Loading...
1 results
Search Results
Now showing 1 - 1 of 1
- Early Diagnosis and Effective Management of Diminished Ovarian Reserve (DOR)Publication . Carvalho, Alexandra Fernandes de; Santos, Ana Teresa Moreira Almeida; Ramos, Mariana Costa Brandão de Moura; Patrício, Ana Cristina Monteiro Ramalhinho TavaresDiminished ovarian reserve (DOR) refers to a reduction in number of available follicles, leading to decreased reproductive potential at an early age (Cohen et al., 2015). This condition affects about 10% of women, with over 50% of these cases being idiopathic (Ferreri et al., 2020; Greene et al., 2014; Nikolaou & Templeton, 2003). A global trend of delayed childbearing particularly impacts women with DOR, often delaying their diagnosis, and making them more likely to require fertility treatments (Barbakadze et al., 2015; Geyter et al., 2020). Furthermore, a significant lack of fertility knowledge (Halleran et al., 2022; Iino et al., 2022; Pedro et al., 2018; Ren et al., 2023), frequently leads to unintended childlessness (Beaujouan, 2020). Interventions to provide information and support fertility management can help mitigate infertility problems; however, they have not been consistently implemented (Bakkensen & Goldman, 2022; Harper et al., 2021). Tailored counselling in reproductive health has also been recommended to help reduce the risk of infertility (Azhar et al., 2015). Once anti-Müllerian hormone (AMH) measurement and antral follicle count (AFC) determination are considered the gold standard methods for assessing ovarian reserve (Tremellen & Savulescu, 2014), they can be used to provide tailored information to women, and support decision-making in reproductive plans. However, at the moment, the Portuguese population lacks comprehensive studies in this particular field. Investigating the genomic profile of a DOR cohort has been suggested to elucidate on the aetiology of this condition, potentially enhancing diagnostics and enabling a targeted therapy (Nesbit et al., 2020). Currently, limited information exists regarding the prevalence of genetic factors contributing to DOR, as most genetic studies have focused on premature ovarian insufficiency (POI) (Huhtaniemi et al., 2018). Single nucleotide polymorphisms (SNPs) in genes encoding hormonal receptors, such as follicle-stimulating hormone receptor (FSHR) or estrogen receptor 1 (ESR1), have been associated with follicle recruitment, development, and maturation (Greene et al., 2014). However, further evidence is required to establish their role in situations of abnormal ovarian reserve. Novel approaches have emerged for rejuvenating ovarian tissue and activating folliculogenesis. Platelet-rich plasma (PRP) has shown potential to stimulate follicle activation and angiogenesis by the presence of growth factors (Alam et al., 2009; Atkinson et al., 2021; Sills & Wood, 2019). While the literature demonstrates a growing interest in the use of PRP to improve outcomes for women with DOR undergoing assisted reproduction (Alam et al., 2009; Cakiroglu et al., 2020; Callejo et al., 2013; Hsu et al., 2020; Melo et al., 2020; Pantos et al., 2019; Sfakianoudis et al., 2018, 2019, 2020; Sills et al., 2018; Sills & Wood, 2019), its efficacy remains under investigation (Reig et al., 2021). Therefore, the most effective strategy for mitigating the effects of DOR has yet to be determined. Our objective was to evaluate different approaches to manage women with DOR, regarding strategies to be implemented before and after the diagnosis. Accordingly, this project was structured into three tasks with their respective objectives: Task 1: intervention in knowledge through general and tailored information on ovarian reserve and diminished ovarian reserve (DOR) Develop and test the impact of two interventions on knowledge about ovarian reserve DOR: 1) providing general leaflet information, and 2) offering tailored information and reproductive counselling based on the assessment of ovarian reserve. Task 2: genetic analysis of infertile populations with abnormal ovarian reserve Review the role of hormonal receptor polymorphisms in ovarian function. Identify genetic variants concerning FSHR rs6166, ESR1 rs2234693 and ESR1 rs9340799 polymorphisms in cases of altered ovarian reserve (DOR and PCOS), among infertile populations. Specifically, investigate the association of polymorphic genetic variants in hormonal receptor genes with ovarian reserve markers in different groups, women with DOR and polycystic ovarian syndrome (PCOS), undergoing fertility treatments. Establish an NGS study protocol to identify genetic variants in the population with DOR, which includes reviewing the genes most relevant to this population and defining the gene panel to be analysed subsequently. Task 3: in vitro study of follicular activation, through the incubation of human ovarian tissue with platelet-rich plasma (PRP) Evaluate the efficacy of PRP supplementation in activating the phosphatidylinositol-3-kinase (PI3K)-protein kinase B (Akt) Pathway and enhancing stromal vascularization using an in vitro culture system with human ovarian tissue. The results are in the order of the three described tasks. An observational longitudinal study was conducted. Our study involved 105 childless women with an average age of 27.33±3.67 years, with the majority (79%) participating by self-proposal. The participants knowledge, reproductive intentions, and attitudes were assessed at three-time-points by questionnaire. Specifically, assessment before any intervention (t0), and after the general information leaflet (t1) and after the tailored information (t2). This group was highly educated, with 55.2% holding a master's degree. Their baseline knowledge about ovarian reserve, assessed by the percentage of correct answers regarding its definition, the factors that affect it, how it decreases, the markers used to evaluate it, and specific situations of DOR and its incidence, was close to 64%. Participants demonstrated significant improvements in their knowledge, after both interventions (p<.001 for to Vs t1 and p=.008 for t1 Vs t2). Most participants were in a relationship (72.38%), even though for 31.4% of them marriage plans are not something that they prioritize. A significant portion of the participants considered having children very important (40%) or extremely important (37.1%), with 58.1% being certain they wanted children in the future. Participants were aware that the ideal age to have their first child is before 30 years, with the ideal age reported as close to 29 years old, at the 3 time-points (p=.129). However, on average, they planned to have their first child at 31 years, consistently expressing a significantly lower mean ideal age for first childbearing compared to the age they actually plan to have children (p<.001). This discrepancy indicates some awareness of the limitations to their reproductive plans. Accordingly, the desired number of children was significantly higher than the number they felt confident they could have, considering factors such as economic situation, age, and personal background. This trend persisted from t0 (p=.004) to t1 (p<.001), and at t2 (p<.001). Participants acknowledged that infertility could limit their parenthood plans, with it being the most considered obstacle at t0 (51.4%) and t1 (54.3%) and the second most considered at t3 (43.8%). If confronted with a diagnosis of DOR, the decision to give up on parenthood remained low and consistent throughout the intervention period (p=.618). Moreover, participants did not adjust their intended childbearing age in response to potential infertility caused by DOR (p=.532). When presented with a hypothetical possibility of infertility, participants showed a preference for ART over adoption (p<.001) and egg donation (p<.001). In addition to evaluating the overall impact of the interventions on knowledge, we also aimed to investigate how the information provided about ovarian reserve assessment affected knowledge, reproductive intentions, and attitudes. To achieve this, we formed subgroups for analysis: women who received reassuring information, with AMH and AFC levels above the 25th percentile for their age (NOR); in comparison to women with either AMH or AFC levels below the 25th percentile for their age (DOR1); women with both AMH and AFC levels below the 25th percentile (DOR2); and women with discrepancies between AMH and AFC levels, where one of these measures was below the 25th percentile (INT). Throughout the study, the only significant change observed was in the preference for oocyte cryopreservation, with a significant decrease in considering social freezing as an option (p=.002). Specifically, between the initial assessment (t0) and the provision of general informational leaflets (t1), the mean value decreased (p=.046). This trend continued between the post-general information stage (t1) and the phase following the provision of tailored information and reproductive counselling (t2) (p=.024). Analysing oocyte freezing across subgroups formed according to ovarian reserve status revealed significant differences when exploring the interplay between time and group (p=.016), particularly noticeable between t1 and t2 (p=.002). Within the DOR1 subgroup, participants considered the option of oocyte cryopreservation less after receiving information (p<.001). This attitude may reflect a more realistic understanding that it is not the most suitable option for them. The knowledge did not improve after providing tailored information about ovarian reserve status, with the interaction between time and group not showing significant changes for the comparisons of DOR1 with NOR subgroups (p=.247), as well as between DOR2, INT, and NOR subgroups (p=.574). Childbearing intentions remained stable when considering the interaction between time and group, regarding the groups formed according to the ovarian reserve testing results. For instance, the number of children participants would like to have (p=.707 and p=.116), the number of children participants consider possible to have (p=.473 and p=.349), the ideal childbearing age (p=.152 and p=.155), and the age at which they plan to have their first child (p=.648 and p=.373) did not show significant changes. Importantly, the message was well-targeted and well-received by participants. In the NOR subgroup, where the information provided was more reassuring, we observed a decrease in the percentage of participants who considered infertility as an obstacle, from 59.3% to 55.9% (p=.004). For INT subgroup, providing information increased the number of participants considering infertility an obstacle, from t0 (47.06%) to t1 (60.78%), p=.039, and this perception was maintained over time: t1 (60.78%) to t2 (54.9%), p=.375. The perception that everything is fine was predominantly chosen by NOR participants (p<.001). On the other hand, participants who received information indicating that their ovarian reserve was below the expected percentile for their age—both in the DOR1 and DOR2 subgroups—frequently assessed their situation as requiring additional time to achieve pregnancy or as necessitating to take action in short- to medium-term (p<.001). Additionally, a minority of DOR1 participants and none of DOR2 believed it was safe to postpone motherhood (p=.015 for DOR1 and NOR, and p=.040 for DOR2, INT and NOR). The interventions did not lead to an increase in anxiety. Anxiety levels showed no significant differences after the key interventions (p=.714 for STAI-S and p=.104 for STAI-T). The interaction between time and groups formed according to the information received also showed no significant differences for STAI-S (p=.272 for DOR1 and NOR, and p= 558 for DOR2, INT, and NOR), nor for STAI-T (p=.344 for DOR1 and NOR, and p=.744 for DOR2, INT, and NOR). The majority of participants (58.1%) rated the evaluation of ovarian reserve as extremely important, and an even larger majority (75.8%) reported having undergone it at the appropriate time in their lives, as noted at the final assessment point of this study. The subsequent tasks (2 and 3) focused on strategic actions to potentially manage DOR after its diagnosis had been established. The study of genetic variants, particularly in gonadotropin receptors, has been suggested to understand situations where there are alterations in ovarian reserve, as well as to optimize the response to controlled ovarian stimulation. Single nucleotide polymorphisms (SNPs) in receptors such as FSHR, luteinizing hormone receptor (LHR), and ESR are described as determinants of follicular development, but their association with ovarian function needs further exploration. From the literature review, it was concluded that the FSHR rs6166, ESR1 rs2234693, and ESR1 rs9340799 polymorphisms are among the most studied in relation to ovarian function. In contrast, polymorphic variation of the LHR is poorly associated with reproductive outcomes. The association of FSHR rs6166 with the response to controlled ovarian stimulation (COS) is extensively studied, with the Ser680 allele often associated with poorer outcomes compared to the Ans680 allele. Although the association of FSHR rs6166 with ovarian reserve markers is less documented in the literature, its potential role in regulating the PI3K-Akt pathway, and affecting follicular activation, remains a topic of investigation and debate. The literature review allowed us to understand that ESR polymorphisms in ovarian function are poorly documented, although there is evidence of the involvement of ESR1 rs2234693 and ESR1 rs9340799 in follicular development and response to COS. The impact of FSHR rs6166, ESR1 rs2234693, and ESR1 rs9340799 polymorphisms in infertile populations with altered ovarian reserve was evaluated in our study, a retrospective observational study. We found no statistically significant differences in the distribution of FSHR rs6166 (p=.807), ESR1 rs2234693 (p=.633), and ESR1 rs9340799 (p=0.672) genetic variants between DOR (N= 88) and the control group (N=219). Similarly, among women under 35 years old, there were no significant differences in the distribution of FSHR rs6166 (p=.301), ESR1 rs2234693 (p=.582), and ESR1 rs9340799 (p=.593) variants, between DOR (N=27) and the control group (N=112). In addition to DOR, PCOS is also an ovarian disorder that impairs female fertility by disrupting normal follicular development. Accordingly, DOR and PCOS are hypothesized to share common dysregulations in key factors affecting this process. In the PCOS population, previous studies showed conflicting data regarding the relationship between FSHR and ESR1 polymorphisms and this condition. Our study found no significant association in the genotype distribution of FSHR rs6166 (p=.522) and ESR1 rs2234693 (p=.697) between PCOS patients (N=88) and controls (N=80). However, the SS genotype of the FSHR rs6166 polymorphism was associated with higher FSH levels on the third day of the menstrual cycle (p=.011). No other differences in AMH, or AFC were found among different FSHR rs6166 and ESR1 rs2234693 genotypes. To deepen the study of the population with DOR, we reviewed the existing literature and have identified Forkhead Box L2 (FOXL2), Forkhead Box O3 (FOXO3), Factor in the Germline Alpha (FIGLA), Phosphatase and Tensin Homolog (PTEN), Insulin-like Growth Factor 1 (IGF1) and Insulin-like Growth Factor 2 (IGF2), Growth Differentiation Factor 9 (GDF9), Bone Morphogenetic Protein 15 (BMP15) and AKT Serine/Threonine Kinase 1 (AKT1) as potential targets, establishing a gene panel for analysis using NGS. This approach avoids the bias of an infertile population and is applicable to blood samples collected during AMH measurement, from participants of Task 1. In summary, the study of genetic variants in both infertile populations and, in the future, in a non-infertile population with altered ovarian reserve aims to identify targets for tailored therapies, such as pharmacogenetics, and improve the response to ovarian stimulation of follicles sensitive to gonadotropins, as well as to provide a more accurate diagnosis of these cases. Additionally, new therapies have been suggested to activate primordial follicles, targeting populations with DOR where COS alone is insufficient. To explore this hypothesis, Task 3 involved optimizing a protocol to obtain PRP and investigating its impact on a pathway involved in follicular activation. PRP has emerged as a promising therapy in regenerative medicine, and its applications have been extended to other areas, including reproductive medicine. However, the lack of standardization in PRP preparation protocols presents a challenge in achieving reproducible and accurate results. We aimed to optimize the PRP preparation protocol by investigating the impact of two different anticoagulants, sodium citrate (SD) and ethylenediamine tetraacetic acid (EDTA), and resuspension media, plasma versus sodium chloride (NaCl). Platelet recovery rate (PRR) was calculated and compared between groups, in addition to platelet activity and vascular endothelial growth factor (VEGF) released into plasma after PRP activation. The PRR was higher with EDTA in comparison to SC (51.04%vs29.85%, p=.005). Platelet activity was also higher, with a higher expression of two platelet antibodies, platelet surface P-38 Selectin (CD62p) and PAC-1 in the EDTA group. The concentration of VEGF was higher with SC in comparison to EDTA (628.73vs265.44 pg/mL, p=.013). PRR and VEGF levels were higher in PRP resuspended in plasma when compared to NaCl (61.60%vs48.61%, p=.011 and 41 363.32vs159.83 pg/mL, p=.005, respectively). After establishing a protocol starting with 15 mL of blood, centrifuged at 300g for 10 minutes followed by 700g for 15 minutes, we proceeded with an experimental in vitro study to evaluate the impact of PRP on human ovarian tissue fragments. Two culture times were tested, 4 and 48 hours (t=4h and t=48h) compared to the tissue evaluated immediately after thawing (t=oh). Additionally, the tissue was analysed with and without PRP (t=4h Vs t=4h+PRP and t=48h Vs t=48h+PRP). Throughout the study, tissue necrosis increased significantly from t=0h to t=48h (p<.001), starting at the 4-hour mark (t=0h vs t=4h, p<.002), but plateaued beyond the initial 4 hours (t=4h vs t=48h, p=.71). Follicular degeneration increased significantly from t=0h to t=48h (p<.001) and from t=4h to t=48h (p<.001), with no significant change between t=0h and t=4h (p=.262). Interstitial edema increased significantly over time, from 4 to 48 hours (p<.001), with no difference between t=0h and t=4h (p=.070). Ki-67 positive follicles increased significantly from t=0h to t=48h (p=.002), while no variation was observed in Ki-67 positive stromal cells (p=.716). Caspase 3 positive follicles and stromal cells showed no significant differences across the three time-points (p=.851 and p=.663, respectively). The vascular density, number of blood vessels per square millimeter (mm2), and the vascular area, the percentage of stromal cells positive for Factor VIII (FVIII) also did not show significant changes over time (p = .937 and p = .284, respectively). PRP supplementation in the culture media significantly impacted the stromal characteristics of ovarian tissue. Tissue necrosis, show a decrease at 4-hour mark (p=.002), while follicular degeneration and interstitial edema decreased with supplementation with PRP, at 48- hour mark (respectively, p=.002 and p<.001). Ki-67 staining increased in stromal cells with PRP supplementation (p<.001) at both 4 hours (p<.001) and 48 hours (p=.007), when compared with the group without PRP. Also, a significant increase in vascular area was noted with PRP supplementation (p=.014), specifically at the 48-hour culture period (p=.024). PRP had no impact on the percentage of caspase 3 positive follicles and stromal cells (p=.898 and p=.986, respectively), nor on vascular density (p=.272). p-Akt expression showed a significant reduction in weak staining and an increase in intense staining over time (both p<.001), with these effects amplified by PRP at both 4 hours (p=.003) and 48 hours (p=.034). In conclusion, this study aimed to address unanswered aspects of DOR, developing strategies from enhancing knowledge of its impact on fertility, to exploring potential post-diagnosis therapies. Investing in information on this topic has proven effective in increasing knowledge, and we advocate for continuing these efforts. Tailored information did not result in any side effects, particularly in terms of post-intervention anxiety. However, the question of its overall value remains to be clarified. This could be addressed in a future follow-up study to better understand its long-term impact on reproductive plans. NGS analysis aligns with our goals to investigate targets for focused diagnosis and personalized therapy in DOR cases, as well as elucidating the condition's aetiology. Lastly, while some details may need further exploration to support the in vitro study of ovarian tissue culture with PRP, we have gained valuable insights into its effect as a stimulant of the PI3K-Akt follicular activation pathway.