Untreated Ovaries Research Articles

P-622 Prospectively randomized study of intraovarian injections of autologous platelet-rich plasma (PRP) into one alternating ovary in Primary Ovarian Insufficiency (POI)

Abstract Study question Does intraovarian injection of autologous PRP affect ovarian function in women with POI? Summary answer Ovaries that were injected with PRP demonstrated a significant increase in follicles >4mm in size compared to the untreated contralateral ovaries. What is known already The injection of PRP into ovaries has been reported to improve subsequent ovarian response to ovulation induction in women with POI and is now widely used in fertility centers in women with POI as well as in women with premature ovarian aging (POA). Prospectively randomized studies are, however, lacking. Study design, size, duration This is a Randomized Controlled Trial with recruitment from July 2018 - November 2023. 40 patients were screened; 34 qualified. POI was defined by amenorrhea among women < 40 years old, with FSH > 30 mIU/mL, undetectable AMH, and no follicles >4mm. For each participant one ovary was randomly selected for treatment ( with the Sealed Envelope Ltd 2022 tool). The study was powered to detect a 20% response in the treated versus untreated ovary. Participants/materials, setting, methods For each patient (n = 34), 10mL of blood was drawn into ReganLab vacutainer and centrifuged. Platelets were re-suspended in 1.5mL of plasma. PRP was injected into the stroma of one randomly selected ovary under iv sedation and ultrasound control. Patients were followed with ultrasounds, estradiol, and FSH q3-4 days for two weeks. Our primary outcome was the development of new follicles >4mm in the treated vs. untreated ovary, using Chi-Square. Main results and the role of chance Study population description: age 33.9±5.5 years (range 21-40.4 years); BMI 23.4±4.2; FSH 96.7±27.6 mIU/mL; AMH 0.019±0.03 ng/mL; length of amenorrhea 2.2 years (3 months-14 years). 10 patients developed no follicles > 4 mm, 21/34 (62%) did develop follicles > 4 mm in treated ovaries; 9/34 (26%) developed a follicle > 4 mm in untreated ovaries (Chi-square = 8.59; P = 0.003). Most follicles developed without significant estradiol production; nine showed sufficient follicle development to initiate ovulation induction with initiation of gonadotropins, four produced eggs and one produced an embryo; one patient had four oocytes banked and resumed menses following PRP treatment for several months. Limitations, reasons for caution N/A Wider implications of the findings This demonstrates the first objective evidence that intraovarian PRP injections into POI ovaries initiate some follicle activation. Though the observed degree of activation was small, findings suggest that remaining follicles in women with POI can be activated through injections into ovaries, suggesting that better agents than PRP may be discovered. Trial registration number N/A

Ovaries of patients recently treated with alkylating agent chemotherapy indicate the presence of acute follicle activation, elucidating its role among other proposed mechanisms of follicle loss

To investigate mechanisms of primordial follicle (PMF) loss invivo in human ovaries shortly after alkylating agent (AA) chemotherapy. Cohort study. Tertiary university medical center. Ninety-six women aged 15-39 years who underwent ovarian tissue cryopreservation for fertility preservation. Fresh ovarian tissue samples were harvested from women treated with AA (n = 24) or non-AA (n = 24) chemotherapy <6 months after treatment and age-matched untreated women (n = 48). Differential follicle counts, time from chemotherapy exposure, immunostaining for apoptosis (cleaved caspase-3) and FOXO3A on tissue harvested within ultrashort time intervals (4-12 days), collagen (Sirius red) and neovascularization (CD34). AA-treated ovaries had significant loss of PMFs, and significant increase in absolute numbers of growing follicles compared with untreated control ovaries. The number of growing follicles was inversely correlated with time from chemotherapy. Representative staining for FOXO3A observed decreased nuclear localization in PMF oocytes in AA-treated ovaries removed within the ultrashort time interval compared with untreated ovaries. Neither significant loss of PMFs, increase in growing follicles, nor decrease in nuclear FOXO3A were observed in non-AA-treated ovaries. No increased expression of cleaved caspase-3 was seen in PMFs within the ultrashort time interval after AA or non-AA chemotherapy. Significant stromal fibrosis and neovascularization were observed in AA-treated ovaries only after follicle loss had already occurred (4-6 months). Follicle activation occurs invivo in ovaries of patients treated with AA, indicating a pathologic mechanism which may contribute to chemotherapy-induced follicle loss.

Whole-ovary decellularization generates an effective 3D bioscaffold for ovarian bioengineering.

To develop a new protocol for whole-ovary decellularization for the production of a 3D bioscaffold suitable for in vitro/ex vivo studies and for the reconstruction of a bioengineered ovary. Porcine ovaries were subjected to the decellularization process (DECELL; n = 20) that involved a freeze-thaw cycle, followed by sequential incubations in 0.5% SDS for 3h, 1% Triton X-100 for 9h, and 2% deoxycholate for 12h. Untreated ovaries were used as a control (CTR; n = 6). Both groups were analyzed to evaluate cell and DNA removal as well as ECM preservation. DECELL bioscaffolds were assessed for cytotoxicity and cell homing ability. DECELL ovaries maintained shape and homogeneity without any deformation, while their color turned from red to white. Histological staining and DNA quantification confirmed a decrease of 98.11% in DNA content, compared with the native tissue (CTR). Histochemical assessments demonstrated the preservation of intact ECM microarchitecture after the decellularization process. This was also confirmed by quantitative analysis of collagen, elastin, and GAG contents. DECELL bioscaffold showed no cytotoxic effects in co-culture and, when re-seeded with homologous fibroblasts, encouraged a rapid cell adhesion and migration, with repopulating cells increasing in number and aggregating in cluster-like structures, consistent with its ability to sustain cell adherence, proliferation, and differentiation. The protocol described allows for the generation of a 3D bioscaffold that may constitute a suitable model for ex vivo culture of ovarian cells and follicles, as well as a promising tool for the reconstruction of a bioengineered ovary.

Expression of Genes Involved in the Non-Neuronal Cholinergic System and Their Possible Functions during Ovarian Follicular Development in Mice

The regulatory roles of the non-neuronal cholinergic system, such as its growth promoting effects on mural granulosa cells during ovarian folliculogenesis, have been reported for several mammalian species; however, its roles in the early stages of follicles are not well-understood. This study was conducted to examine the expression of transcripts involved in the non-neuronal cholinergic system, and the effects of activating acetylcholine (ACh) signaling in mouse ovaries. The transcripts encoding proteins required in processes critical for ACh metabolism and a nicotinic cholinergic receptor, CHRNA7, were detected in ovaries and isolated mural granulosa cells. Stimulation with carbachol, a cholinergic agonist, promoted proliferation of mural granulosa cells in vitro. Moreover, carbachol treatment of neonatal ovaries significantly increased the numbers of primordial follicles compared with control untreated ovaries in organ cultures. These results suggest that a functional non-neuronal cholinergic system exists in mouse ovaries, and that it promotes the proliferation of mural granulosa cells and the formation/survival of primordial follicles.

Biomechanical proposal as a cause of incomplete seed and pericarp development of the sunflower (Helianthus annuus L.) fruits

Absence or underdevelopment of sunflower fruits come usually from failure in fertilization, stresses as well as physiological and morphological defects in the ovary. Thigmomorphogenesis has never been included as a possible cause. We have previously shown that a 2-3 day fertilization shift can occur between neighboring florets in the sunflower capitulum. It is proposed here that those ovaries with advanced fertilization can generate a significant radial and axial compressive stress on ovaries with delayed fertilization. This mechanical stimulus could be strong enough to trigger a thigmo response that affects further ovary development. In vivo tests were performed, isolating ovaries by removing the adjacent flowers and rubbing them several times using a micromanipulator applying a force of 1 to 3 N. Total peroxidases in treated and untreated ovaries, isolated and not isolated were measured 24 h after treatments. Ovary development was studied in control and isolated flowers, both rubbed and not rubbed. Also a mechanical a model was made to simulate the mechanical behavior of an ovary surrounded by advanced growing neighboring ovaries. A meshed 3-D model of a young ovary was constructed and a computer simulation was performed using finite element analysis. Shear stresses generated by the friction of neighboring ovaries in contact with the model, fertilized three days later, were then estimated. After rubbing, isolated ovaries in planta showed a thigmo response that resulted in empty or incompletely developed fruits. Total peroxidase levels (ΔAbs470 min-1.g fresh weight-1) rose from 22 in control ovaries to 72 in rubbed ones. The number of ovaries that did not develop any seed from these incompletely developed fruits rose from 16.2 to 20.0% in the control non-isolated flowers to 61.1 to 86.7% in the rubbed ovaries, but dropped to 6.7 to 7.3% in the non-rubbed but isolated ovaries. From the simulation it was found that the area of contact with the receptacle was prone to show a higher magnitude of stress after deformation induced by shear forces generated by neighboring ovaries. Thigmomorphogenesis can also explain the failures observed at early stages of the sunflower ovary development. The ovary tissue sensitivity at this stage could contribute to the rapid response of the mechanically generated stimulus between neighbouring ovaries.

Paracrine effects of human amniotic epithelial cells protect against chemotherapy-induced ovarian damage

BackgroundHuman amniotic epithelial cells (hAECs) are attractive candidates for regenerative medical therapy, with the potential to replace deficient cells and improve functional recovery after injury. Previous studies have demonstrated that transplantation of hAECs effectively alleviate chemotherapy-induced ovarian damage via inhibiting granulose cells apoptosis in animal models of premature ovarian failure/insufficiency (POF/POI). However, the underlying molecular mechanism accounting for hAECs-mediated ovarian function recovery is not fully understood.MethodsTo investigate whether hAECs-secreting cytokines act as molecular basis to attenuate chemotherapy-induced ovarian injury, hAECs or hAEC-conditioned medium (hAEC-CM) was injected into the unilateral ovary of POF/POI mouse. Follicle development was evaluated by H&E staining at 1, 2 months after hAECs or hAEC-CM treatment. In addition, we performed a cytokine array containing 507 human cytokines on hAECs-derived serum-free conditioned medium. Finally, we further investigated whether hAECs could affect chemotherapy-induced apoptosis in primary human granulosa-lutein (hGL) cells and the tube formation of human umbilical vein endothelial cells (hUVECs) via a co-culture system in vitro.ResultsWe observed the existence of healthy and mature follicles in ovaries treated with hAECs or hAEC-CM, whereas seriously fibrosis and many atretic follicles were found in the contralateral untreated ovaries of the same mouse. To distinguish cytokines involved in the process of hAECs-restored ovarian function, hAEC-CM was analyzed with a human cytokines array. Results revealed that 109 cytokines in hAEC-CM might participate in a variety of biological processes including apoptosis, angiogenesis, cell cycle and immune response. In vitro experiments, hAECs significantly inhibited chemotherapy-induced apoptosis and activated TGF-β/Smad signaling pathway within primary granulosa-lutein cells in paracrine manner. Furthermore, hAEC-CM was shown to promote angiogenesis in the injured ovaries and enhance the tube formation of human umbilical vein endothelial cells (hUVECs) in co-culture system.ConclusionsThese findings demonstrated that paracrine might be a key pathway in the process of hAECs-mediating ovarian function recovery in animal models of premature ovarian failure/insufficiency (POF/POI).

Inhibition of mTOR Signaling Pathway Delays Follicle Formation in Mice

In mammalian ovaries, follicle assembly requires proper germ cell cyst breakdown and the invasion of somatic cells to encapsulate individual oocytes. Abnormalities in this process lead to a number of pathologies such as premature ovarian failure and infertility. As a conserved pathway regulating cell growth and metabolism in response to growth factors and nutrients, the roles of mTOR signaling in follicular development have been extensively studied in recent years. However, its functions during follicle formation remain unknown. In this study, the expression of p-rpS6 (phospho-ribosomal proteinS6), a downstream marker of mTORC1, showed dynamic changes in perinatal ovaries. When E18.5 ovaries, which mainly contained germ cell nests, were incubated with the mTOR inhibitors Rapamycin and Torin1 for 24 h, follicle assembly was delayed with differential somatic cell invasion into germ cell cyst among the groups. After transplanting treated or untreated ovaries into kidney capsules of recipient ovariectomized mice, follicular development was blocked in treated ovaries, as shown by fewer antral follicles and a higher proportion of primordial follicles. Further studies showed a significant decrease in somatic cell proliferation and the expression of marker genes related to follicular development (Kitl, Kit, Gdf9, Bmp15, Zp3, and Amhr2) in treated ovaries. Moreover, the addition of KITL, a growth factor that is mainly produced by pregranulosa cells during germ cell nest breakdown, rescued the extension of follicle formation induced by mTOR inhibitors. These results suggest that KITL functions downstream of mTOR in somatic cells to regulate their communication with oocytes during follicle formation. J. Cell. Physiol. 232: 585-595, 2017. © 2016 Wiley Periodicals, Inc.

Regulation of FOXO3 subcellular localization by Kit ligand in the neonatal mouse ovary.

Foxo3 protein is required in the oocyte nucleus for the maintenance of primordial follicles in a dormant state. PI3K/AKT-dependent phosphorylation of Foxo3 leads to its relocalization to the cytoplasm and subsequent follicular activation. However, the nature of the upstream signals controlling Foxo3 activity and subcellular localization remains unknown. We aimed to study the in vitro effects of Kit ligand (stem cell factor) on the subcellular localization of Foxo3 in primordial follicles within the postnatal mouse ovary. This was an in vitro study using explants of intact neonatal mouse ovaries. The study was performed in laboratory animal facility and basic science research laboratory at a University Hospital. The animals used for this study were FVB mice. Neonatal FVB mice ovaries at postnatal day 7 (PD7) were harvested and incubated in culture medium (DMEM) at 37 °C and 5 % CO(2) for 60-90 min with (n = 3) or without (n = 3) Kit ligand at 150 ng/mL (8 nM). Similar experimental conditions were used to establish a dose-response curve for the effects of Kit ligand and assess the effects of imatinib (small molecule inhibitor of the Kit receptor). Immunofluorescence was used to identify the subcellular location of Foxo3 in oocytes. Proportions of cytoplasmic versus nuclear Foxo3 in primordial follicles were determined. Kit ligand treatment increased the cytoplasmic localization of Foxo3 from 40 % in the untreated ovaries to 74 % in the treated group (p = 0.007 in paired samples and p = 0.03 in unpaired samples). Furthermore, this effect was reversible with imatinib (p = 0.005). A dose-response curve for Kit ligand treatment showed that maximum effect was seen at 150 ng/mL. Kit ligand treatment in vitro increases the proportion of cytoplasmic Foxo3 in primordial follicles at PD7, lending support to the idea that Kit receptor/ligand controls Foxo3 activity in the context of primordial follicle activation.

189 ESTRADIOL PRODUCTION AND GENE UPREGULATION FOLLOWING PRIMORDIAL FOLLICLE ACTIVATION IN VITRO

Activation and growth of dormant follicles completely in vitro, with the production of competent oocytes, would provide not only the opportunity to investigate control mechanisms of follicle growth, but also an instrument by which fertility might be preserved in women with cancer, premature ovarian failure, or advanced maternal age. The objective of this study was to assess the physical culture parameters, as well as the addition of an antibody to the anti-angiogenic isoform of VEGFA (VEGFAxxxb), for the ability to support activation of primordial follicles and subsequent functional follicular growth in vitro. The phosphatase and tensin homologue (PTEN) inhibitor, bpV(pic), was used to activate growth of primordial follicles in neonatal Day 3 mouse ovaries. BpV(pic) (100 µM) was used either alone, or in combination with FSH (0.3 IU mL–1), IGF-1 (10 ng mL–1), EGF (10 ng mL–1), and ITS (insulin-transferrin-selenium; 1x). Ovaries were treated for 24 h and then cultured for an additional 12 to 20 days, either suspended on a membrane or encapsulated in an alginate bead, with or without VEGFAxxxb during culture. Oestradiol (E2) production and expression of Ahr, Amh, Bmp15, Cyp19, and Esr1, genes known to be involved in follicular growth, were evaluated to assess growth and functionality of follicles. The E2 data were analysed with ANOVA; the qPCR data were analysed using the non-parametric Mann–Whitney U t-test (significance at P &lt; 0.05). Forty-eight hours after treatment with bpV(pic), alone or with FSH, IGF-1, EGF, and ITS, resulted in an upregulation of Amh and Esr1, and a tendency for upregulation of Ahr compared with untreated control ovaries. There were no differences in gene expression between the 2 bpV(pic) treatments. Following treatment and in vitro culture, expression of Ahr, Amh, and Bmp15 was upregulated in ovaries treated and cultured in a bead with VEGFAxxxb compared with ovaries treated and cultured in a bead without VEGFAxxxb, and ovaries treated on a membrane and cultured in a bead with VEGFAxxxb. Although ovaries treated on a membrane with bpV(pic) produced more E2 than untreated ovaries (5.1 ± 1.9 pg mL–1 and 2.5 ± 0.8 pg mL–1, respectively), this was not significantly different. Ovaries treated and cultured on a membrane without VEGFAxxxb produced more E2 (22.5 ± 12.2 pg mL–1) than ovaries treated on a membrane and cultured in an alginate bead, with (3.9 ± 1.9 pg mL–1) or without (1.8 ± 1.2 pg mL–1) VEGFAxxxb; this was not significantly different from ovaries treated and cultured on a membrane with VEGFAxxxb (4.6 ± 4.6 pg mL–1). In summary, primordial follicles can be induced to activate and grow in vitro using a PTEN inhibitor, as evidenced by the upregulated expression of follicle growth genes; VEGFAxxxb also increases expression of these genes. Treatment in a bead compared with a membrane appears to be advantageous for gene expression. However, culture on a membrane best supported oestradiol production, regardless of the presence of VEGFxxxb. These are critical pieces in the development of a successful culture system in which primordial follicles can be activated and functional growth supported in vitro.

Impact of Ovarian Sex Steroids on Ovulation and Ovulatory Gene Induction in Aromatase-Null Mice

Female mice deficient in the aromatase gene [aromatase knockout (ArKO)] fail to ovulate owing to an inability to produce estrogens. Here, we demonstrated that sequential administration of adequate amounts of 17β-estradiol (E2), pregnant mare serum gonadotropin, and human chorionic gonadotropin could induce ovulation in immature ArKO mice; nevertheless, significantly fewer oocytes were released into the oviducts in ArKO mice than in wild-type mice. Analysis of ovarian steroids by liquid chromatography coupled with electrospray ionization-tandem mass spectrometry identified a trace amount of E2 in the untreated immature ArKO ovary. The analysis further detected significant increases and decreases in progesterone and testosterone contents, respectively, in addition to an increase of E2 in the ovulation-induced ArKO ovaries compared with the levels in untreated ArKO ovaries. Gene expression analysis demonstrated marked elevation in the mRNA levels of members of the epidermal growth factor family and extracellular matrix-related proteins at 4 h after human chorionic gonadotropin injection in the ovaries of ArKO mice treated for ovulation, as observed in the ovulation-induced wild-type ovaries. Collectively, these findings suggest the vital contribution of the intraovarian milieu of sex steroids to ovulatory regulation in vivo.

Recurrence rate of endometrioma after laparoscopic cystectomy: A comparative randomized trial between post-operative hormonal suppression treatment or dietary therapy vs. placebo

Objective(s) To assess the recurrence rate of endometrioma after laparoscopic cystectomy plus hormonal suppression treatment or plus dietary therapy compared to post-operative placebo. Study design A randomized comparative trial was conducted on 259 consecutive women who underwent laparoscopic unilateral/bilateral cystectomy for endometrioma. Seven days after surgery, the patients were randomly allocated on the basis of a computer-generated randomization sequence, to one of four post-operative management arms as follows: placebo ( n = 65) or gonadotrophin-releasing hormone analogue (tryptorelin or leuprorelin, 3.75 mg every 28 days) ( n = 65) or continuous low-dose monophasic oral contraceptives (ethynilestradiol, 0.03 mg plus gestoden, 0.75 mg) ( n = 64) or dietary therapy (vitamins, minerals salts, lactic ferments, fish oil) ( n = 65) for 6 months. At 18 months’ follow-up after surgery, all patients were monitored with a clinical gynecologic examination, and a transvaginal ultrasonography for possible evidence of endometrioma recurrence. Result(s) At 18 months’ transvaginal ultrasonographic follow-up after surgery, no significant recurrence rate of endometrioma was detected in women who received a postoperative course of hormonal suppression treatment or dietary therapy when compared with placebo (placebo vs. GnRH-a P = 0.316, placebo vs. estroprogestin P = 0.803, placebo vs. dietary therapy P = 0.544). Second-look laparoscopy was performed on a clinical basis and confirmed the ultrasonographic suspicion of recurrence of endometrioma in all cases: 10 (16.6%) in the post-operative placebo group vs. 6 (10.3%) in the post-operative GnRH-a group vs. 9 (15.0%) in the post-operative continuous estroprogestin group vs. 11 (17.8%) in the post-operative dietary therapy group. Of 36 patients with recurrent ovarian endometriosis, 8 had recurrence on the treated ovary, 20 on the contralateral ovary that appeared to be normal at the time of the first-line surgery, and 8 on both the treated and untreated ovaries. Endometrioma recurrences were associated with moderate-to-severe painful symptoms in 14/36 patients (38.8%), while the remaining 22 (61.1%) patients were asymptomatic. Conclusion(s) A 6-month course of hormonal suppression treatment or dietary therapy after laparoscopic cystectomy had no significant effect on the recurrence rate of ovarian endometriosis when compared with surgery plus placebo. So, treatment of endometrioma can be carried out exclusively by laparoscopic cystectomy without post-operative therapy, if a complete excision of ovarian endometriosis has been assured.

Gonadotropin regulation of RIP140 messenger ribonucleic acid expression in the rat ovary

Female mice null for receptor-interacting protein 140 (RIP140) are infertile because of the failure of follicle rupture. The present study examined gonadotropin regulation of RIP140 expression in immature rat ovary. Treatment with PMSG increased ovarian RIP140 mRNA and protein levels. In contrast, hCG treatment rapidly inhibited RIP140 mRNA and protein levels within 1–3 h. RIP140 mRNA was detected in theca cells of growing follicles in untreated ovary and in granulosa cells in PMSG-treated ovary. Interestingly, hCG treatment reduced RIP140 mRNA levels in granulosa cells of preovulatory follicles, but not of growing follicles. Neither treatment of immature rats with diethylstilbestrol in vivo nor of immature granulosa cells with FSH in vitro affected RIP140 mRNA levels. Treatment of immature granulosa cells with 17β-estradiol in vitro, however, stimulated RIP140 mRNA levels. In cultured preovulatory granulosa cells, RIP140 mRNA levels were stimulated at 1 h and then declined to below control levels by 3 h after LH treatment. Treatment with MDL-12,330A, an inhibitor of adenylate cyclase, or chelerythrine chloride, an inhibitor of protein kinase C (PKC), inhibited LH-stimulated RIP140 gene expression. Furthermore, forskolin or TPA treatment for 1 h mimicked the stimulatory action of LH, indicating the involvement of both adenylate cyclase and PKC pathways. These results demonstrate the stimulation by PMSG and inhibition by hCG of RIP140 expression in granulosa cells of preovulatory follicles in the rat ovary.

Expression localisation and functional activity of pituitary adenylate cyclase-activating polypeptide, vasoactive intestinal polypeptide and their receptors in mouse ovary

Pituitary adenylate cyclase-activating polypeptide (PACAP) and vasoactive intestinal polypeptide (VIP) positively affect several parameters correlated with the ovulatory process. PACAP is transiently expressed in rat preovulatory follicles, while VIP is present in nerve fibres at all stages of development. These two peptides act by interacting with three types of receptors: PACAP type I receptor (PAC1-R), which binds with higher affinity to PACAP, and two VIP receptors (VPAC1-R and VPAC2-R), which bind to PACAP and VIP with equal affinity. The aim of the present study was to characterise the PACAP/VIP/receptor system in the mouse ovary. Results obtained by RT-PCR, immunohistochemistry and in situ hybridisation showed that PACAP was transiently expressed in granulosa cells of preovulatory follicles after human chorionic gonadotrophin (hCG) stimulation, while VIP mRNA was never observed. All the receptors were present in 22-day-old untreated mice. In preovulatory follicles, PAC1-R was expressed both in granulosa cells and in residual ovarian tissue but was stimulated by hCG mainly in granulosa cells; VPAC2-R was present in both the cell compartments and was only mildly stimulated; VPAC1-R was present mainly in the residual ovarian tissue and was downregulated by hCG. PACAP and VIP were equipotent in inhibiting apoptosis in granulosa cells, confirming the presence of functional PACAP/VIP receptors. The contemporary induction by hCG of PACAP and PAC1-R in granulosa cells of preovulatory follicles suggests that, also in mouse ovary, PACAP may play a significant role around the time of ovulation. Moreover, the presence of PACAP/VIP receptors in the untreated ovary suggests a possible role for PACAP and VIP during follicle development.

Recurrence of endometriomas after laparoscopic removal: Sonographic and clinical follow-up and indication for second surgery

Study objective This study involved patients who, after laparoscopic surgery, had recurrence of endometriomas detected by sonography. The aim of this study was to evaluate the role of transvaginal sonography (TVS) in the management of recurrent endometriomas and to establish ultrasonographic criteria that would direct the therapy toward additional surgery versus medical or expectant management. Design Retrospective analysis of 62 reproductive-age women who showed recurrence of endometriomas on TVS after laparoscopic removal of an ovarian endometrioma by the stripping technique (Canadian Task Force classification II-1). Setting Obstetrics and Gynecology Department, University of Rome Tor Vergata. Patients Sixty-two patients with recurrent endometriomas after first-line treatment with laparoscopy. Interventions Ultrasonographic follow-up and/or second surgery. Measurements and main results Recurrence of an ovarian endometrioma was defined as the presence of ovarian cysts with the typical sonographic criteria of endometriomas and a diameter of more than 10 mm. The clinical and sonographic postoperative follow-up period lasted from 6 to 97 months (median 24.6) after the first procedure. Of 62 patients with recurrent endometriomas, 50 had recurrence on the treated ovary, 7 on the contralateral untreated ovary, and 5 on both the treated and untreated ovaries. Recurrence of endometriomas was associated with symptoms (pain or infertility) in 47 patients (76%), while the remaining 15 (24%) were asymptomatic. Of the 47 symptomatic patients with recurrence detected by TVS, a second procedure was performed in 15. Second surgery in these patients was indicated by the larger size of the recurrent cysts, a poor response to medical treatment, the presence on TVS of pelvic adhesions and nodules of deep endometriosis, and overall progression of the disease. Symptomatic patients who did not undergo a second procedure (32) had smaller recurrent endometriomas. However of the 31 symptomatic patients with large recurrent endometriomas (>3 cm), only 45% had repeat surgery. Conclusion Recurrent endometriomas, as detected by TVS, can remain asymptomatic and do not necessarily progress in size with or without medical treatment. The decision to reoperate depends less on the endometrioma’s size than on symptoms, in particular severe pain, and failure of medical treatment. However such patients are also more likely to have signs of deep nodules and adnexal/bowel adhesions and larger endometriomas on TVS scan, thus predisposing them to require a second procedure.

Anti-Müllerian hormone inhibits initiation of primordial follicle growth in the mouse ovary.

Recruitment of primordial follicles is essential for female fertility; however, the exact mechanisms regulating this process are largely unknown. Earlier studies using anti-Müllerian hormone (AMH)-deficient mice suggested that AMH is involved in the regulation of primordial follicle recruitment. We tested this hypothesis in a neonatal ovary culture system, in which ovaries from 2-d-old C57Bl/6J mice were cultured for 2 or 4 d in the absence or presence of AMH. Ovaries from 2-d-old mice contain multiple primordial follicles, some naked oocytes, and no follicles at later stages of development. We observed that in the cultured ovaries, either nontreated or AMH-treated, follicular development progressed to the same extent as in in vivo ovaries of comparable age, confirming the validity of our culture system. However, in the presence of AMH, cultured ovaries contained 40% fewer growing follicles compared with control ovaries. A similar reduction was found after 4 d of culture. Consistent with these findings, we noted lower inhibin alpha-subunit expression in AMH-treated ovaries compared with untreated ovaries. In contrast, expression of AMH ligand type II receptor and the expression of oocyte markers growth and differentiation factor 9 and zona pellucida protein 3 were not influenced by AMH. Based on the results, we suggest that AMH inhibits initiation of primordial follicle growth and therefore functions as an inhibitory growth factor in the ovary during these early stages of folliculogenesis.

A Role for Neurotransmitters in Early Follicular Development: Induction of Functional Follicle-Stimulating Hormone Receptors in Newly Formed Follicles of the Rat Ovary

The initiation of follicular growth in the mammalian ovary is a gonadotropin-independent phenomenon. Although some of the intraovarian signaling molecules that control the later phases of this process have been recently identified, the factors involved in the acquisition of gonadotropin receptors by early growing follicles have not been fully defined. In the rat, development of the ovarian innervation precedes the onset of folliculogenesis and occurs before follicles acquire responsiveness to gonadotropins. Because vasoactive intestinal polypeptide (VIP) and norepinephrine (NE), two of the neurotransmitters contained in ovarian nerves, are present in the ovary before the gland becomes responsive to gonadotropins, we sought to determine if VIP and/or NE are able to act on early follicles to facilitate the process of molecular differentiation that leads to gonadotropin dependency. In vitro exposure of 2-day-old rat ovaries to isoproterenol (ISO), a β-adrenoreceptor agonist, or VIP, a neurotransmitter contained in both sympathetic and sensory nerves, increased the steady state levels of the messenger RNAs encoding cytochrome P-450 aromatase (P-450arom) and FSH receptors (FSHR) within 8 h of treatment. A similar effect was observed following forskolin-induced activation of cAMP formation. In situ hybridization experiments revealed that both the P-450arom and FSHR hybridization signals were localized to follicles. The increase in FSHR messenger RNA was accompanied by formation of functional receptor molecules, as demonstrated by the ability of FSH to stimulate cAMP formation in ovaries preexposed to either ISO or VIP, but not in untreated ovaries. The stimulatory effect of ISO and VIP on the formation of FSHR coupled to the cAMP generating system was not reproduced by phenylephrine, an α-adrenergic agonist, or secretin, a member of the VIP family not recognized by ovarian VIP receptors. Treatment of VIP-primed ovaries with FSH resulted in follicular growth, demonstrating that exposure of the gland to the neurotransmitter led to the formation of a functional complement of FSH receptors. These results suggest that ovarian nerves, acting via neurotransmitters coupled to the cAMP generating system, contribute to the differentiation process by which newly formed primary follicles acquire FSH receptors and responsiveness to FSH. Follicles that begin to grow in more densely innervated ovarian regions, may have a selective advantage over those not exposed to neurotransmitter-activated, cAMP-dependent signals and, thus, may become more rapidly subjected to gonadotropin control.

A role for neurotransmitters in early follicular development: induction of functional follicle-stimulating hormone receptors in newly formed follicles of the rat ovary.

The initiation of follicular growth in the mammalian ovary is a gonadotropin-independent phenomenon. Although some of the intraovarian signaling molecules that control the later phases of this process have been recently identified, the factors involved in the acquisition of gonadotropin receptors by early growing follicles have not been fully defined. In the rat, development of the ovarian innervation precedes the onset of folliculogenesis and occurs before follicles acquire responsiveness to gonadotropins. Because vasoactive intestinal polypeptide (VIP) and norepinephrine (NE), two of the neurotransmitters contained in ovarian nerves, are present in the ovary before the gland becomes responsive to gonadotropins, we sought to determine if VIP and/or NE are able to act on early follicles to facilitate the process of molecular differentiation that leads to gonadotropin dependency. In vitro exposure of 2-day-old rat ovaries to isoproterenol (ISO), a beta-adrenoreceptor agonist, or VIP, a neurotransmitter contained in both sympathetic and sensory nerves, increased the steady state levels of the messenger RNAs encoding cytochrome P-450 aromatase (P-450arom) and FSH receptors (FSHR) within 8 h of treatment. A similar effect was observed following forskolin-induced activation of cAMP formation. In situ hybridization experiments revealed that both the P-450arom and FSHR hybridization signals were localized to follicles. The increase in FSHR messenger RNA was accompanied by formation of functional receptor molecules, as demonstrated by the ability of FSH to stimulate cAMP formation in ovaries preexposed to either ISO or VIP, but not in untreated ovaries. The stimulatory effect of ISO and VIP on the formation of FSHR coupled to the cAMP generating system was not reproduced by phenylephrine, an alpha-adrenergic agonist, or secretin, a member of the VIP family not recognized by ovarian VIP receptors. Treatment of VIP-primed ovaries with FSH resulted in follicular growth, demonstrating that exposure of the gland to the neurotransmitter led to the formation of a functional complement of FSH receptors. These results suggest that ovarian nerves, acting via neurotransmitters coupled to the cAMP generating system, contribute to the differentiation process by which newly formed primary follicles acquire FSH receptors and responsiveness to FSH. Follicles that begin to grow in more densely innervated ovarian regions, may have a selective advantage over those not exposed to neurotransmitter-activated, cAMP-dependent signals and, thus, may become more rapidly subjected to gonadotropin control.

The expression of c-myc during follicular growth and luteal formation in the rat ovary in vivo

The processes of folliculogenesis and formation of corpora lutea involve proliferation and differentiation of the follicular cells. The expression of several oncogenes is associated with the proliferative phase in many cell types. The present study examined the expression and hormonal regulation of the c-myc proto-oncogene during follicular development and the luteal phase of pseudopregnancy. Follicular development was initiated by pregnant mare's serum gonadotropin (PMSG) in immature rats followed two days later by the injection of human chorionic gonadotropin (hCG) to induce ovulation and luteal formation. Ovaries were collected at different time points and the content and distribution of c-myc mRNA/protein were examined. C-myc increased rapidly after the administration of both PMSG and hCG, but the effect of PMSG was less pronounced. The increase after PMSG was transient and localized primarily to the granulosa cells of developing follicles. The ovulatory dose of hCG resulted in a rapid and substantial increase of c-myc mRNA and protein with maximal levels at 1 h and 2-4 respectively. At this stage, the c-myc protein was localized to the follicular cells, the surface epithelium and, to some extent, to the interstitial tissue. There was a subsequent decrease prior to ovulation. The luteal phase was characterized by decreasing levels of c-myc with increasing luteal age. In order to examine the involvement of specific hormones in the regulation of c-myc, hypophysectomized, immature rats were injected sequentially with estradiol (E2) and follicle-stimulating hormone (FSH). Hypophysectomy resulted in a decrease of c-myc compared with intact animals. The administration of E2 resulted in an increase of c-myc mRNA and protein. The subsequent treatment with FSH did not result in a further increase and the levels remained at the same level as with E2 only. However, an ovulatory dose of hCG to E2 and FSH primed animals resulted in an additional increase of c-myc mRNA and protein. The levels after E2 and FSH were considerably lower compared with those of untreated ovaries of intact, immature animals, suggesting the involvement of other endocrine and paracrine factors. The presence of proliferating cell nuclear antigen in cell extracts indicated that the expression of c-myc was associated with phases of increased proliferation of follicular cells after hormonal stimulation. The results demonstrate that c-myc is regulated by hormones (E2, gonadotropins) in the rat ovary during follicular development to the preovulatory stage. The pronounced increase prior to ovulation also suggests a role for c-myc in the regulation of proliferative events involved in luteal formation.

Ovarian intrabursal administration of transforming growth factor beta 1 inhibits follicle rupture in gonadotropin-primed mice.

We hypothesized that over-expression and/or activation of latent transforming growth factor betas (TGF betas) by various ovarian cell types may lead to disturbances in ovulation and fertilization. To test this hypothesis, active TGF beta ranging from 1 to 500 ng was administered intrabursally into the ovaries of gonadotropin-primed mice, and the rates of ovum recovery and fertilization were determined. Furthermore, the presence and cellular distribution of endogenous TGF betas and TGF beta type I and type II receptors were determined immunohistochemically in the ovarian tissues of TGF beta 1-treated and untreated groups. The total number of ova recovered per ovary from ovaries treated as pairs or treated singly with TGF beta 1 at 1 to 10 ng/ovary was similar to that from controls, whereas the number recovered from ovaries treated as pairs or singly with 50 or 100 ng of TGF beta 1 per ovary was significantly lower than the number from respective controls (p < 0.05, 0.001). The number of ova recovered per ovary from ovaries treated as pairs or singly with TGF beta 1 at 200 or 500 ng/ovary was similar to the number of ova obtained from ovaries treated with TGF beta 1 at 100 ng/ovary. The rate of in vitro fertilization was low in ova recovered from ovaries treated with 50, 100, 200, and 500 ng/ovary of TGF beta 1, compared to that in ova from untreated ovaries. Histologically, the TGF beta 1-treated ovaries contained large numbers of unruptured follicles, whereas untreated ovaries contained large numbers of corpora lutea. Immunohistochemically, the endogenous TGF beta 1 and TGF beta 2 was localized in theca, granulosa, and luteal cells, without a substantial difference in intensity or distribution, in both TGF beta 1-treated ovaries and in controls. Theca cells were the primary site of immunoreactive TGF beta protein. TGF beta type I and type II receptors were also present in these cells, and their relative immunoreactive intensity was considerably reduced, particularly in granulosa cells in TGF beta 1-treated ovaries compared to controls. The results support our hypothesis and suggest that TGF betas play an important regulatory role in follicular development, oocyte maturation, and the ovulatory process.

Hormonal regulation of the ligand for c-kit in the rat ovary and its effects on spontaneous oocyte meiotic maturation.

Kit ligand (KL, c-kit ligand) mRNA was detected in the ovaries of 26-day-old prepubertal rats using in situ hybridization. In antral follicles there was a gradient in the intensity of the hybridization signal across the layers of granulosa cells, with greatest intensity observed in the cumulus granulosa cells enclosing the oocyte, and less signal occurring in the granulosa cells furthest from the oocyte. In age-matched rats 40 hr after injection of pregnant mare serum gonadotropin (PMSG), the pattern of distribution of KL resembled that in the untreated ovaries, although the intensity of the hybridization signal was greater in the PMSG-primed ovaries. This morphological observation was confirmed using Northern blot analysis, which indicated that granulosa cells of PMSG-treated rats had 3.5-fold greater abundance of KL mRNA compared to untreated rats. The abundance of KL mRNA further increased to 7-fold over control levels at 6 hr after PMSG-primed rats were treated with human chorionic gonadotropin (hCG). By contrast, treatment of rats with diethylstilbestrol to stimulate follicular growth did not cause any change in the abundance of KL transcripts. To investigate a potential role for KL in oocyte meiotic maturation, fully grown oocytes were cultured for 24 hr with or without KL (50 or 500 ng/ml). The presence of KL resulted in a significant, albeit transient, delay in the progression of spontaneous meiotic maturation, using the indices of germinal vesicle breakdown and polar body formation. The inhibitory effects of KL were specifically blocked by ACK2, an antibody to the extracellular domain of the c-kit receptor. These results indicate that KL is produced in rat granulosa cells at particularly high levels in the cells closest to the oocyte and that this production may be regulated directly by gonadotropic hormones. Furthermore, KL inhibits the progression of meiosis in cultured oocytes, which suggests a possible role in the maintenance of meiotic arrest that occurs throughout oocyte growth.