Ovarian Activity Research Articles

Exosomes derived from menstrual blood stromal cells ameliorated premature ovarian insufficiency and granulosa cell apoptosis by regulating SMAD3/AKT/MDM2/P53 pathway via delivery of thrombospondin-1

Premature ovarian insufficiency (POI) is clinically irreversible and seriously damages female fertility. We previously demonstrated that menstrual blood stromal cells (MenSCs)-derived exosomes (EXOs) effectively improved ovarian functions in the POI rat model. In this study, we investigated whether TSP1 is the key component in EXOs to ameliorate ovarian functions and further explored the molecular mechanism of EXOs in improving granulosa cell (GCs) activities. Our results demonstrated that knockdown TSP1 significantly debilitated the therapeutic effect of EXOs on estrous cyclicity, ovarian morphology, follicle numbers and pregnancy outcomes in 4-vinylcyclohexene diepoxide (VCD) induced POI rat model. In addition, EXOs treatment significantly promoted the activities and inhibited the apoptosis of VCD induced granulosa cells in vitro. Moreover, EXOs stimulation markedly activated the phosphorylation of SMAD3(Ser425) and AKT(Ser473), up-regulated the expressions of BCL2 and MDM2 as well as down-regulated the expressions of CASPASE3, CASPASE8, P53 and BAX. All these effects were supressed by SIS3, a inhibitor of TGF1/SMAD3. Our study revealed the key role of TSP1 in EXOs in improving POI pathology, restoring ovarian functions and GCs activities, andprovided a promising basis for EXOs in the treatment of ovarian dysfunction.

COVID-19 lockdown and the rate of central precocious puberty

PurposeThe aim of our study was to compare the incidence of idiopathic central precocious puberty (CPP) in our highly specialized Endocrinological Center before and after the onset of COVID-19 lockdown; we also aimed to identify any potential difference between girls with CPP from the two different time periods.MethodsWe retrospectively analyzed the auxological profile of 49 girls with idiopathic CPP: 30 with pre-lockdown onset and 19 with post-lockdown onset of the disease. We collected patients’ characteristics (medical history, physical examination, baseline and dynamic hormonal assessment, bone age, pelvic ultrasound) and compared them between the two groups.ResultsWe registered an almost threefold increase in CPP incidence in the 2020–2021 period compared to the previous six years. In post-lockdown patients we found a trend for an earlier diagnosis in terms of both chronological age (p 0.0866) and days between the onset of first pubertal signs and diagnosis (p 0.0618). We also found that post-lockdown patients had a significantly lower hypothalamus-pituitary–gonadal axis activation (lower ∆LH% after GnRH test, p 0.0497), a significantly lower increase in bone age calculated at RUS with TW3 method (p 0.0438) and a significantly reduced ovarian activation in females (lower delta-4-androstenedione levels, p 0.0115). Interestingly, post-lockdown patients were born from mothers with an older age at menarche (p 0.0039).ConclusionsBesides confirming a significant increase in new diagnoses of CPP in the post-lockdown period, our findings among Post-lockdown girls also suggest a less progressive form of CPP and a stronger environmental influence compared to genetic background in determining the timing of pubertal onset.

Postpartum longissimus dorsi muscle loss, but not back fat, is associated with resumption of postpartum ovarian activity in dairy cattle

The objectives of this observational cohort study were to assess the effect of body condition score change, back fat depth change, and muscle diameter change on the time to commencement of luteal activity and first estrus in commercial pedigree Holstein cows. One hundred and 40 of 200 commercial pedigree Holstein cows were enrolled in one dairy herd in Somerset, United Kingdom, over 52 weeks in 2021-22. The herd used 4 automatic milking machines with in-line progesterone measurement capability to determine commencement of luteal activity and time to first estrus. Cows were followed until at least 60 d postpartum and milk progesterone measured daily starting from 10 DIM. Body condition scoring and ultrasound measurements of back fat depth and Longissimus dorsi muscle diameter were performed on cows twice: within 7 d of both calving and 60 DIM. Other explanatory variables assessed included parity, 60-d and 305-d milk yield, and subclinical ketosis (β-hydroxybutryate (βHB) ≥ 1.2mmol/L). Occurrence of clinical disease <60 DIM was forced into all models as a binary variable. Data were analyzed using multivariable cox proportionate survival analyses. Muscle loss was associated with commencement of luteal activity and time to first estrus. A reduction in muscle diameter by 1.5 to 5 mm was associated with the shortest time to the start of luteal activity and first estrus. A reduction in muscle diameter >8 mm was associated with the longest times to luteal activity and first estrus. In addition to being affected by muscle loss, commencement of luteal activity was delayed by subclinical ketosis, clinical disease, and failure to gain body condition to 60 DIM. Cows that had a BCS loss of 0.25 or more between calving and 60 DIM were at least 52 ± 22% less likely to have commenced luteal activity compared with those that gained BCS. Interestingly, cows that had no change in body condition score commenced luteal activity later than those that gained body condition score. Muscle loss was associated with time to first estrus irrespective of clinical disease status. Cows that lost > 8 mm of muscle diameter showed estrus behavior later than cows that lost 1.5 to 5 mm. In conclusion, our findings indicate that extensive muscle loss postpartum was associated with a delayed start to luteal activity and first estrus irrespective of body condition change, clinical disease, and subclinical ketosis. Marginal muscle loss and a gain in body condition, however, were associated with an earlier start to luteal activity and first estrus.

MicroRNA-144-3p protects against chemotherapy-induced apoptosis of ovarian granulosa cells and activation of primordial follicles by targeting MAP3K9

Premature ovarian failure (POF) is defined by amenorrhea, ovarian atrophy, hypoestrogenism, elevated gonadotropin level, and infertility under the age of 40. POF is frequently induced by chemotherapeutic agents. However, the underlying mechanisms regarding chemotherapy-mediated damage to ovarian function are unclear. In this study, enhanced apoptosis of granulosa cells (GCs) and aberrant activation of primordial follicles were observed in a POF mouse model induced by cisplatin. We subsequently observed significant downregulation of miR-144-3p and upregulation of mitogen-activated protein kinase kinase kinase 9 (MAP3K9) in primary ovarian GCs from POF mice, as revealed by microarrays. Furthermore, MAP3K9 expression was higher in human ovarian granulosa cells (COV434) treated with cisplatin and was identified as a novel target of miR-144-3p. Functional analysis revealed that miR-144-3p attenuated cisplatin induced apoptosis of GCs via silencing MAP3K9 expression, which suppressed the activity of the downstream p38 mitogen activated protein kinase (MAPK) pathway. Meanwhile, miR-144-3p prevented premature primordial follicle depletion in cisplatin-induced POF mice through targeting Map3k9, which led to a decline in the phosphorylation and activation of the phosphatidylinositol-4,5-bisphosphate 3-kinase (PI3K)/protein kinase b (AKT) pathway. Taken together, this study revealed the protective effects of miR-144-3p on ovarian function and shed light on the epigenetic regulatory mechanism in the development of POF, which might provide new biomarkers for the ovarian reserve.

Influence of an eCG administration on puerperal involution and fertility in Simmentaler cows

In the literature, various methods aiming for a promotion of uterine involution and ovarian function in post-partum dairy cattle have been described. Recently, successful use of equine choriongonadotropin (eCG) in this context has been repeatedly reported, while other studies have failed to demonstrate positive effects of eCG administration on puerperal involution in cows. Due to the contradictory results described in the literature, the aim of this field study was to verify whether the administration of eCG on day 10 post-partum exerts a positive effect on uterine involution, ovarian activity and fertility indices in Simmental cows. In nine dairy farms, 205 Simmental cows were divided by lot into three groups: Group 1 animals received 500 I.U. eCG, group 2 received 50 µg gonadorelin, and group 3 animals received 250 mg butafosfan and 125 µg cyanocobalamin (control group). Treatment was administered on day 10 post-partum in each case. The animals were examined clinically and ultrasonographically on the 10th, 25th and 42nd day post-partum and the time to insemination and pregnancy were recorded. Group comparison with respect to the recorded parameters was initially performed without considering confounding factors by analysis of variance. In the second step, potential influencing variables (lactation, milk yield, change in backfat thickness, retentio secundarium and serverity of the obstetric intervention) were taken into account, as well as the combination of the random factors "farm" and "individual animal within the farm". The administration of eCG exhibited no significant effect on uterine involution, however, the cervical involution was influenced significantly. In the group of cows with eCG treatment, ovarian cysts were detected significantly more frequently on day 25 post-partum (p=0.04). This difference did not persist on the 42nd day post-partum. Neither voluntary waiting period nor the days open were significantly shortened by eCG administration. Lactation number showed a significant effect on uterine involution. Heifers exhibited more rapid involution by day 42 post-partum than cows. Administration of eCG in Simmentaler cows at day 10 post-partum showed no positive effect. The use of eCG in early puerperium to promote fertility in the cow should be avoided.

ASSESSMENT OF PSYCHOLOGICAL PROBLEMS AND COPING STRATEGIES OF PERI-MENOPAUSAL WOMEN IN SELECTED DISTRICT OF WEST BENGAL

Peri-menopause is natural event where ovarian follicular activity is reduced, for this reason women are prone to develop various physical and psychological symptoms. A descriptive study was conducted to detect psychological problems and coping strategies among peri- menopausal women in selected district of West Bengal. 200 participants were selected through non- probability purposive sampling technique to assess psychological problems and coping strategies among them and to find out the association between identified psychological problems and coping strategies with selected demographic variables. Conceptual framework of this study was based on Lazarus &amp; Folkman’s Transactional Model of stress and coping. Semi-structured questionnaire, DASS 21 Questionnaire and Brief COPE Inventory were used to collect data. Result revealed that 63.5% peri- menopausal women had no depression, 2% had extremely severe depression. 71% had no anxiety; 55.5% had no stress and 10% had severe stress. Most of the peri- menopausal women (57.5 %) adopted moderate coping strategies. Chi-square test revealed that there was significant association between depression and educational status (p&lt; 0.05). Coping strategies were also significantly associated with their monthly family income (p&lt;0.05).

The effect of bumble bee gynes’ reproductive status on the response to CO2 narcosis

Many organisms undergo extreme physiological and behavioral changes that dictate their responses to different stimuli during their life cycle. Bumble bees (Bombus sp.) display an annual life cycle, in which the virgin gynes emerge, disperse, mate, and undergo several months of winter diapause before establishing new colonies in the spring. CO2 narcosis induces a direct transition from mating to reproduction, thus diapause can be bypassed. The mechanism underlying the response to CO2 narcosis remains unclear. Here, we used Bombus terrestris gynes in different reproductive statuses (virgin, mated, and post-diapause) to examine the effect of CO2 narcosis on ovarian development, body mass, protein uptake, and metabolic rate. We found that the impact of CO2 narcosis on gynes was inhibited by mating, with virgin gynes showing the strongest effect of CO2 narcosis on ovary activation and protein turnover. We show that mating inhibits the effect of CO2 narcosis prior to the diapause period, suggesting that this effect is upstream to that of CO2 narcosis.

Targeted Glutamate Supply Boosts Insulin Concentrations, Ovarian Activity, and Ovulation Rate in Yearling Goats during the Anestrous Season.

The neuroendocrine regulation of the seasonal reproductive axis requires the integration of internal and external signals to ensure synchronized physiological and behavioral responses. Seasonal reproductive changes contribute to intermittent production, which poses challenges for optimizing goat product yields. Consequently, a significant objective in seasonal reproduction research is to attain continuous reproduction and enhance profitability in goat farming. Glutamate plays a crucial role as a modulator in several reproductive and metabolic processes. Hence, the aim of this study was to evaluate the potential impact of exogenous glutamate administration on serum insulin concentration and ovarian function during the out-of-season period in yearling goats. During the anestrous season, animals were randomly located in individual pens to form two experimental groups: (1) glutamate (n = 10, live weight (LW) = 29.1 ± 1.02 kg, body condition score (BCS) = 3.4 ± 0.2 units) and (2) control (n = 10; LW = 29.2 ± 1.07 kg, BCS = 3.5 ± 0.2), with no differences (p < 0.05) regarding LW and BCS. Then, goats were estrus-synchronized, and blood sampling was carried out for insulin quantification. Ovaries were ultrasonographically scanned to assess ovulation rate (OR), number of antral follicles (AFs), and total ovarian activity (TOA = OR + AF). The research outcomes support our working hypothesis. Certainly, our study confirms that those yearling goats treated with exogenous glutamate displayed the largest (p < 0.05) insulin concentrations across time as well as an augmented (p < 0.05) out-of-season ovarian activity.

Dietary supplementation with different nano and organic selenium doses improves ovarian activity, fertility rate, and progesterone level in Ossimi ewes

This study aimed to investigate the effects of nano and organic selenium on ovarian activity, fertility rate, and progesterone hormone levels in Ossimi ewes. Thirty ewes were treated with intra-vaginal sponges impregnated with medroxyprogesterone acetate (40 mg) for 14 d and simultaneously assigned randomly to three groups. The 1st group ewe served as control (CG). Ewes in the 2nd and 3rd groups were treated orally with organic selenium 3 mg/ewe and Nano-selenium 0.2 mg/ewes during intra-vaginal sponges (IVS) treatment SeG and N-SeG, respectively. The ovarian activity was monitored using an ultrasonic device, and serum samples were harvested for progesterone measurement. The results revealed that ewes in SeG and N-SeG groups had a significantly (p < .05) higher number of small and large follicles after 48 h of IVS withdrawal than in the CG, respectively, on the left ovary. Also, the N-SeG group had a larger diameter of the Corpus luteum on both ovaries’ sides than CG, respectively, at the same time. However, the diameter of large follicles on both ovaries was significantly (p < .05) larger in the N-SeG after 72 h of IVS withdrawal than in SeG and CG. The ewes in N-SeG and SeG came in oestrus earlier (p < .05) than those in CG, and the oestrus durations were longer (p < .05) than that recorded in ewes of CG. The oestrus response and fertility rate were higher (p < .01) in N-SeG and SeG than in CG. Moreover, the progesterone concentrations were significantly higher (p < .05) in the N-SeG and SeG after 48 and 72 h of IVS withdrawal than in the CG. Also, the recorded serum progesterone concentrations in the included ewes non-significantly differed between groups during the first four months of pregnancy. It could be concluded that using nano and organic selenium can improve the quality of ovarian follicles and CL function and enhance the fertility rate in Ossimi ewes.

Telomerase Activity in Somatic Tissues and Ovaries of Diploid and Triploid Rainbow Trout (Oncorhynchus mykiss) Females.

Telomerase activity has been found in the somatic tissues of rainbow trout. The enzyme is essential for maintaining telomere length but also assures homeostasis of the fish organs, playing an important role during tissue regeneration. The unique morphological and physiological characteristics of triploid rainbow trout, when compared to diploid specimens, make them a promising model for studies concerning telomerase activity. Thus, in this study, we examined the expression of the Tert gene in various organs of subadult and adult diploid and triploid rainbow trout females. Upregulated Tert mRNA transcription was observed in all the examined somatic tissues sampled from the triploid fish when compared to diploid individuals. Contrastingly, Tert expression in the ovaries was significantly decreased in the triploid specimens. Within the diploids, the highest expression of Tert was observed in the liver and in the ovaries of the subadult individuals. In the triploids, Tert expression was increased in the somatic tissues, while the ovaries exhibited lower activity of telomerase compared to other organs and decreased compared to the ovaries in the diploids. The ovaries of triploid individuals were underdeveloped, consisting of only a few oocytes. The lack of germ cells, which are usually characterized by high Tert expression, might be responsible for the decrease in telomerase activity in the triploid ovaries. The increase in Tert expression in triploid somatic tissues suggests that they require higher telomerase activity to cope with environmental stress and maintain internal homeostasis.

Effect of melatonin implantation prior to ovsynch plus cidr protocol on subsequent ovarian activity and pregnancy rate in summer anestrous Murrah buffaloes

For assessing the impact of melatonin pre-treatment in summer anestrous Murrah buffaloes on subsequent reproductive activity of a fixed time artificial insemination (FTAI) protocol, 83 anestrous Murrah buffaloes were subcutaneously inserted 2x4 mm size absorbable melatonin (MT) implants (18 mg/50 kg b wt) at the base of left ear and 38 Murrah buffaloes were used as non-implanted controls. On day 35 post-MT, the treatment and control group Murrah buffaloes were subjected to FTAI protocol (Ovsynch plus CIDR). Ovarian ultrasonography and jugular vein blood sampling was carried out on day 7 and 10 of protocol and on day 7-post AI. Overall pregnancy rate on day 90 post-AI in MT group was higher compared to control group (55.4 vs 31.6%, P&lt;0.05), along with the higher (P&gt;0.05) plasma progesterone on day 7 of FTAI protocol in Murrah buffaloes of the former group which conceived subsequently compared to their non-pregnant counterparts. Moreover, MT group had higher (P&gt;0.05) increase in dominant follicle diameter between day 7 and 10 of protocol compared to control group. On day 7 post-AI, the luteal profile in pregnant murrah buffaloes of MT group was better (P&lt;0.05) compared to their counterparts in control group (CL: 14.80±0.70 vs 10.82±0.46 mm, P4: 1.54±0.07 vs 1.04±0.08 ng/ml). In conclusion, melatonin pre-treatment in summer anestrous Murrah buffaloes could be associated with better luteal activity and higher pregnancy rate in the Ovsynch+CIDR protocol.

Correlation of body condition and occurrence of anoestrus in buffaloes

The present study was conducted on 30 buffaloes belonging to department of veterinary gynaecology and obstetrics and village around college of Veterinary Science and Animal Husbandry, Mhow (M.P.), buffalo were selected on the basis of history, which had not exhibited oestrus for 90 days or more postpartum, clinical examination of these buffaloes were made for general health and reproductive soundness, per rectally explored twice 10 days apart to confirm ovarian activity and genital status, the animals is awarded an appropriate body condition score 1 (very thin) to 5 (fat) on practice half score were used for animals whose condition score is intermediate. the experiment was done on 30 buffaloes divided into 6 groups, each group consist of 6 buffaloes. The body condition score on scale 1 (very thin) to 5 (fat) ranged between. Body condition score in anoestrus buffaloes was significantly lower than the normal cycling buffaloes. There is a positive relationship between body condition score and subsequent reproductive performance of the animals.

Sequencing analysis of anti mullerian hormone in polycystic ovarian syndrome and primary ovarian insufficiency

Link of Video Abstract: https://youtu.be/HvI9j8B9vqE Introduction: Polycystic ovarian syndrome (PCOS) is the most common endocrine disorder in women of reproductive age and is the main cause of ovulation disorders. Primary ovarian insufficiency (POI) is a clinical syndrome characterized by loss of ovarian activity before the age of 40 years. All of these disorders lead to the risk of infertility. Therefore, this study aims to assess the AMH gene sequence and the effect of SNP on AMH levels and function. Method: This research was a cross-sectional study. The sample was divided into three groups such as PCOS, POI, and control. This study used ELISA and PCR examination methods. Research data were analyzed using IBM SPSS version 22. The statistical analyses used were the ANOVA, Kruskal-Wallis, chi-square, and Spearman tests. A significant p-value was ≤ 0.05. Results: There were 31 research subjects, 8 POI subjects, 16 PCOS subjects, and 7 control subjects. Overall 30 mutations were found, with two mutations in the gene promoter and 28 mutations in the structure of the AMH gene. Five mutations in the AMH gene structure were missense mutations. 14 SNPs were found in the POI group and 15 SNPs in the PCOS group. There was a significant difference in AMH AMH gene promoter mutation frequency distribution between the POI and PCOS groups at the mutation point 19:g.2249146T&gt;G (p=0.007). In contrast, the mutation point 19:g.2249158T&gt;A was not significantly different (p=0.536). There was no significant correlation between the number of promoter mutations and AMH gene structure with AMH levels in the POI, PCOS and control groups. Conclusion: There were mutations in the promoter and AMH gene structure of the POI, PCOS, and control groups which caused differences in base sequences. However, in this study, the differences in mutations were not significantly different.

Ovarian activity, hormone profile, pro-inflammatory cytokines and reproductive performance of buffalo cows fed diets with different estrogenicity.

Buffalo cows play a vital role in milk and meat production; however, they are characterised by several reproductive disorders. Feeding diets with high oestrogenic activity may be a disrupting factor. This study aimed to evaluate the effects of feeding roughages with different oestrogenic activity on the reproductive performance of early postpartum buffalo cows. A total of 30 buffalo cows were equally stratified into two experimental groups and fed either Trifolium alexandrinum (Berseem clover, phytoestrogenic roughage) or corn silage (nonoestrogenic roughage) for 90 consecutive days. After 35 days from the beginning of the feeding treatments, buffalo cows in both groups were synchronized for oestrus using a double i.m. injection of 2 mL prostaglandin F2α , 11 days apart, subsequently, overt signs of oestrus were observed and recorded. Moreover, ovarian structures, numbers and sizes of follicles and corpora lutea, were ultrasonography examined at day-12 (represents Day 35 of feeding treatment), Day 0 (day of oestrus) and Day 11 after oestrous synchronization (mid-luteal phase). Pregnancy was diagnosed 35 days postinsemination. Blood serum samples were analysed for progesterone (P4 ), estradiol (E2 ), tumor necrosis factor (TNF-α), interlukein-1β (IL-1β) and nitric oxide (NO). The high performance liquid chromatography-analysis of roughages showed the abundance of isoflavones in Berseem clover, with about 58 times higher concentration than that in corn silage group. During the experimental period, the numbers of ovarian follicles of all size categories were higher in the Berseem clover group than that in the corn silage group. No significant difference in the numbers of corpora lutea was observed between both experimental groups, but lower (p < 0.05) diameter of corpus luteum was observed in the Berseem clover group than that in the corn silage group. The Berseem clover group had higher (p < 0.05) overall concentrations of blood serum E2 , IL-1β and TNF-α, but lower (p < 0.05) overall concentrations of blood serum P4 than those recorded in the corn silage group. Oestrous rate, onset of oestrus time and oestrous duration were not significantly affected by the treatment. The conception rate was significantly (p < 0.05) reduced in the Berseem clover group compared with that in the corn silage group. In conclusion, feeding roughage with a high oestrogenic activity such as Berseem clover can negatively affect the conception rate of buffalo cows. This reproductive loss seems to be associated with inadequate luteal function and P4 concentration during early pregnancy.

P-615 Ovarian rejuvenation with human placenta hydrolyzate

Abstract Study question Is it possible to rejuvenate the ovaries with human placenta hydrolyzate? Summary answer Injecting human placenta hydrolyzate into the ovaries can activate dormant follicles and produce mature oocytes in patients with ovarian insufficiency. What is known already Patients with low ovarian reserve and/or ovarian failure are a difficult category for reproductive specialists. Attempts to rejuvenate the ovaries have been made for several years. The use of adjuvant therapy such as PRP can activate dormant follicles. Human placenta hydrolyzate contains growth factors, hormones, trace elements, vitamins. Administration of human placenta hydrolyzate directly to the ovaries can also activate folliculogenesis and oogenesis. Study design, size, duration A cohort study was conducted, which involved 20 women with low ovarian reserve who underwent IVF / ICSI at the Person ICCR in 2022. AMH, FSH, the number of antral follicles, the number of received oocytes were determined before the injection of the human placenta hydrolyzate (JAPAN BIO PRODUCTS Co, LTD, Japan) into the ovaries and 1-3 months after procedures. Participants/materials, setting, methods All patients had previously undergone IVF/ICSI. 20 women with infertility, mean age was 39±6 years. In September-October 2022, we received a human placenta hydrolyzate intraovarially. The human placenta hydrolyzate was injected into the ovaries as in a transvaginal puncture with a 20 G needle, 1 ml into each ovary. All participants gave their voluntary informed consent to the procedure. Main results and the role of chance On the 2-3rd day of the cycle before the procedure, the average level of AMH was 0.11±0.09 ng/ml, FSH 38.11±10.86 mIU/ml, the number of antral follicles 1.18±0, 38, the number of mature oocytes obtained on TVP, 0.68±0.19. After the introduction of the human placenta hydrolyzate into the ovaries, measurements were repeated 1-3 months later: AMH was 0.46±0.18 ng/ml, p = 0.09; FSH was 13.54±7.86 mIU/ml, p = 0.074; CAF 1.96±0.17, p = 0.068; oocytes M2 1.51±0.38, p = 0.058. Due to small cohort size and short follow-up time, we believe that ovarian activation by placental hydrolyzate takes place, this is especially noticeable in the number of obtained mature oocytes that went for freezing or fertilization: 4 patients received embryos suitable for PGT-A, one the embryo (mosaic) was transferred into the uterine cavity and pregnancy occurred. There was no any side effect after the injection. Limitations, reasons for caution Limitations relate to study design. More patients and longer follow-up are also needed. Wider implications of the findings Ovarian rejuvenation is currently an adjuvant therapy, high-level evidence is not yet available. However, for women with POI and POR, any opportunity to improve the chances of obtaining their own oocytes is valuable. Trial registration number NO APPLICABLE

P-427 Control of in vitro follicle activation by inhibition of YAP/TAZ activity in mouse ovaries

Abstract Study question Does verteporfin, an inhibitor of YAP/TAZ activity, prevent from the impacts of chemotherapy exposure on signaling pathways governing follicle activation and survival? Summary answer Verteporfin treatment prevents from the chemotherapy-induced impairments of the Hippo pathway but also of PI3K and survival pathways. What is known already The Hippo pathway is a crucial regulator of the ovarian reserve. Disruption of this pathway occurring in non-physiological contexts, such as ovarian processing during ovarian tissue preservation or after chemotherapy exposure, leads to massive uncoordinated follicular growth and depletion of the ovarian stockpile. To prevent from the harmful impact of chemotherapy exposure, several studies assessed the potential of inhibition of the PI3K pathway, the major signaling pathway involved in physiological follicle activation. However, the protective effect of this inhibition appeared to be moderate in vitro, suggesting the involvement of other disrupted mechanisms, as Hippo pathway. Study design, size, duration This study was performed on post-natal day 3 mouse ovaries. Half-cut ovaries were first treated with 0, 0.2, 1.5 and 3 µM Verteporfin (VERT) during 3 hours in vitro culture to establish the efficient dose of VERT. Whole ovaries were then cultured for 24 and 48 hours and exposed to 10 µM 4-hydroperoxycyclophosphamide (4HC) and/or 3 µM VERT to assess the effect of this inhibitor on follicle activation and survival pathways. (N = 3-5) Participants/materials, setting, methods To assess the impacts of sectioning and VERT treatment on follicle activation, gene expression analyses (RT-qPCR) were used to assess Hippo, PI3K/AKT/mTOR and apoptosis signaling pathways. The potential preventive effect of VERT co-treatment with 4HC exposure was evaluated by gene (RT-qPCR) and protein expression (western blot) analyses of these three pathways, and completed with histological staining for DNA damages (TUNEL). Main results and the role of chance Following exposure to increased concentration of VERT for 3 hours, a significant impact of the inhibitor was observed at 3µM on Ccn2 (p = 0.003) and Cmyc (p = 0.045) expression levels compared to control, without impacting on apoptosis genes expression. Exposure to 10 µM 4HC induced a higher YAP/pYAP protein ratio and Ccn2 expression level compared to control after 24 and 48 hours of culture, confirming the Hippo pathway disruption. VERT co-treatment was able to prevent from this increase of protein levels and gene expression, although the impact on Ccn2 expression was moderated after 48 hours of culture. Assessment of PI3K signaling revealed an induction of mTOR signaling following 4HC exposure at both time of culture as shown by the higher pRPS6/RPS6 ratio compared to control. Surprisingly, VERT co-treatment significantly decreased this level compared to 4HC alone at 24 and 48 hours of culture. This result suggests an indirect impact of this inhibitor on the PI3K pathway. Despite Bax and Bcl2 gene levels remained stable among conditions, ovaries exposed to 4HC had a significant higher level of DNA damages at both culture timepoints compared to control. Notably, VERT co-treatment was able to decrease the 4HC-induced gonadotoxicity at 48 hours of culture. Limitations, reasons for caution This study evaluated the impact of an inhibitor to control follicle activation in mouse model, and was limited to the assessment of two main signaling. Although we completed our study with apoptosis analyses, the results should be interpreted with caution as other pathways may be involved into the activation process. Wider implications of the findings Our results suggest that verteporfin is a promising inhibitor to control acute signaling pathway impairments under unphysiological conditions. Moreover, this study sustains the presence of a potential interaction between the two main signaling pathways regulating follicle activation, PI3K and Hippo pathways. Trial registration number Not applicable

P-801 Human umbilical cord perivascular cells (HUCPVC) reduce ovarian fibrosis and improve pregnancy rates in a mouse model of natural ovarian aging

Abstract Study question Does repeated intravenous administration of human umbilical cord perivascular cells (HUCPVC) during the period of ovarian aging prevent age-related fertility decline in a mouse model? Summary answer Repeated administration of first trimester and term HUCPVC improved pregnancy rates and reduced ovarian fibrosis in a mouse model of natural ovarian aging. What is known already Mesenchymal stromal cells (MSC), such as first trimester (FTM) and term human umbilical cord perivascular cells (HUCPVC), may be good cell candidates to mitigate side-effects of oncotherapy, including infertility, and are also gaining interest in ovarian rejuvenation strategies. The intraovarian or systemic administration of various sources of mesenchymal stromal cells in animal models of advanced reproductive age have been previously reported to improve ovarian reserve, follicle activation and function, via multiple proposed mechanisms. However, mating studies have not previously been performed to demonstrate fertility preservation. In addition, the optimal dose regimen and timing of treatment has not been established. Study design, size, duration Pre-clinical randomized controlled study in a mouse model of natural ovarian aging from 6 months to 12 months, including a negative control group (vehicle-treated), a cell control group (fibroblast-treated) and 3 experimental groups (HUCPVC, 2 sources and 2 dose regimens; n = 10-15 per group). All parameters were compared with young control female mice (6-8 weeks, 6 months). Cell injections and all assessments were blinded. Participants/materials, setting, methods 6-month-old ICR mice randomized into 4 groups received 6 monthly tail vein injections of 1x106 HUCPVC (FTM or term), fibroblast cells or HBSS (vehicle control). A 5th group received a single injection of FTM HUCPVC at 11M (n = 5). Pregnancy and litter size data were collected after breeding trials. The total number of follicles per ovary and picrosirius red (PSR) staining were quantified. Serum anti-Mullerian hormone (AMH) and C-reactive protein (CRP) were analyzed by ELISA. Main results and the role of chance No differences in mortality rates (P = 0.2) and animal weights were observed. The weight gain in FTM and term HUCPVC treated animals was significantly greater than in the other groups, over the duration of the study (P = 0.04). Animals that received 6 injections of FTM and term HUCPVC showed significantly higher pregnancy rates at 12M (100%, 80%) (P = 0.007; P = 0.048, respectively), when compared to animals that received HBSS (40%) and were similar to pregnancy rates of 6-8weeks (100%) and 6M (77%) groups. Fibroblast-treated and 1x FTM HUCPVC-treated groups showed 60% pregnancy rates. Age-associated declines in litter sizes and ovarian reserve indicators were observed as expected, but not rescued by cell treatments, except for levels of AMH which were increased in the group that received 1x FTM HUCPVC injections when compared to HBSS controls (P = 0.04). Age-associated increases in ovarian stroma fibrosis were observed with aging in the control groups (P &amp;lt; 0.0001), and were significantly reduced in 6x FTM HUPCVC, 6x term HUCPVC and 1x FTM HUCPVC groups, when compared to HBSS and fibroblast controls (P &amp;lt; 0.0001; P = 0.01 and P &amp;lt; 0.0001, respectively). An age-associated increase in CRP (P = 0.01) was significantly decreased in 6x FTM HUCPVC-treated groups, when compared to HBSS (P = 0.04). Limitations, reasons for caution Pregnancy rate (the main outcome measured in this study) may confound some of the secondary outcomes analyzed (CRP levels). Use of ovarian tissue for histological analysis and limited serum samples precluded further molecular analyses to assess potential mechanisms for the effects of HUCPVC. Wider implications of the findings FTM HUCPVC have anti-inflammatory and anti-fibrotic effects, and represent a promising source of MSC to prevent fertility decline in women of advanced reproductive age. Trial registration number Not Applicable

O-221 Regenerative endometrial PRGF (plasma rich in growth factors) treatment in patients with thin endometrium, recurrent implantation failure, and recurrent miscarriages: a retrospective, self-controlled, cohort study

Abstract Study question What reproductive outcomes are observed in patients submitted to endometrial PRGF therapy due to thin endometrium (ThE), recurrent implantation failure (RIF) and recurrent miscarriages (RM)? Summary answer Following endometrial PRGF treatment, success rates were significantly increased in ThE and RIF patients, whereas in the RM group pregnancy loss rates were not affected. What is known already Adequate development of a functional endometrium is a prerequisite for successful embryo implantation in ART cycles. In contrast, the management of patients with thin endometrial lining or recurrent implantation failure has been an ongoing challenge with none of the proposed surgical, hormonal, and pharmacological interventions achieving satisfactory results. In recent years, the use of plasma rich in growth factors (PRGF) - successfully applied in other medical fields - has become a novel treatment option in reproductive medicine. It was used experimentally both for ovarian follicle activation and to enhance endometrial receptivity although published studies are small with often contradictory results. Study design, size, duration All consecutive patients (n = 107) who underwent endometrial PRGF treatment (n = 150) in a single, private centre between 2016-2022 were included in this retrospective analysis. Patients were recruited into ThE (n = 64), RIF (n = 36) and RM (n = 7) groups, respectively. Live birth / ongoing pregnancy rates per embryo transfer were compared to success rates obtained in previous embryo transfers preceding the endometrial PRGF intervention. External Ethics Committee approval was obtained for a comparative pilot study including above patient groups. Participants/materials, setting, methods PRGF was obtained by processing the patients’ autologous blood sample in an in-house validated open system. Endometrial PRGF interventions were performed using a thawed PRGF sample with a series of intrauterine instillations (43%) or with a combined approach performing hysteroscopic subendometrial infiltration during early follicular phase (57%). Endometrial preparation was conducted using an artificial hormone replacement protocol with high-dose oral estrogens and vaginal progesterone. Most embryo transfers involved the replacement of a single, vitrified-thawed blastocyst. Main results and the role of chance A total of 107 patients underwent 150 endometrial PRGF treatments and 131 subsequent embryo transfers. Altogether 19 (13%) embryo transfers were cancelled, higher in ThE than in the RIF group (16 vs 7.1%). In the ThE group (64 patients, 98 PRGF cycles and 107 controls), positive pregnancy (41 vs 32%, NS), clinical pregnancy (35 vs 22%, p = 0.049) and ongoing pregnancy/live birth rates (24 vs 4.7%, p &amp;lt; 0.0001) per embryo transfer were significantly higher in the endometrial PRGF treatment group compared to previous embryo transfers. In the RIF group (36 patients, 42 PRGF cycles and 101 controls), positive pregnancy (59 vs 20%, p &amp;lt; 0.0001), clinical pregnancy (44 vs 11%, p &amp;lt; 0.0001) and ongoing pregnancy/live birth rates (33 vs 7.9%, p &amp;lt; 0.0001) per embryo transfer were significantly higher in the endometrial PRGF treatment group compared to previous embryo transfers. In the RM group (7 patients, 10 PRGF cycles and 15 controls), positive pregnancy (50 vs 60%), clinical pregnancy (20 vs 27%) and ongoing pregnancy/live birth rates (0 vs 0%) per embryo transfer were not significantly different and no ongoing pregnancies were achieved. So far, 20 singletons and 1 set of twins have been confirmed to be born from the above PRGF cycles (12 pregnancies still ongoing). Limitations, reasons for caution Heterogeneity of clinical severity between included ThE patients could affect observed reproductive outcomes. The self-controlled design of the study might have influenced the comparison between pre-, and post-intervention pregnancy rates, although this also highlights the poor-prognostic nature of included participants. RM patients were too few to evaluate pregnancy loss rates. Wider implications of the findings This preliminary, retrospective study has shown that regenerative therapy using autologous PRGF is a safe, affordable, and efficient treatment option for ThE and RIF patients. Further randomized studies are warranted, although they are hampered by patient selection issues and the lack of applicable efficient treatment options in the non-intervention group. Trial registration number not applicable

P-395 Protective effect of metformin on the ovarian reserve of prepubertal mice under cyclophosphamide chemotherapy

Abstract Study question Can metformin reduce the side effects of cyclophosphamide on the ovaries of prepubertal mice treated with cyclophosphamide? Summary answer Yes, metformin, as an inhibitor of mTOR and, by affecting the HIPPO pathway, can exert its protective effect in the chemotherapy mice model. What is known already PI3K/AKT and HIPPO pathways are influential pathways in the process of activation of primordial follicles and proliferation of cell growth, which are affected by various agents and drugs such as cyclophosphamide. One of the side effects of cyclophosphamide is the reduction of the ovarian reserve through abnormal changes in the PI3K/AKT and HIPPO pathways. Metformin is a drug that is used to treat type 2 diabetes and at the same time regulates PI3K/AKT and HIPPO pathways and prohibits the proliferation of cancer cells. Study design, size, duration Controlled experimental study. After metformin and cyclophosphamide dose adjustment, 12 female NMRI 14-day-old mice were randomly divided into four groups: the control group (CONT), metformin group (MET), cyclophosphamide group (CYC) and metformin with cyclophosphamide group (MET-CYC). Participants/materials, setting, methods Mice were treated by intraperitoneal injection of a dose of 150mg/kg of metformin for 11 consecutive days, and/or a dose of 65mg/kg of cyclophosphamide every three days (The third, sixth and ninth days). The mice were sacrificed 24 hours after the last metformin injection or 3 days after the last cyclophosphamide injection. The ovaries were collected for stereological evaluation, and expression of Pten, Mtor, Yap-1, p53, Bcl-2, and Bax genes by qRT-PCR method. Main results and the role of chance The stereological evaluation showed that the ovarian volume and primordial follicle number in the CYC group were decreased compared to the other groups (P &amp;lt; 0.05), and also in the MET-CYC group reduced compared to the CONT and MET groups, but increased significantly compared to the CYC group (P &amp;lt; 0.05). A significantly increased number of growing follicles was observed in the CYC group compared to the other groups (P &amp;lt; 0.05). The molecular analysis showed that the Pten expression in the CYC group was lower than CONT and MET groups (P &amp;lt; 0.05). Mtor expression in the CYC group was significantly increased compared to other groups (P &amp;lt; 0.05). The expression of Yap-1 was significantly decreased in the MET group compared to other groups (P &amp;lt; 0.05), and was lower in the MET-CYC than in the CYC group (P &amp;lt; 0.05). The of P53 expression in the MET-CYC group was higher than CONT and MET groups (P &amp;lt; 0.05), and was increased in the CYC compared to the CONT group (P &amp;lt; 0.05). Bcl-2 expression was higher in CONT and MET groups than in MET-CYC and CYC groups (P &amp;lt; 0.05). The expression of Bax in the CYC group was significantly increased compared to CONT and MET groups (P &amp;lt; 0.05). Limitations, reasons for caution Due to ethical issues in less use of mice, dose of metformin was obtained according to the articles used in adult mice and also by using the human to animal dose conversion formula. Wider implications of the findings It can be concluded that metformin during cyclophosphamide treatment can maintains ovarian volume, reserve, and activity in prepubertal mice by regulating PI3K/AKT and HIPPO pathways. Considering the relatively acceptable cost and long-term safety of metformin, it is a promising factor to be used to preserve fertility during chemotherapy. Trial registration number not applicable

Quality improvement project in primary care to improve management of women with premature ovarian insufficiency

Background: Premature Ovarian Insufficiency (POI) is defined as the loss of ovarian activity before the age of 40 years. The prevalence in the general population is an estimated 1% in the UK. If untreated, POI can cause serious long term health consequences, including increased risk of cardiovascular disease, osteoporosis, impaired cognition, reduced psychological wellbeing, and quality of life. To lower the risk of these, hormone replacement therapy (HRT) is advised until the natural age of the menopause. Investigations are recommended by ESHRE to look at primary causes as well as a baseline DEXA scan. We present results from a study exploring prevalence, diagnosis, and management of POI in an urban Primary Care Practice in East London.