study is to assess the follicular development in the frozen-thawed ovarian tissues following heterotopic transplantation in both female and male bodies.Methods: Cluster of differentiation 1 (CD1) mice (6-8 weeks) were used in this study as ovarian tissue donors and foster mothers for embryo transfer. Sperm from CD1 male mice were used for insemination by intracytoplasmic sperm injection (ICSI). Nude severe combined immunodeficiency mice (8 weeks) were employed as recipients of ovarian tissue transplantation. The frozen-thawed ovarian tissues were transplanted to 4 sites on each mouse, female and male, subcutaneously. After 3 months, both female and male mice were injected with 5.0 IU gonadotropins intraperitoneally. Post 48 hours of injection, the mouse was killed for ovarian transplant collection. Only fully grown oocytes with contacted cumulus cells (cumulus-oocyte complexes) were then selected for maturationin vitro.In vitro matured oocytes were inseminated with fresh sperm by ICSI, and the developed blastocysts were frozen using the vitrification method and stored until embryo transfer. After thawing, the thawed blastocysts were incubated for at least 2 hours before the transfer. The foster mice mothers mated with vasectomised male 3 days previously. Live birth was monitored at 19 days after transfer, and the resulted offspring was raised for fertility test.Results: The relatively high recovery rates of the transplanted ovarian tissues were collected in both frozen-thawed and fresh ovarian tissue transplants from both female and male bodies. The fully grown immature oocytes became maturein vitro and the fertilized zygotes developed to blastocyst stage. There are no differences between frozen-thawed and fresh ovarian transplants in term of oocyte quality and embryo development to blastocyst rates. Nineteen-day post-transfer, 3 foster mothers from the frozen-thawed ovarian tissue transplant group delivered 13 pups and the 4 foster mothers of the fresh ovarian tissue transplant group delivered 12 live pups. The produced offspring were normal in appearance and grew healthy and fertile.Conclusions: Our results attest that the follicles can survive and develop in the frozen-thawed ovarian tissues following the subcutaneous transplant to adult male mouse’s body regardless of basal endocrinal environment. Those fully grown oocytes can produce healthy and fertile offspring which will provide the possibility for further mechanistic understanding of endocrinology of folliculogenesis.

An experimental study on autologous transplantation of fresh ovarian tissue in sheep

Objective:To investigate current autologous transplantation methods and sites of ovarian tissues in sheep.Methods:Sheep ovaries were resected.Ovarian cortices were sliced and transplanted orthotopically into the ovarian mesangial latum and heterotopically into the greater omentum and under groin skin.The grafts were removed two months after transplantation and examined to evaluate the survival of follicles(hematoxylin-eosin staining)and help determining feasible graft sites and transplantation methods.Results:Graft nodules were found in the transplanted sites.HE staining of the grafts showed that multiple primordial follicles were able to survive in the grafts on both sides of the ovarian mesangial latum,the right side of the greater omentum,and the left inguinal subcutaneous tissue.Secondary or cystic follicles were found in almost all of the grafts.Conclusion:The ovarian mesangial latum,the greater omentum and the inguinal subcutaneous tissue can be used as autologous transplantation sites,where sheep ovarian tissue can survive and the follicles grow and develop in good condition.

Examining the Effect of Hydroalcoholic Extract of Dracaena cinnabari on Sex Hormones and Ovarian and Uterine Tissues of Rats

Introduction: Dracaena cinnabari is considered a rich source of phytochemicals used widely in traditional medicine. In the present study, the effect of D. cinnabari hydraulic extract on the reproductive system of female rats was investigated. Methods: The samples were randomly divided into four groups(six samples in each group), including three treatment groups and one control group, and all samples were kept at the same conditions. Hydraulic extract of D. cinnabari and injected intraperitoneally daily for 10 days, while physiological serum was used for injection in to the control group. After 10 days of injection, estrogen and progesterone levels were measured by enzyme immunoassay technique. After dissection, the ovaries and uterine tissues were isolated for histological examination, and tissue changes were carefully examined. Results: The results revealed that the levels of estrogen and progesterone in experimental Groups 2 and 3 had a significant increase(P < 0.001). Regarding tissue changes, a significant increase was observed in epithelial thickness(P < 0.001), number of corpus luteum(P < 0.01), and Graafian follicle(P < 0.01) in doses of 100 and 150 mg/kg. Conclusions: Based on the results, it seems that D. cinnabari extract has an effect on the ovarian follicles.

Gonadal damage and options for fertility preservation in female and male cancer survivors

It is estimated that in 2010, 1 in every 250 adults will be a childhood cancer survivor. Today, oncological surgery, radiotherapy and chemotherapy achieve relatively high rates of remission and long-term survival, yet are often detrimental to fertility. Quality of life is increasingly important to long-term survivors of cancer, and one of the major quality-of-life issues is the ability to produce and raise normal children. Developments in the near future in the emerging field of fertility preservation in cancer survivors promise to be very exciting. This article reviews the published literature, discusses the effects of cancer treatment on fertility and presents the options available today thanks to advances in assisted-reproduction technology for maintaining fertility in male and female patients undergoing this type of treatment. The various diagnostic methods of assessing the fertility potential and the efficacy of in vitro fertilization （IVF） after cancer treatment are also presented.

Female Fertility： Is it Safe to Freeze.

Objective： To evaluate the safety and risk of cryopreservation in female fertility preservation. Data sources： The data analyzed in this review were the English articles from 1980 to 2013 from journal databases, primarily PubMed and Google scholar. The criteria used in the literature search show as tbllowing： （ 1 ） human; embryo; cryopreservation/freezing/vitrification, （2） human; oocyte/immature oocyte; cryopreservation/freezing/vitrification, （3） human; ovarian tissue transplantation; cryopreservation/ freezing/vitrification, （4） human; aneuploidy/DNA damage/epigenetic; cryopreservation/freezing/vitrification, and （5） human; fertility preservation; maternal age. Study selection： The risk ratios based on survival rate, maturation rate, fertilization rate, cleavage rate, implantation rate. pregnancy rate. and clinical risk rate were acquired from relevant meta-analysis studies. These studies included randomized controlled trials or studies with one of tile primary outcome measures covering cryopreservation of human mature oocytes, embryos, and ovarian tissues within the last 7 years （from 2006 to 2013. since the pregnancy rates of oocyte vitrification were significantly increased due to the improved techniques）. The data involving immature oocyte cryopreservation obtained from individual studies was also reviewed by the at, thors. Results： Vitrifications of mature oocytes and embryos obtained better clinical outcomes and did not increase the risks of DNA damage, spindle configuration, embryonic aneuploidy, and genomic imprinting as compared with fresh and slow-freezing procedures, respectively. Conclusions： Both embryo and oocyte vitrifications are safe applications in female fertility preservation.

Chinese expert consensus on clinical practice of female fertility preservation

Many female fertility preservation-related technologies have recently been developed in response to increasing demand for such treatments. To establish standard practices of female fertility preservation in China, the Chinese Maternal and Child Health Association Affiliated Fertility Preservation Professional Committee assembled specialists to construct a consensus, referring to the current clinical guidelines of some countries combined with clinical practice and expert opinions. The consensus includes two parts: (1) indications for female fertility preservation and related techniques, in which we sought to be inclusive regarding the indications for fertility preservation;and (2) practical guidance for the clinical application of the female fertility preservation technologies.

Fertility Preservation in Cancer Patients

Traditional radiotherapy and chemotherapy often cause irreversible damage to the fertility and endocrine function of cancer patients.The current methods of fertility preservation include freezing the sperms of adult and adolescent males after puberty;freezing the embryos,oocytes,and ovarian tissue of females;and drug intervention and fertility preservation surgery.This article reviews fertility preservation in cancer patients with respect to current methods,indications,and some more recently developed methods that remain under investigation.