Molecular Mechanisms Determining Mammalian Oocyte Quality with the Treatment of Cancer Therapy.

Cancer is a global public health issue and remains one of the leading causes of death in the United States (Siegel et al. CA Cancer J Clin. 72:7-33, 2022). It is estimated in the US in 2022, about 935,000 new cases of cancer will be diagnosed in women, and the probability of developing invasive cancer is 5.8% for females younger than 50 years old (Siegel et al. CA Cancer J Clin. 72:7-33, 2022). However, advances in screening programs, diagnostic methods, and therapeutic options have greatly increased the five-year survival rate in reproductive-age women with a variety of cancers. Given the clinical consequences of gonadotoxic cancer therapies, young, female cancer survivors may face compromised fertility, premature ovarian insufficiency, early-onset menopause, and endocrine dysregulation (Bedoschi et al. Future Oncol. 12:2333-44, 2016). Gonadotoxic side effects may include decreased oocyte quality within surviving follicles, loss of ovarian follicles, and impaired ovarian function. In reproductive-age women, oocyte quality is an important element for successful clinical pregnancies and healthy offspring as poor-quality oocytes may be a cause of infertility (McClam et al. Biol Reprod. 106:328-37, 2022; Marteil et al. Reprod Biol. 9:203-24, 2009; Krisher. J Anim Sci. 82: E14-E23, 2004). Thus, it is critical to determine the quantity and quality of surviving follicles in the ovary after cancer treatment and to assess oocyte quality within those surviving follicles as these are markers for determining the capacity for ovarian function restoration and future fertility, especially for young cancer survivors (Xu et al. Nat Med. 17:1562-3, 2011). The long-term effects of cancer therapeutics on oocyte quality are influenced by factors including, but not limited to, individual patient characteristics (e.g. age, health history, comorbidities, etc.), disease type, or treatment regimen (Marci et al. Reprod Biol Endocrinol. 16:1-112, 2018). These effects may translate clinically into an impaired production of viable oocytes and compromised fertility (Garutti et al. ESMO Open. 6:100276, 2021).

Hypoxia Leads to Diminished Ovarian Reserve in an Age-Dependent Manner.

OBJECTIVES: Perinatal hypoxia causes premature activation and initiation of growth in dormant follicles, leading to diminished ovarian reserve. An indirect mechanism such as the release of stress-related hormones may influence ovarian follicle recruitment under hypoxic conditions. We wanted to determine whether hypoxic ovarian damage results from increased follicle growth and "burnout" or from increased apoptosis and whether this damage is age-dependent. DESIGN: Animal study was conducted. PARTICIPANTS/MATERIALS, SETTING, METHODS: Using adult 6-week-old (n = 8) and one-day-old newborn (n = 20) ICR (CD-1) female mice, ovarian follicular counts were conducted on H&E-stained sections. METHODS: Immunohistochemistry was performed on sections stained with Ki-67, anti-Caspase 3, and anti-FOXO3A. RESULTS: Exposure to hypoxia resulted in significantly reduced proportion of primordial follicles versus normoxia in both adult dams and newborn pups (3.17 ± 2.75 vs. 17.89 ± 4.4%; p = 0.004; 40.59 ± 14.88 vs. 81.92 ± 31.56%, p = 0.001, respectively), concomitant with increased growing-primary and secondary follicles, and more pronounced in adult dams versus newborn pups (6-fold vs. 2-fold, respectively). Ki67 staining revealed higher scores of cell proliferation in follicular granulosa cells after exposure to hypoxia than normoxia. However, Caspase 3 and Foxo3A staining did not show any differences in these markers of apoptosis in oocytes, granulosa cells, theca cells, or stromal cells when exposed to hypoxia versus normoxia. LIMITATIONS: The current study has several limitations; first, the sample size for each group is relatively small, which could limit the generalizability of the findings. Second, the study used an ex vivo culture system, which may not fully capture the complex interactions that occur in the whole animal. Third, the exposure to hypoxia only lasted for 3 h, which may not be long enough to observe all the potential effects. In addition, the study only analyzed specific markers of apoptosis in a few cell types, and other cell types or apoptotic pathways might be involved. Lastly, the study provides evidence for accelerated follicular activation and decreased ovarian reserve, but the underlying mechanisms are not fully explored. CONCLUSIONS: Direct tissue hypoxia led to premature activation and initiation of growth in dormant follicles leading to diminished ovarian reserve. Hypoxic damage is age-dependent, with adult ovaries more susceptible than newborn ovaries. These findings support the possibility of follicular "burn out" as a potential mechanism responsible for hypoxia-induced loss of ovarian reserve.

Loss of ERß Disrupts Gene Regulation in Primordial and Primary Follicles.

Loss of ERß increases primordial follicle growth activation (PFGA), leading to premature ovarian follicle reserve depletion. We determined the expression and gene regulatory functions of ERß in dormant primordial follicles (PdFs) and activated primary follicles (PrFs) using mouse models. PdFs and PrFs were isolated from 3-week-old Erß knockout (Erßnull) mouse ovaries, and their transcriptomes were compared with those of control Erßfl/fl mice. We observed a significant (≥2-fold change; FDR p-value ≤ 0.05) deregulation of approximately 5% of genes (866 out of 16,940 genes, TPM ≥ 5) in Erßnull PdFs; ~60% (521 out of 866) of the differentially expressed genes (DEGs) were upregulated, and 40% were downregulated, indicating that ERß has both transcriptional enhancing as well as repressing roles in dormant PdFs. Such deregulation of genes may make the Erßnull PdFs more susceptible to increased PFGA. When the PdFs undergo PFGA and form PrFs, many new genes are activated. During PFGA of Erßfl/fl follicles, we detected a differential expression of ~24% genes (4909 out of 20,743; ≥2-fold change; FDR p-value ≤ 0.05; TPM ≥ 5); 56% upregulated and 44% downregulated, indicating the gene enhancing and repressing roles of Erß-activated PrFs. In contrast, we detected a differential expression of only 824 genes in Erßnull follicles during PFGA (≥2-fold change; FDR p-value ≤ 0.05; TPM ≥ 5). Moreover, most (~93%; 770 out of 824) of these DEGs in activated Erßnull PrFs were downregulated. Such deregulation of genes in Erßnull activated follicles may impair their inhibitory role on PFGA. Notably, in both Erßnull PdFs and PrFs, we detected a significant number of epigenetic regulators and transcription factors to be differentially expressed, which suggests that lack of ERß either directly or indirectly deregulates the gene expression in PdFs and PrFs, leading to increased PFGA.

Why is there an "oversupply" of human ovarian follicles?†.

Women are born with hundreds of thousands to over a million primordial ovarian follicles (PFs) in their ovarian reserve. However, only a few hundred PFs will ever ovulate and produce a mature egg. Why are hundreds of thousands of PFs endowed around the time of birth when far fewer follicles are required for ongoing ovarian endocrine function and only a few hundred will survive to ovulate? Recent experimental, bioinformatics, and mathematical analyses support the hypothesis that PF growth activation (PFGA) is inherently stochastic. In this paper, we propose that the oversupply of PFs at birth enables a simple stochastic PFGA mechanism to yield a steady supply of growing follicles that lasts for several decades. Assuming stochastic PFGA, we apply extreme value theory to histological PF count data to show that the supply of growing follicles is remarkably robust to a variety of perturbations and that the timing of ovarian function cessation (age of natural menopause) is surprisingly tightly controlled. Though stochasticity is often viewed as an obstacle in physiology and PF oversupply has been called "wasteful," this analysis suggests that stochastic PFGA and PF oversupply function together to ensure robust and reliable female reproductive aging.

Effect of direct therapeutic ultrasound exposure of ovaries on histopathology, inflammatory response, and oxidative stress in dogs.

BACKGROUND: This research was designed to evaluate the effects of therapeutic ultrasound waves on ovarian germinal tissue and inflammatory cytokines (interleukin-6 (IL-6), IL1ß, tumor necrosis factor-α (TNF-α)), acute phase proteins (serum amyloid A (SAA), C reactive protein (CRP)) and oxidative stress (total antioxidant capacity (TAC), and malondialdehyde (MDA)) in dogs. Twenty-six clinically healthy adult mix-breed female dogs were aligned into three groups. Laparotomy was performed in control (n = 6) and treatment (T5, n = 10; T10, n = 10) groups. The ultrasonic exposure of ovaries in treatment groups was performed during laparotomy by round motions of the therapeutic ultrasonic transducer on both ovaries (1 MHz frequency, 1.5 W/cm2) for 5 min in the T5 group and for 10 min in the T10 group. Blood samples were collected from the jugular vein into a plain glass tube on days 0 (before laparotomy), 3, 6, and 9 after surgery. All control and treatment groups' dogs were ovariectomized for histological evaluation on day 60 after laparotomy or laparotomy + ultrasound exposure. RESULTS: Direct exposure of ovaries with therapeutic ultrasound waves induced inflammation and oxidative stress comparison with the control group. Histopathological evaluation of treated ovaries with ultrasound waves indicated a decreased number of primordial follicles (ovarian reserve) and oocyte preservation scores compared with ovaries in the control group. CONCLUSIONS: These changes may cause subfertility in the long term. It seems that inflammatory response and oxidative stress are factors in the permanent damage of ovarian tissue.

Co-culture of human cryopreserved fragmented ovarian tissue with theca progenitor cells derived from theca stem cells.

PURPOSE: Despite the significant advances in the in vitro development of human primordial follicles, it is still a challenging approach with great potential for improvements. Therefore, the present study aimed to investigate the effect of a feeder layer of human theca progenitor cells (hTPCs) on the development of primordial follicles embedded in human ovarian tissue. METHODS: Fragments of frozen-thawed ovarian tissue were activated using the vanadate-derivative dipotassium bisperoxo (5-hydroxy-pyridine-2-carboxylic) oxovanadate (V) and kit ligand for 24 h. Then, the specimens were divided into the co-culture and mono-culture groups and were cultured with and without a hTPC feeder layer for 6 days, respectively. Afterward, the follicles were counted and classified, and the hormone levels and expression levels of apoptosis- and folliculogenesis-related genes were assessed. RESULTS: Both culture groups showed significant follicle growth (P < 0.05). However, the co-culture group had a significantly higher number of growing follicles compared to the other group (P < 0.05). Moreover, the expression levels of ZP1, ZP2, ZP3, BMP-7, AMH, and GDF9 were significantly higher in the co-culture group compared to the other group (P < 0.05), while the expression levels of P53 and CASP3 were significantly lower (P < 0.05). Also, the concentrations of estradiol, progesterone, testosterone, and androstenedione were significantly higher in the co-culture group compared to the other group (P < 0.05). CONCLUSION: The present study results provided novel evidence on the direct role of hTPCs in the growth and development of human primordial follicles. However, there is a need for future studies to illustrate the underlying mechanisms. Schematic summary of the results. According to our results, the expression of ZP1, ZP2, ZP3, and GDF9 in the oocytes, AMH in the granulosa cells, and BMP4 in the theca cells of the co-culture group were significantly higher than those of the mono-culture and non-culture groups, while the expression of apoptotic genes (BAX, CASP3, and P53) was significantly lower. Moreover, the co-culture group showed significantly increased levels of estradiol, progesterone, testosterone, and androstenedione in its culture media compared to the mono-culture groups.

Retention of higher fertility depending on ovarian follicle reserve in cystine-glutamate transporter gene-deficient mice.

The cystine-glutamate transporter (xCT) is responsible for the transport of cystine into cells. We recently found that xCT-deficient (xCTKO) aged mice maintained a higher rate of ovulation and ovarian weight compared with wild-type (WT) mice. It has been reported that a xCT deficiency in cultured cells induces autophagy through the suppression of mTOR survival pathways. We have previously reported that starvation in neonatal mice increases the number of primordial follicles with concomitant autophagy activation. Therefore, we investigated age-related changes in follicle reserve and fertility in xCTKO mice and clarified whether the PI3K/AKT/mTOR signaling pathway contributes to this. The numbers of offspring in the xCTKO mice aged 10 and 12 months were significantly higher than those in the WT mice. The primordial follicle numbers in xCTKO neonatal mice tended to be higher than WT mice during all times evaluated. In contrast, the primary follicle number was significantly lower in the xCTKO mice at 60 h after birth. The expression of p-AKT, which promotes follicle development, was significantly lower in xCTKO mice than that in WT mice, whereas the expression ratios of LC3-II/LC3-I were significantly higher. The xCTKO mice had significantly more primordial follicles than WT mice at 2 months of age and showed a similar trend at 13-15 months of age. These results suggest that the maintenance of fertility in aged xCTKO mice can be attributed to high follicle reserve after puberty by suppression of follicle activation during the neonatal period.

Interference of fixatives and fixation period on the morphologic analysis of ovarian preantral follicles.

Ovine ovarian fragments (3 × 3 × 1 mm) were fixed in neutral buffered formalin (NBF), Carnoy's solution (CAR), Davidson's solution (DAV), or paraformaldehyde (PFA) for 12 h or 24 h. After this fixation time, each fragment was prepared for histological analysis. Although fixative and fixation period did not affect follicular and stromal cells density, the percentages of morphologically normal primordial and primary follicles was affected by the fixative type and period of fixation. Paraformaldehyde was not indicated as a fixative for ovarian fragments. Formalin was a suitable fixative only when the period of fixation was 12 h, while Carnoy was efficient after a fixation period of 24 h. In conclusion, the most indicated fixative for the morphological evaluation of ovarian preantral follicles was DAV, regardless of the fixation period, that is 12 or 24 h.

Automatic Evaluation for Bioengineering of Human Artificial Ovary: A Model for Fertility Preservation for Prepubertal Female Patients with a Malignant Tumor.

INTRODUCTION: The in vitro culture of primordial follicles is the only available option for preserving fertility in prepubertal girls with malignant tumors. The cultivation of primordial follicles in scaffolds as artificial ovaries is a promising approach for this. METHODS: Dissociated follicles were placed into an artificial ovarian scaffold composed of fibrinogen and thrombin. The follicles were cultured in a dish dedicated to live cell imaging and observed for growth using immunofluorescence and development via optical microscopy. The morphology of the follicles in the scaffold was three-dimensionally reconstructed using the Imaris software. Growth and development were also quantified. RESULTS: The morphology of artificial ovaries began to degrade over time. Within approximately 7 days, primordial follicles were activated and grew into secondary follicles. A comparison of optical and confocal microscopy results revealed the superior detection of live cells using confocal microscopy. The three-dimensional reconstruction of the confocal microscopy data enabled the automatic enumeration and evaluation of the overall morphology of many follicles. CONCLUSIONS: The novel artificial ovary-enabled primordial follicles to enter the growth cycle after activation and grow into secondary follicles. The use of a fibrin scaffold as a carrier preserves the developmental potential of primordial germ cells and is a potentially effective method for preserving fertility in prepubertal girls.

Intestinal Flora Changes Induced by a High-Fat Diet Promote Activation of Primordial Follicles through Macrophage Infiltration and Inflammatory Factor Secretion in Mouse Ovaries.

Obesity induced by a high-fat diet (HFD) leads to the excessive consumption of primordial follicles (PFs) in the ovaries. There is systemic chronic inflammation under HFD conditions, but no previous studies have explored whether there is a certain causal relationship between HFD-induced chronic inflammation and the overactivation of PFs. Here, we showed that HFD causes disorders of intestinal microflora in mice, with five Gram-negative bacteria showing the most profound increase at the genus level compared to the normal diet (ND) groups and contributes to the production of endotoxin. Endotoxin promotes M1 macrophage infiltration in the ovaries, where they exhibit proinflammatory actions by secreting cytokines IL-6, IL-8, and TNFα. These cytokines then boost the activation of PFs by activating Signal Transducer and Activator of Transcription 3 (STAT3) signaling in follicles. Interestingly, transplantation of the HFD intestinal microflora to the ND mice partly replicates ovarian macrophage infiltration, proinflammation, and the overactivation of PFs. Conversely, transplanting the ND fecal microbiota to the HFD mice can alleviate ovarian inflammation and rescue the excessive consumption of PFs. Our findings uncover a novel and critical function of gut microbes in the process of PF overactivation under HFD conditions, and may provide a new theoretical basis for the microbial treatment of patients with premature ovarian insufficiency caused by HFD.

ELAVL2-directed RNA regulatory network drives the formation of quiescent primordial follicles.

Formation of primordial follicles is a fundamental, early process in mammalian oogenesis. However, little is known about the underlying mechanisms. We herein report that the RNA-binding proteins ELAVL2 and DDX6 are indispensable for the formation of quiescent primordial follicles in mouse ovaries. We show that Elavl2 knockout females are infertile due to defective primordial follicle formation. ELAVL2 associates with mRNAs encoding components of P-bodies (cytoplasmic RNP granules involved in the decay and storage of RNA) and directs the assembly of P-body-like granules by promoting the translation of DDX6 in oocytes prior to the formation of primordial follicles. Deletion of Ddx6 disturbs the assembly of P-body-like granules and severely impairs the formation of primordial follicles, indicating the potential importance of P-body-like granules in the formation of primordial follicles. Furthermore, Ddx6-deficient oocytes are abnormally enlarged due to misregulated PI3K-AKT signaling. Our data reveal that an ELAVL2-directed post-transcriptional network is essential for the formation of quiescent primordial follicles.

Adipose tissue-derived stem cells protect the primordial follicle pool from both direct follicle death and abnormal activation after ovarian tissue transplantation.

PURPOSE: To investigate whether adipose tissue-derived stem cells (ASCs) protect the primordial follicle pool, not only by decreasing direct follicle loss but also by modulating follicle activation pathways. METHODS: Twenty nude mice were grafted with frozen-thawed human ovarian tissue from 5 patients. Ten mice underwent standard ovarian tissue transplantation (OT group), while the remaining ten were transplanted with ASCs and ovarian tissue (2-step/ASCs+OT group). Ovarian grafts were retrieved on days 3 (n = 5) and 10 (n = 5). Analyses included histology (follicle count and classification), immunohistochemistry (c-caspase-3 for apoptosis and LC3B for autophagy), and immunofluorescence (FOXO1 for PI3K/Akt activation and YAP for Hippo pathway disruption). Subcellular localization was determined in primordial follicles on high-resolution images using structured illumination microscopy. RESULTS: The ASCs+OT group showed significantly higher follicle density than the OT group (p = 0.01). Significantly increased follicle atresia (p < 0.001) and apoptosis (p = 0.001) were observed only in the OT group. In primordial follicles, there was a significant shift in FOXO1 to a cytoplasmic localization in the OT group on days 3 (p = 0.01) and 10 (p = 0.03), indicating follicle activation, while the ASCs+OT group and non-grafted controls maintained nuclear localization, indicating quiescence. Hippo pathway disruption was encountered in primordial follicles irrespective of transplantation, with nuclear YAP localized in their granulosa cells. CONCLUSION: We demonstrate that ASCs exert positive effects on the ovarian reserve, not only by protecting primordial follicles from direct death but also by maintaining their quiescence through modulation of the PI3K/Akt pathway.

PI3K/PTEN/AKT Signaling Pathways in Germ Cell Development and Their Involvement in Germ Cell Tumors and Ovarian Dysfunctions.

Several studies indicate that the PI3K/PTEN/AKT signaling pathways are critical regulators of ovarian function including the formation of the germ cell precursors, termed primordial germ cells, and the follicular pool maintenance. This article reviews the current state of knowledge of the functional role of the PI3K/PTEN/AKT pathways during primordial germ cell development and the dynamics of the ovarian primordial follicle reserve and how dysregulation of these signaling pathways may contribute to the development of some types of germ cell tumors and ovarian dysfunctions.

Activation of autophagy in early neonatal mice increases primordial follicle number and improves lifelong fertility†.

The number of stockpiled primordial follicles is thought to be responsible for the fate of female fertility and reproductive lifetime. We previously reported that starvation in nonsuckling early neonatal mice increases the number of primordial follicles with concomitant autophagy activation, suggesting that autophagy may accelerate the formation of primordial follicles. In this study, we attempted to upregulate the numbers of primordial follicles by administering an autophagy inducer and evaluated the progress of primordial follicle formation and their fertility during the life of the mice. To induce autophagy, mice were intraperitoneally injected with the Tat-beclin1 D-11 peptide (0.02 mg/g body weight) at 6-54 h or 60-84 h after birth. In animals that received Tat-beclin 1 D-11 by 54 h after birth, the primordial follicle numbers were significantly increased compared with the control group at 60 h. The ratio of expressed LC3-II/LC3-I proteins was also significantly greater. The numbers of littermates from pregnant females that had been treated with Tat-beclin 1 D-11 were maintained at remarkably greater levels until 10 months old. These results were supported by an abundance of primordial follicles at even 13-15 months old.

Follicle inhibition at the primordial stage without increasing apoptosis, with a combination of everolimus, verapamil.

The aim of the present study was to test whether inhibition of ovarian primordial follicles and subsequent activation can be achieved by transient mTOR inhibition. In this preclinical investigation, forty-five female immature Wistar rats were randomized in 5 groups. The control group received subcutaneous saline injections. The other groups received Everolimus, Everolimus plus Verapamil, Everolimus plus Fisetin, and Fisetin alone. Primary and secondary outcomes were measured in the left ovary after a treatment period of 8 weeks. Ten days later, animals received 35 IU FSH for 4 days and 35 IU of hCG on the 5th day. The same parameters were examined in the right ovary. AMH, estradiol, and progesterone levels were assessed at the end of both interventions. Significantly, more primordial and less atretic follicles were observed in the Everolimus plus Verapamil group. AMH and progesterone levels were substantially lower in the Everolimus group. Interestingly, after ovarian stimulation higher levels of AMH and progesterone were observed in the Everolimus plus Verapamil group. Immunoblot analysis of ovarian extracts revealed that the administration of Everolimus led to a significant reduction in the mTORC1-mediated phosphorylation of the 70-kDa ribosomal protein S6 kinase 1. This decrease was reversed in the presence of FSH after stopping drug administration. The expression of the anti-apoptotic molecule Bcl2 as well as of LC3-II and ATG12 was increased after removal of the Everolimus plus Verapamil combination, indicating reduced apoptosis and increased autophagy, whereas the levels of the proliferation marker PCNA in the granulosa cells were elevated, consistent with initiation of follicular growth.Thus, the combination of Everolimus plus Verapamil is capable of increasing the number of competent primordial follicles while reducing atresia.

Activation of goat primordial follicles in vitro: Influence of alginate and ovarian tissue.

The present study aimed to evaluate the effect of three culture systems on caprine primordial follicle activation in vitro: follicles cultured either in the isolated form within alginate (Isolated follicles + Alginate treatment), or enclosed in ovarian tissue (in situ), with or without alginate (Fragment + Alginate, and Fragment alone treatments, respectively). After culture, the Isolated follicles + Alginate treatment presented a percentage of morphologically normal follicles (MNF) similar to both the non-cultured control and the Fragment Alone treatments. Nevertheless, Fragment + Alginate treatment showed a significant reduction in the number of MNF when compared to the other treatments. Regarding follicle development, our results showed that regardless of the alginate, the presence of ovarian tissue limited primordial follicle activation during in vitro culture. Remarkably, the Isolated primordial follicle + Alginate treatment was the only one that significantly promoted follicle activation and increased both follicle and oocyte diameters during IVFC, pointing out a higher cell proliferation. In conclusion, the presence of ovarian tissue with or without alginate limited follicle development (activation) after culture. Nevertheless, when primordial follicles were isolated and encapsulated in alginate they presented suitable survival rates, higher rates of follicle activation and continued to grow throughout the culture period.

Histology of the normal ovary in premenopausal patients.

Detailed descriptions of ovarian histology are rare. We reviewed in detail 57 cases of normal ovaries in premenopausal patients, when the ovaries are active and primordial follicles are found. We also proposed updated definitions to more clearly distinguish inclusion cysts, which do not have a known relationship with any disease process, from endosalpingiosis, a lesion closely associated with low grade serous neoplasia of the ovary. The most interesting findings were the significant variation in the histologic features including the variation in the amount and the distribution of primordial follicles, follicular cysts, and endosalpingiosis, within the ovary and between both ovaries in the same patient, the frequent presence of primordial follicles in the medulla, specifically in cases of multiple follicular cysts, and the frequent presence of endosalpingiosis. We believe that to confirm a pathologic process in the ovary, we need to become familiar with the histologic features of the normal ovary and their variations.

Formation and activation induction of primordial follicles using granulosa and cumulus cells conditioned media.

Fertility preservation of prepubertal girls subjected to invasive cancer therapy necessitates defining protocols for activation of isolated primordial follicles. Granulosa (GCs) and cumulus cells (CCs) play pivotal role in oocyte development. Although GCs and CCs share some similarities, they differ in growth factors production. The current study was conducted to evaluate the effects of GCs, CCs and their conditioned media on mice primordial follicles activation. One-day-old mice ovaries were subjected to 6-day culture with base medium (BM), GC conditioned medium (GCCM), GC coculture (GCCC), CC conditioned medium (CCCM) or CC coculture (CCCC). Follicular growth and primordial to primary follicle transition was observed during 6-day culture, and follicular activation rate tended to be greater in GCCM than other groups (0.05

Disruptions in the reproductive system of female rats after prenatal lipopolysaccharide-induced immunological stress: role of sex steroids.

Stress signals during fetal or early postnatal periods may disorganize reproductive axis development at different levels. This study was aimed to test the hypothesis that prenatal immunological stress induced by bacterial endotoxin, lipopolysaccharide (LPS), has impact on structure and function of the reproductive system in female offspring. Adult female Wistar rats were divided into two groups, a control group (n = 5) and a LPS group (n = 12). Rats were injected with LPS 50 µg/kg body or 0.9% saline intraperitoneally on the 12th day of pregnancy. After birth the female pups (n = 20 in each group) were divided into four groups: (group 1) 0.9% saline prenatally, sesame oil (vehicle) postnatally; (group 2) LPS prenatally, sesame oil postnatally; (group 3) LPS prenatally, fulvestrant postnatally; (group 4) LPS prenatally, flutamide postnatally. Pups were injected subcutaneously into the neck with fulvestrant (estrogen receptor antagonist), 1.5 mg/kg in sesame oil, from postnatal day (PND) 5 to PND14; or flutamide (androgen receptor antagonist), 20 mg/kg in sesame oil, from PND14 to PND30. Rats of the control group were injected with sesame oil during the same time period. Parameters were evaluated by ELISA (serum estradiol and testosterone) and ovarian histology. The main findings were: (1) prenatal stress during the critical period resulted in delayed vaginal opening, decreased body weight and serum concentrations of sex steroids, and significant disorders in ovarian development; (2) postnatal estradiol and testosterone antagonist treatments decreased follicular atresia through increasing the number of healthy follicles and restored endogenous steroid production. Lay summaryImmunological stress, caused by simulating infection through exposure to a bacterial toxin (LPS), during a critical period of fetal development in laboratory rats results in delayed reproductive maturity, decreased body weight and decreased secretion of sex steroids in female offspring, and abnormalities in the ovaries like those in polycystic ovarian syndrome. These prenatally toxin-induced sexual disorders in females could be corrected by estradiol/testosterone antagonists during the postnatal period.

Future developments: In vitro growth (IVG) of human ovarian follicles.

Removal and storage of ovarian cortical tissue is currently offered to young female cancer patients undergoing potentially sterilizing chemotherapy and/or radiotherapy. For patients at high risk of reintroduction of malignancy through auto-transplantation, the ultimate aim is to achieve complete oocyte development from this tissue in vitro. The ability to develop human oocytes from the earliest follicular stages through to maturation and fertilization in vitro would revolutionize fertility preservation practice. This has been achieved in mice where in vitro grown oocytes from primordial follicles have resulted in the production of live offspring. Systems that support growth and development of oocytes from human ovarian cortex are being developed by several groups. This review focuses on the steps required to recapitulate in vitro the process of human oocyte development from the primordial stage and the systems currently available to support this.