Primary Follicles Research Articles

Expression of voltage-gated Ca2+ channels, Insp3Rs, and RyRs in the immature mouse ovary

BackgroundThe postnatal mammalian ovary undergoes a series of changes to ensure the maturation of sufficient follicles to support ovulation and fecundation over the reproductive life. It is well known that intracellular [Ca2+]i signals are necessary for ovulation, fertilization, and egg activation. However, we lack detailed knowledge of the molecular identity, cellular distribution, and functional role of Ca2+ channels expressed during folliculogenesis. In the neonatal period, ovarian maturation is controlled by protein growth factors released from the oocyte and granulosa cells. Conversely, during the early infantile period, maturation becomes gonadotropin-dependent and is controlled by granulosa and theca cells. The significance of intracellular Ca2+ signaling in folliculogenesis is supported by the observation that mice lacking the expression of Ca2+/calmodulin-dependent kinase IV in granulosa cells suffer abnormal follicular development and impaired fertility.ResultsUsing immunofluorescence in frozen ovarian sections and confocal microscopy, we assessed the expression of high-voltage activated Ca2+ channel alpha subunits and InsP3 and ryanodine receptors in the postnatal period from 3 to 16 days. During the neonatal stage, oocytes from primordial and primary follicles show high expression of various Ca2+-selective channels, with granulosa and stroma cells expressing significantly less. These channels are likely involved in supporting Ca2+-dependent secretion of peptide growth factors. In contrast, during the early and late infantile periods, Ca2+ channel expression in the oocyte diminishes, increasing significantly in the granulosa and particularly in immature theca cells surrounding secondary follicles.ConclusionsThe developmental switch of Ca2+ channel expression from the oocytes to the perifollicular cells likely reflects the vanishing role of the oocytes once granulosa and theca cells take control of folliculogenesis in response to gonadotropins acting on their receptors.

726 The Wnt-inhibitor Dkk4 is required for primary hair follicle induction and patterning

Androgenetic alopecia is a common form of pattern hair loss, characterized by miniaturized hair follicles (HFs) at the front and parietal scalp, while hairs on the occipital scalp are preserved. Moreover, different sites of the human body exhibit distinct types and patterns of HFs. Understanding the molecular basis of this heterogeneity will help to design targeted treatment strategies. The Wnt signalling pathway and its Dickkopf (Dkk) inhibitors have been suggested to regulate HF induction and patterning.

Update in Diagnosis and Management of Primary Cutaneous B-Cell Lymphomas.

Primary cutaneous lymphomas are a rare group of diseases, with an estimated incidence of 0.5-1 case per 100,000 people per year. Primary cutaneous B-cell lymphomas (pCBCLs) represent 25-30% of all primary cutaneous lymphomas. There are three main subtypes of pCBCL: primary cutaneous marginal zone lymphoma, primary cutaneous follicle center lymphoma, and primary cutaneous diffuse large B-cell lymphoma, leg type. Cutaneous B-cell lymphomas have a broad spectrum of clinical presentations, which makes diagnostic and therapeutic strategies challenging. To date, treatment recommendations for cutaneous B-cell lymphomas have been largely based on small retrospective studies and institutional experience. Recently, the pharmacotherapeutic landscape has expanded to include drugs that may modify the underlying disease pathology of pCBCLs, representing new therapeutic modalities for this rare group of diseases. Novel therapies used for other systemic B-cell lymphomas show promise for the treatment of pCBCLs and are being increasingly considered. These new therapies are divided into five main groups: monoclonal antibodies, immune checkpoint inhibitors, small-molecule inhibitors, bispecific T-cell engaging, and chimeric antigen receptor T cell. In this review, we discuss the clinical, histopathological, molecular, and cytogenetic features of the most common pCBCL subtypes with a focus on current and innovative therapeutic developments in their management. These emerging treatment strategies for B-cell lymphomas and cutaneous B-cell lymphomas may represent novel first-line options for the management of these rare diseases.

A prospective, observational study of chemotherapy-induced ovarian damage on follicular reserve and maturation.

Chemotherapy negatively affects gonadal function, often resulting in premature ovarian failure (POF) due to ovarian reserve depletion. Mechanisms of gonadotoxicity, such as primordial follicle overactivation and "burnout", remain to be established. Ovarian tissue cryopreservation (OTC) before treatment plays an important role in safeguarding fertility. This is a prospective observational study that aims to evaluate the feasibility of OTC after chemotherapeutic treatment initiation. Patients were divided into 2 groups depending on whether they received chemotherapy before the harvesting procedure (Group 1) or not (Group 2). The main outcomes of this study are serum anti-Mullerian hormone (AMH) levels and histological follicular counts on ovarian tissue biopsies. Between 2012 and 2020, 79 patients underwent OTC at our Hospital. Follicular counts from the ovarian biopsies of 30 post-pubertal patients and respective serum AMH levels were included in the analysis. AMH levels did not significantly differ between the 2 groups (P = 0.70) as well as the number of primordial follicles (P = 0.73). Ovarian biopsies of patients from Group 1 showed a higher number of primary follicles (P = 0.04) and atretic follicles (P = 0.05) with respect to Group 2. In conclusion, OTC appears to be feasible even after the start of chemotherapeutic treatment, since in treated patients, the main ovarian reserve indicators (number of primordial follicles and serum AMH levels) were not significantly reduced compared to untreated patients. The "burnout" theory of chemotherapeutic damage to the ovary seems to be supported by the higher number of primary follicles found in the ovaries of patients who received chemotherapy before OTC.

The expression of IGFBP-5 in the reproductive axis and effect on the onset of puberty in female rats

Insulin-like growth factor-binding protein-5 (IGFBP-5) has recently been shown to alter the reproductive capacity by regulating insulin-like growth factor (IGF) bioavailability or IGF-independent effects. The present study aimed to investigate the effect and mechanism of IGFBP-5 on the onset of puberty in female rats. Immunofluorescence and real-time quantitative PCR were used to determine the expression and location of IGFBP-5 mRNA and protein distribution in the infant's hypothalamus-pituitary-ovary (HPO) axis prepuberty, peripuberty, puberty and adult female rats. Prepubertal rats with IGFBP-5 intracerebroventricular (ICV) were injected to determine the puberty-related genes expression and the concentrations of reproductive hormones. Primary hypothalamic cells were treated with IGFBP-5 to determine the expression of puberty-related genes and the Akt and mTOR proteins. Results showed that Igfbp-5 mRNA and protein were present on the HPO axis. The addition of IGFBP-5 to primary hypothalamic cells inhibited the expression of Gnrh and Igf-1 mRNAs (P < 0.05) and increased the expression of AKT and mTOR protein (P < 0.01). IGFBP-5 ICV-injection delayed the onset of puberty, reduced Gnrh, Igf-1, and Fshβ mRNAs, and decreased the concentrations of E2, P4, FSH,serum LH levels and the ovaries weight (P < 0.05). More corpus luteum and fewer primary follicles were found after IGFBP-5 injection (P < 0.05).

Investigation of the Apoptotic Effect of Levofloxacin on Ovarian Follicles of NMRI Mice and its Anticancer Effect on Human Ovarian Cancer Cell Line

Background: Levofloxacin is a fluoroquinolone antibiotic with extensive anti-bacterial effects. Levofloxacin is widely used for treatment of urinary and vaginal infections. Objectives: This research surveyed the cytotoxicity effects of levofloxacin on ovarian follicles of mice in both in vitro and in vivo conditions, as well as its anticancer effect on human ovarian cancer cell line (SKOV3). Methods: For in vitro study, the ovaries of animals were isolated and treated with levofloxacin at doses of 1, 2, 5, and 10 µg/ml for 6 days. For in vivo study, animals were treated with levofloxacin at concentrations of 100, 200, 400, and 800 mg/kg for 24 days. Histopathological and morphological examinations of ovarian tissues were performed. Real-time PCR and Western Blot techniques were performed to analyze apoptosis-related genes and proteins of ovarian tissues. Results: Levofloxacin at higher concentrations caused morphological changes and remarkably decreased the number of primary, secondary, and adult follicles compared to the control group. The percentage of viable SKOV3 cells was 10.12%, 7.63%, and 2.17% following exposure to levofloxacin (at a concentration of 800 μg/ml) for 24, 48, and 72 h, respectively. The half-maximal inhibitory concentration of levofloxacin against SKOV3 was found to be 181.1, 74.84, and 27.58 μg/ml at 24, 48, and 72 h, respectively. The percentage of SKOV3 cells apoptosis following exposure to levofloxacin after 72 h at 20, 80, and 200 μg/ml doses was 11%, 42%, and 52%, respectively. Real-time PCR revealed up-regulation of Bax and Caspase-3 genes after exposure to levofloxacin in SKOV3 cells, whereas the expression of Bcl2 was significantly decreased in a concentration-depended manner. Conclusion: The present study demonstrates that levofloxacin induces apoptosis of both ovarian follicles and human ovarian cancer cell lines.

An investigation of the effects of chronic zonisamide, sultiam, lacosamide, clobazam, and rufinamide anti-seizure medications on foliculogenesis in ovarian tissue in prepubertal non-epileptic rats.

We aimed to determine the morphological and histological effects of zonisamide, sultiam, lacosamide, clobazam, and rufinamide on ovarian folliculogenesis in rats. Sixty female Wistar rats were divided into six experimental groups as control, zonisamide, sultiam, lacosamide, clobazam, and rufinamide groups; control solution and drugs were administered by gavage for 90 days. The number of healthy follicles in the control group was significantly higher than in the anti-medication groups (p< 0.001), and the number of corpus luteum was significantly lower (p< 0.001). There was a significant difference in the number of TUNEL positive apoptotic follicles between the control and drug groups (p< 0.001). With EGF, IGF-1, and GDF-9 staining, a very strong immunoreaction was observed in the ovarian multilaminar primary follicle granulosa cells and oocytes in the control group compared to the drug group (p< 0.001). Long-term anti-seizure medication with zonisamide, sultiam, lacosamide, clobazam, and rufinamide from prepubertal to adulthood causes apoptosis and disruption of folliculogenesis in the ovarian follicles of nonepileptic rats.

Multiple skin-colored facial papules in a child

Multiple skin-colored facial papules in a child

Leukemia inhibitory factor's effect on the growth and survival of sheep's follicles of ovarian tissue during vitrification.

One of the experimental programs for fertility protection in women includes protective cryopreservation. Vitroficasion of ovarian tissue is one of the protective cryopreservation methods that use high concentrations of antifreeze and faster cooling. To reduce its complications, LIF (Leukemia inhibitory factor) was used as a pretreatment in this study. In this study, the ovaries were randomly divided into 8 groups. In NCN (without pretreatment and LIF in culture media), NCP (without pretreatment and with LIF in culture media), PCP (with pretreatment and LIF in culture media), and PCN (with pretreatment and without LIF in culture media) groups, vitrification and reversal were not performed. In the groups NVN (without pretreatment and LIF in culture media), NVP (without pretreatment and with LIF in culture media) PV, PVP (with pretreatment and LIF in culture media), and PVN (with pretreatment and without LIF in culture medium) groups, vitrification and tissue reversal were performed. All groups were cultured and histological, cellular, and molecular evaluations were performed. The results of the present study showed that LIF in the culture medium reduced the number of abnormal, primordial, primary, and secondary follicles, and DNA breakage compared to the group without LIF (P < 0.05) and increases the growth of follicles and expression of GDF9, BMP, AMH, KITLG genes (P < 0.05). The use of LIF pretreatment before vitrification and melting of sheep ovary tissue in its culture medium reduces the damage caused by it and increases the growth and development of ovarian follicles while maintaining their function.

Induction of Ovulation using Low-Level Laser Biostimulation

Abstract&#x0D; Aim: Low level laser therapy (LLLT) has been used in biostimulation of rat ovaries using a fibreoptic laser introduced directly through the vagina to deliver laser doses, which is a new modality of laser induction of ovulation, compared with conventional medical induction of ovulation using clomiphene citrate and with non-interventional controls. &#x0D; Study design: A Prospective experimental study carried out from January 2014 to February 2016.&#x0D; Setting: University-based photobiology laboratory.&#x0D; Materials and methods: Seventy-two (72) Female-Wistar Albino rats were used in this study, divided into three groups: 17 rats used as a control group, 19 rats received clomiphene citrate (Clomid group) and 36 rats exposed to diode laser 660 nm wavelength (laser group). Histopathological assessment of rat ovaries was done in the three study Groups.&#x0D; Results: Higher number of follicles was found in laser group compared to controls, the difference was significant in the Primordial follicles. Non-significant increase in the number of Primordial follicles in laser group was found compared to the clomid group. Clomid group showed a non-significant increase in primary, secondary and Antral follicles.&#x0D; Conclusion: This study shows that ovarian laser biostimulation is a new encouraging method for induction of ovulation.

Insulin improves ovarian function during the ovarian cycle in adult mice

Folliculogenesis is controlled by numerous inside and outside ovarian factors such as endocrine, paracrine, and autocrine signals. Among these factors, insulin is an important molecule that regulates processes in the female reproductive system. So, the purpose of this study was to determine the impact of insulin treatment on ovary tissue during the ovarian cycle. For this probe, 18 adult female NMRI mice were randomly divided into two groups: control and insulin (100 μL with a 72-hour interval by intraperitoneal injection for 30 days). Blood samples for hormonal evaluation were obtained from the heart, and the serum levels of FSH, LH, progesterone, and estradiol were measured. Then, right and left ovaries were extracted for real-time PCR and stereology, respectively. According to our results, insulin administration increased mRNA levels of INS-R, Ki67, while reduced Caspase-3, IGF-1, and FOXO-1. Insulin administration augmented the concentrations of female reproductive hormones. Histological analyses showed that the total volume of ovary in mice receiving insulin was significantly reduced. Likewise, a significant decrease in the number of antral follicles, Graafian follicles, and the number of corpora lutea were seen following the use of insulin. In contrast, the results showed a significant growth in the number of primordial and primary follicles in the insulin group compared with the control group. Briefly, insulin administration is able to increase the expression of genes related to insulin receptor and cell proliferation, as well as the amount of female reproductive hormones, but simultaneously, has a dual effect on the histological parameters of the ovary.

P-467 Optimizing in vitro growth of immature follicles from cryopreserved vs fresh human cortical ovarian tissue

Abstract Study question Can cryopreserved-thawed human ovarian cortex be cultured in vitro to produce growing follicles as efficiently as from fresh samples? Summary answer In contrast to fresh samples, cryopreserved-thawed ovarian cortex did not develop morphologically normal growing follicles, even when adding follicular growth stimulators. What is known already A multistep culture system has been shown to produce mature oocytes from immature follicles present in the human cortex, without the addition of follicular growth stimulators. However, these promising results were achieved using fresh human ovarian cortex tissue donated from cisgender women, which is not available for the purpose of fertility preservation. It remains unclear whether this multistep culture system can be used to mature follicles from cryopreserved-thawed human ovarian cortex. In addition, the applicability of the culture system to mature follicles in vitro has not been investigated using ovarian tissue from transmasculine people. Study design, size, duration Fresh and cryopreserved ovarian cortex tissue isolated from the same transmasculine people were cultured during 8 days (first step of the culture system) with or without the addition of a PI3K/Akt promotor, Sphingosine-1-Phosphate (S1P). The ovarian cortex fragments were collected a day 0 and day 8 for downstream morphological and quantitative analysis of the follicular population. This study also included data of in vitro culture of cryopreserved samples from oncological cisgender women. Participants/materials, setting, methods Ovarian cortex tissue was collected from six testosterone user transmasculine people (25,8 ± 4,9 years) and three oncological cisgender women (24,0 ± 6,0 years). Fresh cortex was either directly cultured or previously cryopreserved. All ovarian fragments were cultured in the same conditions for 8 days. After the culture period, follicular population was quantified on histological sections (haematoxylin-eosin) and compared to samples of day 0. Follicular cell types, cell proliferation and apoptosis were assessed by immunofluorescence. Main results and the role of chance Fresh ovarian cortex tissue isolated from transmasculine people showed morphologically normal primary follicles (PFs) and secondary follicles (SFs) after 8 days of culture and no antral follicles were observed. Moreover, the addition of the follicular growth stimulator, S1P, to the culture medium significantly increased the number of growing follicles (PFs, P &amp;lt; 0.05; SFs, P &amp;lt; 0.01). The addition of S1P stimulated the emergence of SFs that showed expression of anti-mullerian hormone (AMH), contained proliferative (PCNA+) granulosa cells and showed low levels of apoptotic cells (TUNEL assay). Structurally, these in vitro grown SFs showed an intact basement membrane assessed by the expression of collagen IV. By contrast, using the same culture protocol but starting from cryopreserved-thawed cortex tissue from the same patients resulted in a very low number of viable growing follicles (PFs and SFs) even in the presence of S1P. Lastly, when using the same culture protocol on cryopreserved-thawed samples from oncological cisgender women, we observed a low efficiency of follicular growth, comparable to that observed in cryopreserved ovarian tissue from (age-matched) transmasculine people. Limitations, reasons for caution Limitations of this study are: small sample size and lack of fresh cortex tissue from oncological cisgender women. Due to the high variability of follicular density among patients, the use of a larger number of samples is recommended. Wider implications of the findings Optimization of culture systems to grow and mature follicles in the ovarian cortex of cryopreserved samples (both from cis and trans persons) is urgently needed. Current culture protocols can be applied to fresh samples, but further characterization is needed to consider this as an alternative in fertility preservation treatment. Trial registration number Not applicable

O-265 Luteinizing hormone is able to protect reproductive health in cancer patients

Abstract Study question Is luteinizing hormone (LH) able to protect ovarian follicles against cyclophosphamide (CPM)-induced damage in women? Summary answer LH significantly reduces primordial follicle (PMF) loss in ovarian cortical strips cultured in vitro with phosphoramide mustard (PM), the active metabolite of CPM. What is known already Cancer therapies are cause of premature ovarian insufficiency (POI) in female patients, since they induce a severe reduction of PMFs. For this reason, several compounds have been analyzed as adjuvant therapies to protect the ovarian reserve without interfering with cancer treatment on tumor cells. We recently demonstrated the protective effect of LH against ovotoxicity induced by PM and cisplatin on the ovary of prepuberal and adult mice. These results suggest the possibility to use LH prior or in concomitance with anticancer drugs in order to preserve oocytes in human patients, therefore preventing the early onset of menopause and/or infertility. Study design, size, duration Ovarian cortical tissues were collected from nine patients (age ± SD: 15.33 ± 4.50) who have cryopreserved their tissue before receiving anticancer treatment. For each patient, ovarian cortical strips were thawed and randomly assigned to the experimental conditions: Control (CTRL), PM and PM+LH. LH was added 1 hour before the treatment with PM. Samples were analyzed after 8, 16, 24 and 48 hrs of treatment. Participants/materials, setting, methods Ovarian cortial strips were placed onto culture plate insert and cultured for a maximum of 48 hours, in αMEM with 10% Serum Substitute Supplement with/out 200mIU/ml LH and/or 10μM PM. The samples were processed for: - Histology, for PMFs and primary follicles (PFs) analysis; - Immunohistochemistry, for the expression of protein involved in DNA damage, apoptosis and follicle activation; - Real-Time PCR, for the expression of apoptotic and inflammation related genes. Main results and the role of chance The follicle density in the untreated group varied from 233.03 to 3420.27 PMFs/mm3 between the nine patients analysed. Relative follicular density (%) was performed to analyse statistical differences between the groups. Relative PMFs density (%) was significantly reduced in PM vs CTRL either after 24 and 48 hrs, while this reduction was significantly counteracted by LH (24 hrs: CTRL=76.19±2.12; PM = 44.95±5.06; PM+LH=73.97±11.64. 48 hrs: CTRL=66.47±3.96; PM = 36.76±3.96; PM+LH=55.74±8.72). To investigate the mechanism underlying the observed effects, the expression of markers involved in DNA damage (gH2AX), apoptosis (Cleaved caspase 3, NOXA, PUMA), follicle activation (p-AKT, FOXO3a), cell cycle arrest (p21, Ki67), and inflammation (IL1β, TNFα) were analysed. The results showed that LH did not prevent DNA damage induced by PM, since gH2AX positive oocytes were seen both in PM and PH+LH group at 16h; at 24hrs however we observed a downregulation of the gH2AX expression in the PH+LH group (PM@ 100% vs PM+LH@35%). LH also counteracts the activation of chemotherapy-induced apoptotic processes, by reducing the levels of pro-apoptotic factors such as NOXA, PUMA and CC3, and follicles activation lowering AKT-FOXO3a signaling axis. Moreover, PM treatment creates a proinflammatory microenvironment, as shown by increased IL1β and TNFα gene expression, partially counteracted by LH pretreatment. Limitations, reasons for caution Experiments are carried out in vitro; the functionality of the cortex in xenografts should be evaluated. Careful evaluation of which patients might use this protector is needed. Wider implications of the findings These findings demonstrate that LH is able to reduce PMFs loss in human ovarian cortex exposed to PM. These results encourage thinking about the use of the hormone as a ferto-protector agent in clinical trials to prevent the premature onset of menopause and/or infertility in women undergoing anticancer treatments. Trial registration number not applicable

P-614 Predicting if or when ovarian activation occurs after Drug-Free in vitro Activation (IVA) in primary ovarian insufficiency (POI) patients

Abstract Study question Can we predict if or when ovarian activation resumption occurs after Drug-Free in vitro Activation (IVA) in primary ovarian insufficiency (POI) patients? Summary answer Duration of amenorrhea alone could predict if ovarian activity resumption occurs. Regarding to when: as early as 21 days until 330 days after surgery. What is known already Primary ovarian insufficiency occurs before age 40, leading patients to infertility. Although there are a few remaining follicles, their only chance to achieve pregnancy is through egg donation. Recently, studies have been focusing on the determinants and intracellular signaling pathways involved in the activation of primordial follicles, as the maintenance of a correct ovarian reserve. A new surgical technique known as Drug-Free IVA would activate ovarian function by mechanical fragmentation. For selected patients whose ovaries still contain residual secondary follicles, the fragmentation step alone, which implies a Hippo signaling pathway disruption, would be sufficient to promote follicle growth. Study design, size, duration Our study is a prospective observational cohort of patients recruited in a tertiary-care university hospital approved by the Ethics Research Committee from Hospital Clinic of Barcelona (HCB/2017/0856). The diagnostic criteria of POI followed the European Society of Human Reproduction and Embryology (ESHRE) Guideline from 2016. All patients provided written informed consent.Thirty-two women included in our study underwent Drug-Free IVA by laparoscopy between January 2018 and December 2019. After surgery, a year-long follow-up was carried out. Participants/materials, setting, methods All patients were younger than 40 years of age; presented oligomenorrhoea or amenorrhea for at least 4 months; and FSH level greater than 25 IU/mL . We also restrict inclusion criteria to low estradiol serum levels (&amp;lt;20 pg/ml) absence of antral follicles in transvaginal ultrasound; and anti-Müllerian hormone (AMH) levels less than 0.10 ng/mL. Regardless of the time from POI diagnosis, all patients had duration of amenorrhea greater than 1 year. Main results and the role of chance As for prediction of ovarian activation, duration of amenorrhea was the only factor that could significantly differentiate patients' response to Drug-Free IVA. Since a shorter time of amenorrhea (less than 2 years) was related with ovarian activation during a year follow-up. No differences were found in age at Drug-free IVA, age of menarche, baseline FSH or AMH levels; nor presence of follicles in ovarian tissue biopsy. Regarding to time to response, in 12 patients ovarian activation occurred in the first 6 months after surgery with a total of 5 pregnancies achieved (22.7%)- resulting in 4 heathy live births and, unfortunately, one miscarriage. The other 10 patients showed ovarian activation resumption once the 180 days passed, resulting in 3 pregnancies (pregnancy rate of 30%): 2 healthy live births and one neonatal death due to extreme prematurity. An important concept here is that activation before 6 months after surgery could mean an effect on primary and/or secondary follicles, while beyond 6 months the effect might occurred on primordial follicles. Sugesting that maybe the disruption of the Hippo pathway would not only be able to act in the activation of the secondary follicles but also act in the earlier phases of the folliculogenesis. Limitations, reasons for caution The lack of a control group is the main limitation of our study because it would be interesting to seek for the placebo effect or intensive medical attention/care consequences in those patients, whose reported pregnancy rate is as low as 4% in observational studies. Wider implications of the findings Our findings define the clinical profile of patients with POI in which Drug-Free IVA would be more effective and report late ovarian follicular resumption. Meaning that disruption of the Hippo signaling pathway by ovarian fragmentation could be capable of acting in the initial phases of folliculogenesis prior to secondary follicles. Trial registration number not applicable

P-439 Human Platelet Lysate improve the growth and survival of isolated human pre-antral follicles in vitro

Abstract Study question Can human Platelet Lysate (hPL) and umbilical cord plasma (UCP) be used as alternative serum sources in the culture of isolated human pre-antral follicles? Summary answer hPL significantly increased the growth and survival of human follicles cultured for 8 days compared to fetal bovine serum (FBS) and human serum albumin (HSA). What is known already Culture of human ovarian follicles is a potential new source of mature oocytes for fertility preservation and an important model system to study basic biology. However, only a few studies have produced mature human oocytes after culturing pre-antral stage follicles. Moreover, optimising culture systems is challenging due to the scarcity of human material. Platelet-rich plasma solutions have been used extensively in regenerative medicine due to their high platelet content, which upon activation, release a multitude of growth factors, hormones, and cytokines. Clinically cell-based therapies have used platelet-rich solutions, such as hPL or UCP, successfully as an animal-free serum. Study design, size, duration Human pre-antral follicles (n = 378; mean diameter: 78 µm; range: 46-237 µm) were isolated from ovarian medulla tissue. The follicles were encapsulated in 0.5% alginate and cultured for 8 days in media supplemented with one of four sources of serum; 5% FBS (n = 74), 2.5% HSA (n = 74), 5% hPL (n = 140), and 5% UCP (n = 90). The primary endpoints were follicular growth and survival. Secondary endpoints included follicular gene expression analysis and media hormone concentrations. Participants/materials, setting, methods Ovarian surplus tissue was donated by 7 women (aged 19-32 years) undergoing unilateral oophorectomy and ovarian tissue cryopreservation for fertility preservation. Pre-antral follicles were isolated enzymatically and growth and survival were assessed every second day during culture by microscopy. AMH and Estradiol concentrations were measured by ELISA in media collected at day 4 and 8. At day 8, surviving follicles were snap-frozen and the expression of AMH, FSHR, GDF9, and BMP15 was analysed by qPCR. Main results and the role of chance After 8 days in culture, the follicle survival rate in the hPL group (86%; n = 120/140) was significantly higher compared to the FBS group (60%; n = 45/74; p &amp;lt; 0.0003), but comparable to the HSA group (76%; n = 56/74; p = 0.292). On the contrary, the follicle survival rate in the UCP group (33%; n = 30/90) was significantly lower compared to any of the other groups (p &amp;lt; 0.0001 for all three groups). The average diameter of surviving follicles was statistically significantly larger in the hPL and UCP group compared to the FBS and HSA groups (hPL: 149 ±4.6 µm; UCP: 138 ±7.6 μm; FBS: 110 ±3.8 μm; HSA: 115 ±4.2 μm; p &amp;lt; 0.001 for hPL compared to FBS and HSA; p = 0.0129 for UCP compared to HSA). The relative growth of the follicles compared to their initial diameter was 45% and 33% higher in the hPL group compared to the FBS and HSA groups, respectively, which was statically significant in both groups (p &amp;lt; 0.001). In the UCP group, follicle growth was also significantly higher by 25% compared to the FBS group (p &amp;lt; 0.001) and by 14% compared to the HSA group (p = 0.0376). Hormone measurements from the culture media and follicle gene expression analysis are awaited. Limitations, reasons for caution One limitation is the variability in the initial follicle sizes. Extrapolating results according to primordial, primary, and secondary follicles would require more follicles but could provide valuable insight. Moreover, long term culture studies are needed to evaluate the effects of hPL and UCP on antral follicle development and oocyte maturation. Wider implications of the findings Our findings show that hPL can be used as an alternative serum source in the culture of human pre-antral follicles as it increased follicle growth and survival compared to FBS and HSA. UCP also increased follicle growth but had detrimental effects on follicle survival which needs further studies. Trial registration number Not applicable

P-458 Testosterone treatment induces changes in stromal collagen and elastin content of the ovaries of transgender men

Abstract Study question Does gender-affirming testosterone therapy alter the composition of the extra-cellular matrix (ECM) within the ovarian stroma and subsequently affect follicle activation in vivo Summary answer Ovarian stroma of trans men is more collagenous and less elastic, indicating fibrotic change. This may affect in vivo follicle growth activation What is known already Changes in the ovarian stroma have been demonstrated in the ovaries of transgender men taking testosterone, including thickening of the tunica albuginea, stromal cell hyperplasia and stromal cell luteinisation. Ovaries of trans men also have increased cortical stiffness. These changes are similar to those seen in female patients with PCOS and in physiological ovarian aging, which has been attributed to accumulation of collagen in the ECM. Increasing stiffness of the supportive follicular microenvironment has been shown to reduce follicle growth activation in vitro Study design, size, duration Whole ovaries were obtained from transgender men (mean age 27.6 ± 1.7 years, n = 8) with informed consent at oophorectomy. All patients had received 1000mg testosterone undecanoate intramuscularly at 12-16 week intervals for a minimum of 18 months pre-operatively (range 18 months-10 years). Cortical tissue was dissected into small fragments (≈1x1x0.5mm) and fixed for histological and immunohistochemical analysis. Testosterone-treated ovaries were compared to cortical biopsies from age-matched healthy women obtained at caesarean section (mean age 31.8±1.5, n = 8). Participants/materials, setting, methods Follicle number, classification of developmental stage, non-growing follicle density (NGFD) and stromal cell density were evaluated by histological analysis of ovarian cortical tissue. Sections were stained with Picrosirius red (PSR) to analyse total collagen content using brightfield microscopy. Polarised light was also used to analyse the collagen birefringence, which allows quantification of collagen fibre thickness into thick, medium or thin. Total elastin content was evaluated using immunofluorescence. Main results and the role of chance 4526 follicles were analysed. Transgender ovary showed a higher proportion of non-growing follicles found compared to control (93.9±1.2% vs 84.6±1.5% p &amp;lt; 0.05): the proportions of primary (4.7±0.9% vs 10.6±1.5%, p = 0.2) and secondary (1.4±0.4% vs 4.6±0.7%, p = 0.1) follicles tended to be lower. Stromal cell density was significantly higher in transgender ovarian cortex than control (2.5±0.1 x106cells/mm3 vs 1.7±0.1 x106cells/mm3), indicating stromal cell hyperplasia. Combined data from control and transgender groups showed a positive correlation between NGFD and stromal density (r = 0.64, p = 0.01). Transgender ovary had a higher total collagen content (77.2±1.2%) compared to control (31.3±3.3%, p &amp;lt; 0.005). Analysis of collagen birefringence showed that transgender ovaries had similar quantities of thick collagen fibres (0.014±0.005 vs 0.010±0.009, p = 0.1), more medium thickness collagen fibres (45.1±6.6%vs 14.4±4.9%, p &amp;lt; 0.05) and fewer thinner fibres (41.5±9.6% vs 27.7±2.8%, p = 0.08) than control. The total elastin content in transgender ovaries was lower than control (1.3±0.1% vs 3.6±0.6%, p &amp;lt; 0.005) and subsequently, the collagen/elastin ratio was significantly higher (63.1±7.9 vs 10±1.3, p &amp;lt; 0.005). Limitations, reasons for caution The impact of these findings on in vivo follicle growth are unclear. The effect of duration of testosterone treatment has not investigated. Wider implications of the findings More collagenous, less elastic ovarian stroma in trans men indicates fibrotic change; these findings are similar to women with PCOS and with reproductive ageing. These stromal changes may alter follicle growth activation and may contribute value to our understanding of the regulation of follicle function in a range of conditions. Trial registration number nil

O-127 Higher probability of live birth after testosterone pretreatment in women with poor ovarian response undergoing IVF: a systematic review and meta-analysis

Abstract Study question Does testosterone pretreatment increase the probability of pregnancy in poor responders undergoing ovarian stimulation with gonadotrophin-releasing hormone (GnRH) analogues and gonadotrophins for in-vitro fertilization (IVF)? Summary answer Testosterone pretreatment increases clinical pregnancy and live birth rates in poor responders undergoing ovarian stimulation for IVF. What is known already Androgens have been shown to stimulate early stages of follicular growth, increase the number of primary, pre-antral and antral follicles as well as increase ovarian sensitivity to follicle stimulating hormone (FSH). Although androgen supplementation has been evaluated in several randomized controlled trials (RCTs) and meta-analyzed in six systematic reviews until today currently no solid conclusions can be drawn regarding its effectiveness. Study design, size, duration A literature search was performed until September 2021 aiming to identify RCTs evaluating testosterone pretreatment in poor responders. Outcome measures included achievement of pregnancy, total dose of gonadotrophins required, duration of stimulation, estradiol levels, endometrial thickness and number of follicles ≥17 mm on the day of triggering final oocyte maturation, number of cumulus–oocyte complexes (COCs) retrieved, embryos transferred, metaphase II (MII) and 2-pronuclei oocytes (2pn) and the proportion of patients having an embryo transfer (ET). Participants/materials, setting, methods Eight RCTs published between 2006 and 2021 were analyzed, including 760 women. Pretreatment with transdermal testosterone gel was performed in all studies with a dose ranging from 10 to 12.5 mg/day for 10 to 56 days. In dichotomous data, estimates were expressed as risk ratio (RR) with 95% confidence intervals (CIs), using the fixed or random effects method. In continuous data, differences were pooled across resulting in a weighted mean difference (WMD) with 95% CI. Main results and the role of chance Testosterone pretreatment was associated with a significantly higher live birth (RR: 2.07, 95%CI: 1.09 to 3.92) and clinical pregnancy rate (RR: 2.25, 95%CI: 1.54) in women with POR undergoing IVF, while there was also a significant increase in the number of COCs retrieved (WMD: +0.88, 95% CI: +0.22 to + 1.54). Significantly less days to complete ovarian stimulation (WMD: -0.81 days, 95% CI: -1.46 to −0.16), a lower total dose of gonadotrophins (WMD: -368.8 IUs, 95% CI: −612.4 to -125.2), a thicker endometrium on the day of triggering final oocyte maturation (WMD: +0.83 mm, 95% CI: +0.13 to + 1.53) and a lower cancellation rate due to poor ovarian response (RR: 0.37, 95%CI: 0.20 to 0.71) were observed. No significant differences were observed in estradiol levels (WMD: -8.12 pg/mL, 95% CI: -118.2 to + 101.96), in the numbers of follicles ≥17 mm on the day of triggering final oocyte maturation (WMD: +0.82, 95%CI: -0.11 to + 1.74), of MII oocytes (WMD: +0.50, 95% CI: -0.17 to + 1.17), of 2pn oocytes (WMD: +0.49, 95% CI: -0.11 to + 1.10), of embryos transferred (WMD: +0.21, 95%CI: -0.07 to + 0.49) and in the proportion of patients with ET (RR: 1.00, 95% CI: 0.96 to 1.04). Limitations, reasons for caution The definition of poor ovarian response varied among studies and a considerable heterogeneity regarding the type, dose and duration of testosterone pretreatment was present. Although the present study is currently the largest meta-analysis evaluating testosterone pretreatment, the total number of patients is still not large enough to draw solid conclusions. Wider implications of the findings The current study suggests that the probability of pregnancy is increased in poor responders pretreated with transdermal testosterone. This increase, in the absence of other proven beneficial interventions in these patients, justifies the conduction of further relevant RCTs. Trial registration number CRD42021262098

P-686 Molecular mechanism of Zishen Yutai Pills protecting ovarian reserve in premature ovarian insufficiency: a multi-omics analysis

Abstract Study question What is the potential molecular mechanism of Zishen Yutai Pills when compared to Hormone Replacement Therapies in treating premature ovarian insufficiency through protecting ovarian reserve? Summary answer Compared to estrogen, the cAMP signaling pathway and steroid hormone biosynthesis may be the potential molecular mechanisms for Zishen Yutai Pills preserving ovarian function. What is known already Premature ovarian insufficiency (POI) is a major reproductive disease that greatly affects ovarian function. It is a complex disorder with multiple pathogenic factors and high etiological heterogeneity. The conventional treatment for POI is hormone replacement therapies (HRT), which could alleviate the low estrogen syndromes. Compared to HRT, Zishen Yutai Pills (ZSYTP), a patented formula that is widely used for treating POI, can induce follicle development and stimulate ovulation. The combination of omics, including proteomics, metabolomics and transcriptomics, could promote the comprehensive understanding of the potential molecular mechanism involved in both effective treatments for POI in the rat model. Study design, size, duration 20 female Sprague-Dawley rats were randomized to 4 groups: the normal control group (NC), POI model group (POI), ZSYTP group and E2 Gavage group(E2). Intraperitoneally injected with cyclophosphamide for 15 days to establish a POI rat model, follow up with intragastric administration with ZSYTP or E2 daily for 3 weeks. The combined analysis of the proteomics, metabolomics and transcriptomics of rat ovarian tissues shows the potential pathways and molecular targets involved in ZSYTP and E2. Participants/materials, setting, methods All protocols of animal experiments were approved by the Institutional Animal Care and Ethics Committee of Zhuhai BestTest (IAC (S) 202108005). Rat ovary tissues were weighed and quickly frozen after dissection. Muti-omics analysis was performed based on the results from proteomics, metabolomics and transcriptomics analysis, in which Correlation and KEGG pathway analysis were conducted. Statistical analyses were performed with SPSS; statistical significance was set to P &amp;lt; 0.05. Main results and the role of chance Both the E2 and ZSYTP groups show elevated serum estrogen levels compared with the POI group(P&amp;lt;0.05). In the histological assessment, a significantly elevated amount of primary follicles was observed in the ZSYTP group compared to the E2 POI group, indicating that ZSYTP improves ovarian follicle development. Under electron microscopy, fewer necrosis cells were observed in the ZSYTP group, which shows that ZSYTP could improve ovarian repair function and reserve. A combined analysis of ovary proteomics, metabolomics and transcriptomics in ZSYTP vs. E2 shows that both interventions share molecular functions, including steroid binding, protein digestion and absorption, and carbon metabolism. In the KEGG pathway enrichment analysis, the cAMP signaling pathway and steroid hormone biosynthesis were enriched. Limitations, reasons for caution A larger sample size of animal experiments is needed for future study. Quantitative expression analyses of specific genes disclosed in this multi-omics analysis are the next stage of the study. Wider implications of the findings This is the first study to conjoint analyses multi-omics to explore the two different treatments on POI. Zishen Yutai Pills could alleviate low estrogen syndrome and preserve ovarian function through regulating the cAMP signaling pathway and steroid hormone biosynthesis. Zishen Yutai Pills could serve as an effective treatment for POI. Trial registration number Not applicable

Immunolocalization of melatonin receptors in bovine ovarian follicles and in vitro effects of melatonin on growth, viability and gene expression in secondary follicles

This study aims to investigate the (1) expression of melatonin receptors types 1A/B (MTNR1A/B) in bovine ovaries and (2) the in vitro effects of melatonin on secondary follicle development, antrum formation, viability, and expression of messenger ribonucleic acid (mRNA) for superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase-1 (GPX1) and peroxiredoxin 6 (PRDX6). The expression of MTNR1A/B in bovine ovarian follicles was demonstrated by immunohistochemistry. To choose the most effective concentration of melatonin on follicular growth and viability, isolated secondary follicles were cultured individually at 38.5°C, with 5% CO2 in air, for 18 d in TCM-199+ alone or supplemented with 10−11, 10−9, 10−7 or 10−5 M melatonin. Then, melatonin receptor antagonist, luzindole, was tested to further evaluate the mechanisms of actions of melatonin, that is, the follicles were cultured in control medium alone or supplemented with 10−7 M melatonin, 10 µM luzindole and both 10−7 M melatonin and 10 µM luzindole. Follicular growth, morphology and antrum formation were evaluated at days 6, 12 and 18. At the end of culture, viability of secondary follicles was analyzed by calcein-AM and ethidium homodimer-1, and the relative levels of mRNA for SOD, CAT, GPX1 and PRDX6 were evaluated by real time polymerase chain reaction. Immunohistochemistry results showed expression of MTNR1A/B in oocyte and granulosa cells of primordial, primary, secondary and antral follicles. Secondary follicles cultured in medium supplemented with melatonin at different concentrations had well preserved follicles after 18 d of culture. Furthermore, follicles cultured in presence of 10−7 M melatonin presented significantly higher diameters than those cultured in other treatments. The presence of melatonin receptor antagonist, luzindole, blocked the effects of melatonin on follicular growth and viability. In addition, follicles cultured in medium containing only melatonin had significantly higher rates of antrum formation. Follicles cultured in medium containing only melatonin had higher relative levels of mRNA for CAT, SOD and PRDX-6 than those cultured with both melatonin and luzindole. Follicles cultured with luzindole only or both melatonin and luzindole had lower relative levels of mRNA for PRDX6 and GPX1 than those cultured control medium. In conclusion, melatonin promotes growth of bovine secondary follicles through its membrane-coupled receptors, while luzindole blocks the effects of melatonin on follicle growth and reduces the expression of antioxidant enzymes in cultured follicles.

Single-cell transcriptomics of staged oocytes and somatic cells reveal novel regulators of follicle activation.

In briefProper development of ovarian follicles, comprised of an oocyte and surrounding somatic cells, is essential to support female fertility and endocrine health. Here, we describe a method to isolate single oocytes and somatic cells from the earliest stage follicles, called primordial follicles, and we characterize signals that drive their activation.Primordial follicles are the first class of follicles formed in the mammalian ovary and are comprised of an oocyte surrounded by a layer of squamous pre-granulosa cells. This developmental class remains in a non-growing state until individual follicles activate to initiate folliculogenesis. What regulates the timing of follicle activation and the upstream signals that govern these processes are major unanswered questions in ovarian biology. This is partly due to the paucity of data on staged follicle cells since isolating and manipulating individual oocytes and somatic cells from early follicle stages are challenging. To date, most studies on isolated primordial follicles have been conducted on cells collected from animal-age- or oocyte size-specific samples, which encompass multiple follicular stages. Here, we report a method for collecting primordial follicles and their associated oocytes and somatic cells from neonatal murine ovaries using liberase, DNase I, and Accutase. This methodology allows for the identification and collection of follicles immediately post-activation enabling unprecedented interrogation of the primordial-to-primary follicle transition. Molecular profiling by single-cell RNA sequencing revealed that processes including organelle disassembly and cadherin binding were enriched in oocytes and somatic cells as they transitioned from primordial to the primary follicle stage. Furthermore, targets including WNT4, TGFB1, FOXO3, and a network of transcription factors were identified in the transitioning oocytes and somatic cells as potential upstream regulators that collectively may drive follicle activation. Taken together, we have developed a more precise characterization and selection method for studying staged-follicle cells, revealing several novel regulators of early folliculogenesis.