Follicle Quality Research Articles

Can frozen-thawed human ovary withstand refreezing-rethawing in the form of cortical strips?

The aim of this study was to elucidate whether ovarian tissue is able to withstand a double freezing-thawing procedure. Human ovarian cortical biopsies from 4 thawed whole ovaries were divided into 4 experimental subgroups: (a) frozen-thawed non-grafted group, (b) frozen-thawed xenografted group, (c) refrozen-rethawed non-grafted group, and (d) refrozen-rethawed xenografted group. Xenografting was performed using 8 severe combined immunodeficient mice for a total duration of 21days. The following analyses were conducted: classic hematoxylin and eosin staining, Ki67 immunolabeling, transmission electron microscopy, Masson's green trichrome, and double CD34 immunostaining. Morphologically normal preantral follicles were detected in all groups. We observed a dramatic decline of more than 65% in early preantral follicle survival rates after grafting of both frozen-thawed (p< 0.0001) and refrozen-rethawed (p< 0.0001) ovarian tissue. However, mean follicle densities remained comparable between the frozen-thawed and refrozen-rethawed non-grafted groups, as well as both grafted groups. Equivalent proportions of proliferating early preantral follicles were identified in frozen-thawed and refrozen-rethawed samples, whether the tissue was grafted or not. Furthermore, we did not observe any significant difference in atretic follicle rates between any of the four groups, and the ultrastructural quality of follicles appeared unaffected by the refreezing procedure. Similar proportions of fibrosis were noted in the frozen-thawed and refrozen-rethawed groups, irrespective of grafting. Finally, no significant differences were witnessed in terms of vascularization. We were able to demonstrate, for the first time, that refrozen-rethawed ovarian tissue has the same functional characteristics as frozen-thawed ovarian tissue.

Dynamic Characterization of the Biomechanical Behaviour of Bovine Ovarian Cortical Tissue and Its Short-Term Effect on Ovarian Tissue and Follicles.

The ovary is a dynamic mechanoresponsive organ. In vitro, tissue biomechanics was reported to affect follicle activation mainly through the Hippo pathway. Only recently, ovary responsiveness to mechanical signals was exploited for reproductive purposes. Unfortunately, poor characterization of ovarian cortex biomechanics and of the mechanical challenge hampers reproducible and effective treatments, and prevention of tissue damages. In this study the biomechanical response of ovarian cortical tissue from abattoir bovines was characterized for the first time. Ovarian cortical tissue fragments were subjected to uniaxial dynamic testing at frequencies up to 30 Hz, and at increasing average stresses. Tissue structure prior to and after testing was characterized by histology, with established fixation and staining protocols, to assess follicle quality and stage. Tissue properties largely varied with the donor. Bovine ovarian cortical tissue consistently exhibited a nonlinear viscoelastic behavior, with dominant elastic characteristics, in the low range of other reproductive tissues, and significant creep. Strain rate was independent of the applied stress. Histological analysis prior to and after mechanical tests showed that the short-term dynamic mechanical test used for the study did not cause significant tissue tear, nor follicle expulsion or cell damage.

Glutathione-dependent enzymes in the follicular fluid of the first-retrieved oocyte and their impact on oocyte and embryos in polycystic ovary syndrome: A cross-sectional study.

Background Oxidative stress and GSH-dependent antioxidant system plays a key role in the pathogenesis of polycystic ovary syndrome (PCOS).Objective We compared glutathione peroxidase (GPx) and glutathione reductase activities and reduced glutathione (GSH) levels in serum and follicular fluid (FF) of the first-retrieved follicle and their impact on quality of oocyte and embryo in PCOS women undergoing IVF.Materials and Methods This cross sectional study was conducted on 80 pairs of blood samples and FF of the first-retrieved follicle from PCOS women, at the Infertility center of Ghadir Mother and Child Hospital. The mean activity of GPx and GR, also GSH levels in the serum and FF were compared to the quality of the first follicle and resultant embryo.ResultsRetrieved oocytes included 53 (66.25%) MII, 17 (21.25%) MI, and 10 (12.5%) germinal vesicles; after IVF 42 (52.50%) embryos with grade I and 11 (13.75%) with grade II were produced. The mean values for all three antioxidants were higher in the FF compared to serum (p 0.001). Also all of the mean measured levels were significantly higher in the FF of the MII oocytes compared to that of oocytes with lower grades (p = 0.012, 0.006 and 0.012, respectively). The mean GPX activity and GSH levels were significantly higher in the serum (p = 0.016 and 0.012, respectively) and FF (p = 0.001 for both) of the high-quality grade I embryos.Conclusion GSH-dependent antioxidant system functions more efficiently in the FF of oocytes and embryos with higher quality.

Tetracaine combined with propofol for painless oocyte retrieval: from a single center study.

In vitro fertilization-embryo transfer is a main assisted reproductive technique that can improve the success rate of conception. At present, the ultrasound-guided oocyte retrieval is often employed in clinics, but this process will cause pain and fear in outpatients. This study explored the safety and satisfaction of patients who received egg retrieval under vaginal topical tetracaine anesthesia combined with intravenous propofol anesthesia. Patients who underwent elective egg retrieval in the Reproductive Center of Shanghai Tenth People's Hospital were recruited from January 2017 to August 2018 [tetracaine + propofol group (n=53); propofol group (n=48)]. Results showed that tetracaine combined with propofol anesthesia could effectively reduce the dose of propofol during surgery, ensure the quality of follicles, effectively reduce the postoperative pain and improve the operational satisfaction without affecting the prognosis. Our findings provide evidence for further clinical applications of this technique.

Impact of Dietary Selenium on Modulation of Expression of Several Non-Selenoprotein Genes Related to Key Ovarian Functions, Female Fertility, and Proteostasis: a Transcriptome-Based Analysis of the Aging Mice Ovaries.

Female reproductive (ovarian) aging is characterized by a marked decline in quantity and quality of follicles and oocytes, as well as alterations in the surrounding ovarian stroma. In our previous report, we have shown that dietary selenium (Se) insufficiency and supplementation differentially impacted the reproductive efficiency in aging mice; however, the precise understanding of such modulation is still incomplete. In the present study, we sought to determine the impact of low (mildly low level) and moderately high (medium level) Se diets on expression profile of non-selenoprotein genes in the ovaries of aging mice. For this purpose, the aged mice were divided in two groups and fed either a low Se (Se-L; 0.08mg Se/kg) diet or a moderately high Se (Se-M; 0.33mg Se/kg) diet. RNA-seq analysis revealed that a total of 168 genes were differentially expressed between the two groups. From these, 72 and 96 differentially expressed genes (DEGs) were found to be upregulated and downregulated, respectively. Gene Ontology (GO) and pathways enrichment (KEGG) analyses revealed that these DEGs were enriched in several key GO terms and biological pathways including PI3K-Akt signaling pathway, steroid hormone biosynthesis, signaling pathways regulating pluripotency of stem cells, Hippo signaling pathway, ovarian steroidogenesis, and Wnt signaling pathway. Further filtering of RNA-seq data revealed that several DEGs such as Star, Hsd3b6, Scd1, Bmp7, Aqp8, Gas1, Fzd1, and Wwc1 were implicated in key ovarian- and fertility-related functions. In addition, some of the DEGs were related to ER homeostasis and/or proteostasis. These results highlight that dietary low and moderately high (medium level) Se diets, in addition to modulation of selenoproteins, can also have an impact on expression of several non-selenoprotein genes in the ovaries of aging mice. To sum up, these findings add more value to our understanding of Se modulation of ovarian functions and female fertility and will pave a way for the focused mechanistic and functional studies in this domain.

Comparison of Bovine Small Antral Follicle Development in Two- and Three-Dimensional Culture Systems.

To compare the effects of two-(2D, microplate) and three-dimensional (3D, alginate) culture systems on the in vitro growth of small antral follicles in cattle, individual follicles were separately cultured in the two culture systems for 8 days. Half of the culture medium was replaced by fresh medium every 2 days; the former medium was used to assess the amount of follicular hormone secretion using ELISA. Individual follicle morphology, diameter, and survival rate were recorded every alternate day. The results showed that in 4 days, there was no significant difference between the two systems, except that the growth rate of follicles in 2D system was relatively faster. After 4 days, estradiol concentration in 3D system was higher than that in 2D system. However, progesterone concentration was lower than that in the 2D system. The survival rate and oocyte quality of follicles in 2D system were significantly lower than those in 3D system on day 8. The follicle diameter slightly increased (30-60 μm) in the entire process. Taken together, for in vitro culture of follicles within 4 days, the 2D culture system is more suitable. However, when the culture duration is >4 days, the 3D culture system is more suitable.

Dietary Selenium Supplementation Ameliorates Female Reproductive Efficiency in Aging Mice.

Female reproductive (ovarian) aging is distinctively characterized by a markedly reduced reproductive function due to a remarkable decline in quality and quantity of follicles and oocytes. Selenium (Se) has been implicated in playing many important biological roles in male fertility and reproduction; however, its potential roles in female reproduction, particularly in aging subjects, remain poorly elucidated. Therefore, in the current study we used a murine model of female reproductive aging and elucidated how different Se-levels might affect the reproductive efficiency in aging females. Our results showed that at the end of an 8-week dietary trial, whole-blood Se concentration and blood total antioxidant capacity (TAOC) were significantly reduced in Se-deficient (0.08 mg Se/kg; Se-D) mice, whereas both of these biomarkers were significantly higher in inorganic (0.33 mg/kg; ISe-S) and organic (0.33 mg/kg; OSe-S) Se-supplemented groups. Similarly, compared to the Se-D group, Se supplementation significantly ameliorated the maintenance of follicles and reduced the rate of apoptosis in ovaries. Meanwhile, the rate of in vitro-produced embryos resulting from germinal vesicle (GV) oocytes was also significantly improved in Se-supplemented (ISe-S and OSe-S) groups compared to the Se-D mice, in which none of the embryos developed to the hatched blastocyst stage. RT-qPCR results revealed that mRNA expression of Gpx1, Gpx3, Gpx4, Selenof, p21, and Bcl-2 genes in ovaries of aging mice was differentially modulated by dietary Se levels. A considerably higher mRNA expression of Gpx1, Gpx3, Gpx4, and Selenof was observed in Se-supplemented groups compared to the Se-D group. Similarly, mRNA expression of Bcl-2 and p21 was significantly lower in Se-supplemented groups. Immunohistochemical assay also revealed a significantly higher expression of GPX4 in Se-supplemented mice. Our results reasonably indicate that Se deficiency (or marginal levels) can negatively impact the fertility and reproduction in females, particularly those of an advancing age, and that the Se supplementation (inorganic and organic) can substantiate ovarian function and overall reproductive efficiency in aging females.

Follicular fluid steroid profile in sows: relationship to follicle size and oocyte quality†.

Identification of reliable characteristics of follicle quality and developmental competence has been pursued in numerous studies, but with inconsistent outcomes. Here, we aimed to identify these characteristics by analysis of the follicular fluid (FF) steroid profile in relation to cumulus-oocyte complex (COC) morphology and follicle size, followed by molecular substantiation. Multiparous sows at weaning were used to facilitate analysis at the start of the follicular phase of the oestrus cycle. Sows with a higher average follicle size (≥5 mm vs. < 5 mm) had a higher follicular fluid β-estradiol concentration, but did not differ in other measured steroids. Sows with high compared to low percentage high-quality COCs (<70% vs. ≥70% high-quality) had follicular fluid with a higher concentration of β-estradiol, 19-norandrostenedione, progesterone, and α-testosterone, while the concentration of cortisol was lower. Transcriptome analysis of granulosa cells of healthy follicles of sows with a high percentage high-quality COCs showed higher abundance of transcripts involved in ovarian steroidogenesis (e.g., CYP19A2 and 3, POR, VEGFA) and growth (IGF1) and differential abundance of transcripts involved in granulosa cell apoptosis (e.g., GADD45A, INHBB). Differences in aromatase transcript abundance (CYP19A1, 2 and 3) were confirmed at the protein level. In addition, sows with a high percentage high-quality COCs lost less weight during lactation and had higher plasma IGF1 concentration at weaning, which may have affected COC quality. To the best of our knowledge, this study is also the first to report the relation between FF steroid profile and COC quality.

The antioxidant curcumin postpones ovarian aging in young and middle-aged mice.

Reproductive senescence is accompanied by a reduced number and quality of ovarian follicles in response to the accumulation of free radicals and the process of apoptosis. Having selected mice as models, we examined the hypothesis that curcumin as an antioxidant and anti-inflammatory agent might prevent or retard ovarian aging. Female NMRI 21-day-old mice were divided into control, vehicle and curcumin groups. In the treatment group the mice received curcumin at 100mgkg-1day-1 intraperitoneally. After 6, 12 and 33 weeks several parameters were examined including ovarian reserve, oocyte quality, oxidative status, invitro fertilisation and expression of ovulation-related (growth differentiation factor 9 (GDF-9) and bone morphogenetic protein 15 (BMP-15)) and anti-aging-related (sirtuin 1 (SIRT-1) and SIRT-3) genes. Curcumin treatment up to 12 and 33 weeks resulted in increased ovarian volume and number of follicles and was associated with elevated anti-Müllerian hormone and oestrogen and diminished FSH serum levels. Furthermore, enhanced oocyte maturation, fertilisation and embryo development plus reduced oxidative stress were seen in the curcumin group. Also, the expression of GDF-9, BMP-15, SIRT-1 and SIRT-3 genes was increased in the curcumin group. Concerning gestational age, the findings of the study suggested that administration of curcumin could delay the process of oocyte aging in a mouse model.

Impact of first-line cancer treatment on the follicle quality in cryopreserved ovarian samples from girls and young women.

STUDY QUESTIONDoes first-line chemotherapy affect the quality of ovarian pre-antral follicles and stromal tissue in a population of young patients?SUMMARY ANSWERExposure to first-line chemotherapy significantly impacts follicle viability, size of residual intact follicles, steroid secretion in culture and quality of the stromal compartment.WHAT IS KNOWN ALREADYFirst-line chemotherapy is considered to have a low gonadotoxic potential, and as such, does not represent an indication for fertility preservation. Studies investigating the effects of chemotherapy on the quality of ovarian tissue stored for fertility preservation in young patients are limited and the results sometimes contradictory.STUDY DESIGN, SIZE, DURATIONWe conducted a retrospective cohort study including young patients referred to three centers (Helsinki, Oslo and Tampere) to perform ovarian tissue cryopreservation for fertility preservation between 2003 and 2018.PARTICIPANTS/MATERIALS, SETTING, METHODSA total of 43 patients (age 1–24 years) were included in the study. A total of 25 were exposed to first-line chemotherapy before cryopreservation, whereas 18 patients were not. Density and size of follicles divided by developmental stages, prevalence of atretic follicles, health of the stromal compartment and functionality of the tissue in culture were evaluated and related to age and chemotherapy exposure. Activation of dormant follicles and DNA damage were also assessed.MAIN RESULTS AND THE ROLE OF CHANCEPatients exposed to first-line chemotherapy showed a significantly higher density of atretic primordial and intermediary follicles than untreated patients. The intact primordial and intermediary follicles were significantly smaller in size in patients exposed to chemotherapy. Production of steroids in culture was also significantly impaired and a higher content of collagen and DNA damage was observed in the stromal compartment of treated patients. Collectively, these observations may indicate reduced quality and developmental capacity of follicles as a consequence of first-line chemotherapy exposure. Neither increased activation of dormant follicles nor elevated levels of DNA damage in oocyte nuclei were found in patients exposed to chemotherapy.LIMITATIONS, REASONS FOR CAUTIONThe two groups were not homogeneous in terms of age and the patients were exposed to different treatments, which did not allow us to distinguish the effect of specific agents. The limited material availability did not allow us to perform all the analyses on the entire set of patients.WIDER IMPLICATION OF THE FINDINGSThis study provides for the first time a comprehensive analysis of the effects of first-line chemotherapy on the health, density and functionality of follicles categorized according to the developmental stage in patients under 24 years of age. When exposed to these treatments, patients were considered at low/medium risk of infertility. Our data suggest a profound impact of these relatively safe therapies on ovarian health and encourages further exploration of this effect in follow-up studies in order to optimize fertility preservation for young cancer patients.STUDY FUNDING/COMPETING INTEREST(S)This study was funded by the Swedish Childhood Cancer Foundation, the Finnish Cancer Society, the Finnish Pediatric Research Foundation, the Väre Foundation for Pediatric Cancer Research, The Swedish Research Council, the Stockholm County Council (ALF project) and Karolinska Institutet. The authors have no conflict of interest to declare.

Effect of exogenous gonadotropin on the transcriptome of human granulosa cells and follicular fluid hormone profiles

BackgroundSuperovulation treatment had some adverse effects on maturity and development of oocytes. Can superovulation dose of gonadotropins (Gns) affect the transcriptome of granulosa cells and follicular fluid (FF) hormone levels?MethodsOne leading pre-ovulatory follicle per subject was used from three natural-cycle and four Gn-stimulated patients. Granulosa cells and FF samples were collected from the same leading follicle of each patient. RNA was extracted from granulosa cells and subjected to deep sequencing and analysis. Follicle-stimulating hormone (FSH), estradiol (E2), androstenedione (AND), testosterone (T), luteinizing hormone (LH), and progesterone (P4) levels in FF were measured by immunoassays. Student’s t test was used for statistical analysis.ResultsA total of 715 genes were up-regulated, and 287 genes were down-regulated, in the Gn-stimulated group relative to the control group. Gene Ontology analysis revealed that the down-regulated genes were enriched in cell cycle and meiosis pathways, primarily those associated with follicle or oocyte maturation and quality. On the other hand, the up-regulated genes were enriched in functions related to immunity and cytokine–cytokine receptor interactions. Compared to the follicles of natural cycle, the E2 and LH concentrations were significantly reduced (P < 0.001), the P4 concentration was significantly increased (P = 0.003), and the concentrations of FSH, T and AND had no difference in the follicles of Gn-stimulated cycle.ConclusionsCell cycle– and meiosis-associated genes were down-regulated by Gns stimulation, whereas immune- and cytokine-associated genes were up-regulated. Hormone levels were also affected by Gns stimulation. Compared with natural-cycle follicles,putative markers associated with oocyte quality and follicle maturation were significantly different from those in Gn-stimulated follicles. Hormone levels in follicles were compatible with the steroidogenic patterns of granulosa cell, which reflects the follicle maturation and oocyte quality.

What Changed on the Folliculogenesis in the Process of Mouse Ovarian Aging?

There are about 1-2 million follicles presented in the ovary at birth, while only around 1000 primordial follicles are left at menopause. The ovarian function also decreases in parallel with aging. Folliculogenesis is vital for ovarian function, no matter the synthesis of female hormones or ovulation, yet the mechanisms for its changing with increasing age are not fully understood. Early follicle growth up to the large preantral stage is independent of gonadotropins in rodents and relies on intraovarian factors. To further understand the age-related molecular changes in the process of folliculogenesis, we performed microarray gene expression profile analysis using total RNA extracted from young (9 weeks old) and old (32 weeks old) mouse ovarian secondary follicles. The results of our current microarray study revealed that there were 371 (≥2-fold, q-value ≤0.05) genes differentially expressed in which 174 genes were upregulated and 197 genes were downregulated in old mouse ovarian secondary follicles compared to young mouse ovarian secondary follicles. The gene ontology and KEGG pathway analysis of differentially expressed genes uncovered critical biological functions such as immune system process, aging, transcription, DNA replication, DNA repair, protein stabilization, and apoptotic process were affected in the process of aging. The considerable changes in gene expression profile may have an adverse influence on follicle quality and folliculogenesis. Our study provided information on the processes that may contribute to age-related decline in ovarian function.

Slush nitrogen vitrification of human ovarian tissue does not alter gene expression and improves follicle health and progression in long-term in vitro culture

To study whether slush nitrogen (SN) vs. liquid nitrogen (LN) vitrification affects human ovarian tissue gene expression and preserves follicle health during extended invitro culture. Randomized experimental study. University research laboratory. Ovarian biopsies collected by laparoscopic surgery from patients with benign gynaecologic conditions. None. Ovarian strips were vitrified with LN or SN, warmed, and analyzed before or after culture for 9days (d9) in gas-permeable dishes. Expression of genes involved in stress and toxicity pathways was analyzed in fresh and warmed strips by polymerase chain reaction (PCR) array and quantitative real-time-PCR. Fresh and vitrified/warmed strips were analyzed for follicle quality, progression, and viability before or after culture. The SN vitrification preserved follicle quality better than LN (% grade 1 follicles: fresh control, 54.2; LN, 29.3; SN, 48.8). Quantitative reverse transcription-PCR demonstrated a noticeable up-regulation of 13 genes in LN samples (range, 10-35) and a markedly lower up-regulation of only 5 genes (range, 3.6-7.8) in SN samples. Long-term invitro culture evidenced worse follicle quality and viability in LN samples than in both fresh and SN samples (% grade 1 follicle: fresh d0, 51.5; fresh d9, 41; LN d9, 16.4; SN d9, 55) and a highly significant reduction of primordial follicles and a concomitant increase of primary and secondary follicles in all samples. Follicle growth to the secondary stage was significantly higher in vitrified tissue than in fresh tissue, being better in SN than in LN vitrified tissue. Follicle quality, gene expression, viability, and progression are better preserved after SN vitrification.

Optimization of medium for in vitro culture of sheep ovarian tissue

Creation of genetic cryobanks is a way of preserving animal genetic materials. The gene bank can be used actively for increasing populations as well as minimization of interrelated crossing. One of the ways of preserving animal genetic material is ovarian tissue cryopreservation. After cryopreservation and storage, part of the ovarian tissue can be thawed and cultured in vitro in order to check a quality of follicles and stromal cells. The viability rate indicates whether or not it is possible to restore the reproductive function of the animal. Our study aimed to search and test a new medium for in vitro culture of sheep ovarian tissue. Ovarian pieces from 32 ewes (n = 320 and n = 180 in two series of experiments) were cultured during 16 days in three basic media: Tissue Culture Medium (TCM) 199, Buffered Tissue Culture Medium (TCM-HEPES), and Dulbecco Modified Eagle Medium (DMEM). These basic culture media used in different experimental groups (12 and 6) were supplemented with fetal calf serum (FCS), ewe estrous serum (EES) as well as follicles stimulating hormone and luteinizing hormone (FSH and LH, respectively). After the cultures of ovarian tissue and histological treatment, respectively, developmental rates of primordial, primary and secondary follicles as well as the secretion of hormones by tissues (progesterone and estradiol 17ß) were evaluated. Maximal developmental rate of follicles and hormonal activity were observed in the group with the culture medium TCM-HEPES + EES + FSH + LH. For in vitro culture of sheep ovarian tissue, the following medium is recommended: TCM-HEPES (supplemented with 100 μg/mL penicillin-streptomycin, 50 μg/mL gentamicin, and 2 mM l-Glutamine) +10% EES + 5 μg/mL FSH + 5 μg/mL LH.

Progesterone supplementation improves fertility of lactating Holstein cows without a corpus luteum at initiation of the timed AI protocol

Ovulation synchronization and timed AI programs have been adopted in many dairy herds for managing reproduction in cows. Timed AI programs ensure maximum submission rates. However, pregnancy per AI is reduced when cows do not have a functional CL at initiation of the timed AI protocol. Suboptimal progesterone concentrations as observed in cows without a CL at initiation of timed AI negatively affects the quality of ovulatory follicle, oocyte competence, embryo growth, and endometrial functions. Ultrasound evaluation of the ovaries at initiation of the timed AI protocol combined with targeted progesterone supplementation can improve fertility in dairy cows lacking CL initiation of the timed AI protocol. Intravaginal controlled internal drug release (CIDR) devices offer a practical method to increase plasma progesterone concentrations in cows without a CL at initiation of the timed AI protocol. Our objectives were to evaluate the effects of progesterone supplementation using 1 or 2 intravaginal progesterone inserts on fertility of first service lactating dairy cows without a CL at initiation of the timed AI protocol.

Effects of tongyuan acupuncture on pregnancy outcomes of ovarian induction period with clomiphene citrate: a retrospective analysis

To observe the clinical efficacy and action mechanism of tongyuan acupuncture for ovulatory disorder infertility of ovarian induction period with clomiphene citrate (CC). Through retrospective analysis, 36 patients with ovulatory disorder infertility were divided into CC group (16 cases) and tongyuan acupuoture group (20 cases) by method. The patients in the CC group were treated with oral administration of CC (50 mg) for 5 days from the 5th day of menstruation; based on the treatment of CC, patients in the tongyuan acupuncture group were treated with tongyuan acupuncture; the group A of acupoint was mainly consisted of acupoints of the conception vessel and the three yin meridians and front-mu acupoints in abdomen; the group B of acupoint was mainly consisted of acupoints of the governor vessel and back-shu acupoints. The two groups of the acupoints were selected alternately, combined with the si-guan points (Taichong (LR 3)、Hegu (LI 4)) and five-shu points. The needles were inserted with flying needling method, and the needles were retained for 30 min per treatment; acupuncture was given once every other day. All the patients were treated for 2 months, and treatment stopped if pregnancy occurred. The pregnancy rate, cycle ovulation rate, maximum diameter of follicle, endometrial thickness and adverse effects were compared between the two groups. The pregnancy rate of the tongyuan acupuncture group was 75.0% (15/20), which was superior to 37.5% (6/16) in the CC group (P<0.05). The cycle ovulation rates of two groups were 70.4% (19/27, P>0.05). The maximum diameter of follicle and the endometrium thickness in the tongyuan acupuncture group were superior to those in the CC group (both P<0.05). The incidence of adverse effects in the tongyuan acupuncture group was 0% (0/20), which was lower than 18.8% (3/16) in the CC group (P<0.05). Tongyuan acupuncture combined with CC could improve the quality of follicle and the receptivity of endometrium in patients with ovulatory disorder infertility, so as to improve the pregnancy rate, which could be used as a safe and effective means to cooperate with modern assisted reproductive technology.

Morphological and chemical information in fresh and vitrified ovarian tissues revealed by X-ray Microscopy and Fluorescence: observational study

Many clinical circumstances impose the necessity of collection and prolonged storage of gametes and/or ovarian tissue in order to preserve the reproduction potential of subjects. This is particularly appropriate in the case of young women and pre-pubertal girls undergoing chemotherapeutic treatments. The success of later assisted fertilization will depend on the suitable cooling protocols minimizing cryo-damages and preserving their biological function. The freeze-thaw processes of cryopreservation may induce, in fact, morphological and structural damages of oocytes and tissue mainly due to the formation of intracellular ice and to the toxicity of cryoprotectant. The most used cryo-protocol is the slow freezing procedure, but recently many authors have proposed vitrification as an alternative, because of its simplicity. The damage extent and the quality of follicles after cryopreservation are usually evaluated morphologically by conventional histological procedures, light and electron microscopy. Our laboratory, to further improve the evaluation and to better investigate damages, is adopting a combination of Synchrotron soft X-ray Microscopy (at TwinMic – Elettra) and XRF at different incident energies (at TwinMic – Elettra and ID21 – ESRF). X-ray techniques were performed on histological sections at micro and sub-micron resolution. Phase contrast and absorption images revealed changes in the compactness of the tissues, as well as cellular abnormalities revealed at sub-micrometric resolution. The distributions of the elements detected at 7.3 and 1.5 keV were compared and particularly Cl resulted to be indicative of follicle integrity. The results demonstrate the utility and the potential of X-ray microscopy and fluorescence in this research field.

Effects of Heyan Kuntai Capsule () on Follicular Development and Oocyte Cohesin Levels in Aged Mice.

To evaluate the effect of Heyan Kuntai Capsule (, HYKT) on the ovarian function of aged mice and expressions of cohesion complexes in oocytes. Twenty-five 9-month-old female C57BL/6J mice were randomly divided into 5 groups by block randomization method (n=5 per group), including the control group (saline), 17β-estradiol group [E2, 100 μg/(kg•d)], and low-, medium-, and highdose of HYKT groups [0.3, 0.9, 2.7 g/(kg•d), respectively]. All mice were treated by intragastric administration for 4 weeks. Hematoxylin and eosin staining and anti-VASA staining were used to detect the amounts of follicles. The apoptosis of follicles was measured by anti-gamma H2A histone family member X (γH2AX) staining and TdT-mediated dUTP Nick-End Labeling (TUNEL) assay. The density of cohesin subunits, REC8 meiotic recombination protein (REC8), structural maintenance of chromosome (SMC) 1β and SMC3 in oocytes were evaluated by immunofluorescent staining. After administration of E2 and high-dose of HYKT, the total number of follicles as well as the number of primordial and primary follicles were significantly increased (P<0.05). Anti-γH2AX staining and TUNEL assay demonstrated that high-dose of HYKT and E2 partly suppressed the apoptosis of follicles (P<0.05). Furthermore, it showed an increased trend in the levels of REC8 and SMC1β, after administration with E2 and HYKT, and no obvious change in the level of SMC3. HYKT could enhance the number of follicles, suppress apoptosis of oocytes and have a trend to elevate the meiotic-specific cohesin subunits (REC8 and SMC1β) in oocytes of aged mice, indicating a beneficial effect on the ovarian function in terms of the quantity and quality of follicles.

Is oxygen availability a limiting factor for in vitro folliculogenesis?

Transplantation of ovarian tissue for the preservation of fertility in oncological patients is becoming an accepted clinical practice. However, the risk of re-introducing tumour cells at transplantation has stirred an increased interest for complete in vitro folliculogenesis. This has not yet been achieved in humans possibly for the lack of knowledge on the environmental milieu that orchestrates folliculogenesis in vivo. The main aim of this study was to investigate the effect of oxygen availability on follicle health and growth during in vitro culture of ovarian tissue strips. To this end, a model was developed to predict the dissolved oxygen concentration in tissue under varying culture conditions. Ovarian cortical strips of bovine, adopted as an animal model, and human tissue were cultured in conventional (CD) and gas permeable (PD) dishes under different media column heights and gaseous oxygen tensions for 3, 6 and 9 days. Follicle quality, activation of primordial follicles to the primary stage, and progression to the secondary stage were analysed through histology. Follicle viability was assessed through a live-dead assay at the confocal scanning laser microscope. Findings showed a higher follicle quality and viability after culture of bovine ovarian strips in PD in adequate medium height and oxygen tensions. The best culture conditions found in the bovine were adopted for human ovarian strip culture and promoted a higher follicle quality, viability and progression. Overall, data demonstrated that modulation of oxygen availability in tissue plays a key role in maintaining follicles’ health and their ability to survive and progress to the secondary stage during ovarian tissue in vitro culture. Such culture conditions could increase the yield of healthy secondary follicles for subsequent dissection and individual culture to obtain competent oocytes.

A modified and tailored human follicle isolation procedure improves follicle recovery and survival

BackgroundOvarian tissue cryopreservation followed by transplantation after cancer remission is the most commonly applied fertility restoration approach in very young girls and women who require immediate cancer therapy. However, clinicians strongly advise against reimplantation of one’s own ovarian tissue when there is a high risk of recurrence after grafting. For these patients, development of an alternative strategy, namely a transplantable artificial ovary, offers future hope of conceiving. The first essential requirement for an artificial ovary is the set-up of a safe and effective follicle isolation procedure. Despite encouraging results with different variants of this technique, none of them take into the account the physiology and great variability in follicular density inside individual tissue fragments and between different patients. The goal of this study was to improve our previously applied follicle isolation procedure in order to develop a tailored isolation procedure for human follicles according to individual tissue properties. To this end, enzymatic digestion was divided into three time intervals in order to initially recover the first follicles to be isolated, and then further dissociate undigested fragments of tissue containing entrapped follicles.ResultsAfter thawing frozen human ovarian tissue using a modified and tailored follicle isolation method, already 35% of follicles were fully isolated and recovered after 30 min of enzymatic digestion. Indeed, this protocol resulted in a higher follicle yield (p < 0.01) and greater numbers of primordial and primary follicles (p < 0.05) than the previous approach. However, no significant difference was found in caspase-3-positive and Ki67-positive staining between the two isolation protocols. In addition, greater follicle quality was demonstrated. When human follicles isolated using the modified protocol were encapsulated in a fibrin matrix with high concentrations of fibrinogen and thrombin and xenografted to a SCID mouse, more follicles were found to be healthy after one week of transplantation than in a previous our study.ConclusionsWith the modified follicle isolation method, we were able to maximize the number and quality of isolated primordial and primary follicles, and develop a tailored follicle isolation procedure according to individual tissue properties. Moreover, improved follicle survival inside an artificial ovary prototype was detected after one week of xenografting.