Patients Undergoing IVF Treatment Research Articles

Optimizing oocyte yield utilizing a machine learning model for dose and trigger decisions, a multi-center, prospective study

The objective of this study was to evaluate clinical outcomes for patients undergoing IVF treatment where an artificial intelligence (AI) platform was utilized by clinicians to help determine the optimal starting dose of FSH and timing of trigger injection. This was a prospective clinical trial with historical control arm. Four physicians from two assisted reproductive technology treatment centers in the United States participated in the study. The treatment arm included patients undergoing autologous IVF cycles between December 2022–April 2023 where the physician use AI to help select starting dose of follicle stimulating hormone (FSH) and trigger injection timing (N = 291). The control arm included historical patients treated where the same doctor did not use AI between September 2021 and September 2022. The main outcome measures were total FSH used and average number of mature metaphase II (MII) oocytes. There was a non-significant trend towards improved patient outcomes and a reduction in FSH with physician use of AI. Overall, the average number of MIIs in the treatment vs. control arm was 12.20 vs 11.24 (improvement = 0.96, p = 0.16). The average number of oocytes retrieved in the treatment vs. control arm was 16.01 vs 14.54 (improvement = 1.47, p = 0.08). The average total FSH in the treatment arm was 3671.95 IUs and the average in the control arm was 3846.29 IUs (difference = −174.35 IUs, p = 0.13). These results suggests that AI can safely assist in refining the starting dose of FSH while narrowing down the timing of the trigger injection during ovarian stimulation, benefiting the patient in optimizing the count of MII oocytes retrieved.

P-095 Towards deciphering the molecular architecture of the human sperm connecting piece

Abstract Study question How can we make the human sperm connecting piece accessible for structural analysis by cryo-electron tomography (cryo-ET)? Summary answer Preparation of sperm by chemical thinning or production of lamellae via cryo-focused ion beam (cryo-FIB) milling allows direct imaging of the human sperm connecting piece. What is known already Previous studies from our group and others have demonstrated the versatility of cryo-ET to advance our understanding of sperm biology. Structures of macromolecular complexes have been resolved in vitro and in situ, enabling a functional assessment at molecular or even atomic level. Resolving structures at high resolution further allowed identification of proteins previously not known to have a role in mediating male fertility. Such insights are instrumental to further improve diagnosis and treatment of male infertility. Study design, size, duration In our research study we use sperm samples from patients undergoing IVF treatment or donor material, which we receive through an ongoing collaboration with the Division of Woman and Baby, University Medical Centre Utrecht (UMCU). These samples are processed in our own laboratory and subsequently imaged by cryo-ET at the Electron Microscopy Centre of Utrecht University. Participants/materials, setting, methods Due to its thickness, the connecting piece cannot be directly analyzed by cryo-ET. Thus, we designed two strategies to achieve thinning of this area and allow collection of tomograms for structural analysis. While the first approach involves incubation of sperm with a detergent, a reducing agent and heparin, the second approach utilizes cryo-FIB milling to produce thin lamellae of the connecting piece. Main results and the role of chance Both strategies developed to access the connecting piece with cryo-ET are effective and allowed us a glimpse of the molecular landscape of the link between sperm head and tail. Chemical thinning of the connecting piece enabled imaging of the proximal and distal centriole while still embedded in the segmented columns or outer dense fibers, respectively. Furthermore, electron density within the microtubules of both centrioles indicates that they are decorated by microtubule inner proteins. Data obtained from lamellae produced by cryo-FIB milling complements these findings, displaying regular densities along the microtubules extending from the proximal centriole. Analyzing tomograms collected from cryo-FIB lamellae we further found a regular protein density that appears to be connecting the nuclear envelope to the base plate. Moreover, cryo-ET of lamellae allowed a detailed view of nuclear pore complexes and the postacrosomal sheath in their native environment. Limitations, reasons for caution Altering the native state of the sperm cell by chemical preparation is a limitation of this approach. Cryo-FIB milling retains the native state of the cell but is limited in throughput. To improve our cryo-FIB milling approach, we will implement automated milling procedures. Wider implications of the findings Chemical preparation and cryo-FIB milling of sperm enable the in-depth analysis of the connecting piece by cryo-ET. These strategies can now be applied to provide structural details at the molecular level and thus advance our understanding of male infertility related to defects of the head-tail junction. Trial registration number not applicable

P-086 Consequences of elevated levels of DNA fragmentation and its influence on pregnancy – A multicentric cohort analysis

Abstract Study question Does an abnormal DNA Fragmentation Index impact the clinical outcomes of patients undergoing IVF treatment? Summary answer Statistical differences were seen in biochemical, implantation, clinical, and ongoing pregnancy rate in sperms with high and low DFI scores with autologous and donated oocytes. What is known already Sperm chromatin integrity is associated with embryo development and clinical outcomes of an IVF cycle. DNA Fragmentation Index (DFI) is a diagnostic tool for the detection of spermatozoa with damaged chromosomes in the ejaculate. DNA fragmentation is a significant cause of day 3 embryo arrest and the development of poor-quality embryos. The interventions associated with elevated levels of DFI are ICSI, lifestyle changes and antioxidant therapies. However, the published data regarding the potential of embryo development with high DFI is limited to the sample size thus, encouraging a need for more research in this area. Study design, size, duration The study is population-based and included 768 male patients who underwent DFI score detection across 44 centers at Indira IVF Hospital Private Limited from January 2023 to September 2023. The abstinence period for all the patients was recorded during the DFI detection. After the DFI assessment patients were classified into normal DFI score ( < =15%) (n = 203) and abnormal DFI score (>15%) (n = 565). Participants/materials, setting, methods The retrospective cohort analysis was performed on our databank on the male patients regardless of their age (21-50), ethnicity, and medical history. DFI was calculated using a Sperm Chromatin Dispersion assay. Patients were categorized into two groups: Normal DFI ( < =15%) and Abnormal DFI (>15%). The characteristics of population along with clinical outcomes were evaluated after transferring embryos fertilized with normal DFI sperm and abnormal DFI sperm. The data was analyzed and estimated by SPSS software. Main results and the role of chance Our analyses discovered that embryos fertilized with sperms of normal DFI score have significantly enhanced the likelihood of implantation compared with embryos fertilized with sperms of abnormal DFI (50.27% v/s 44.40%; p = 0.047). Simultaneously, early pregnancy was evaluated by determining the values of beta-HCG in blood, also known as biochemical pregnancy rate (BPR). The embryos fertilized with sperms of normal DFI score significantly enhanced the BPR compared with embryos fertilised with sperms of abnormal DFI (67% v/s 59.1%; p = 0.048). Clinical Pregnancy Rate (CPR) defined as presence of a heartbeat of gestational sac observed during the 6-week scan. Furthermore, it was evaluated that the CPR showed a significant enhancement in embryos fertilized with sperms of normal DFI score in comparison with embryos fertilized with sperms of abnormal DFI (65.5% v/s 57.9%; p = 0.034). However, it was noticed that male DFI score had no impact on the miscarriage rate of embryos inseminated with normal DFI score and abnormal DFI score (7.52% v/s 7.34%; p = 0.687). Moreover, the pregnancy was tracked, and it was revealed that embryos inseminated with sperms of normal DFI score significantly enhanced the ongoing pregnancy till 12 weeks compared with embryos inseminated with sperms of abnormal DFI score (60.6% v/s 53.6%; p = 0.039). Limitations, reasons for caution Embryos are known to have a self-correct mechanism to correct the presence of mosaicism in the embryos. Hence, the oocyte quality also affects the outcomes of the IVF cycle. Therefore, using a high grade of the oocyte for the patients with abnormal DFI score can be useful. Wider implications of the findings Our data suggest that abnormal DFI have an impact on the clinical outcomes of an IVF cycle. Therefore, examining the levels of DNA Fragmentation is utmost imperative for a successful pregnancy. Trial registration number not applicable

Oocyte retrieval in social fertility preservation: challenging traditional beliefs on gonadotropin dosing and follicular size

ObjectivesTo investigate treatment approaches for fertility preservation patients, with a focus on timing of oocyte retrieval, and to determine whether their characteristics differ from those of other IVF patients. Additionally, to evaluate the significance of follicle size on triggering day in the context of fertility preservation.MethodsThis retrospective cohort study was conducted in a tertiary, university-affiliated medical center. It compared 140 matched patients undergoing social fertility preservation to 140 patients undergoing IVF treatment due to male factor infertility.ResultsPatients undergoing fertility preservation received a higher initial gonadotropin dose and had more oocytes retrieved than the control group. Within the fertility preservation cohort, a negative correlation was observed between the rate of large follicles and the number of retrieved oocytes. While there was no significant association between rate of large follicles and oocyte maturation rate in the entire group, age-stratified analysis revealed a negative relationship. Analysis revealed that although traditional treatment determinants such as follicular size and gonadotropin dosing were considered, peak estradiol levels were consistently identified as significant predictors of treatment outcomes.ConclusionsPhysicians may modify treatments for fertility preservation, emphasizing a higher gonadotropin dosage to maximize oocyte retrieval. Elevated estradiol levels can serve as a real-time predictive marker for the number of mature oocytes. While treatment strategies can influence outcomes, intrinsic patient factors, particularly baseline ovarian function, remain crucial. These results challenge beliefs regarding the importance of larger follicles and suggest the need for a tailored approach, considering patient age and specific fertility preservation objectives.

EGF-like growth factors upregulate pentraxin 3 expression in human granulosa-lutein cells

The epidermal growth factor (EGF)-like factors, comprising amphiregulin (AREG), betacellulin (BTC), and epiregulin (EREG), play a critical role in regulating the ovulatory process. Pentraxin 3 (PTX3), an essential ovulatory protein, is necessary for maintaining extracellular matrix (ECM) stability during cumulus expansion. The aim of this study was to investigate the impact of EGF-like factors, AREG, BTC, and EREG on the expression and production of PTX3 in human granulosa-lutein (hGL) cells and the molecular mechanisms involved. Our results demonstrated that AREG, BTC, and EREG could regulate follicular function by upregulating the expression and increasing the production of PTX3 in both primary (obtained from 20 consenting patients undergoing IVF treatment) and immortalized hGL cells. The upregulation of PTX3 expression was primarily facilitated by the activation of the extracellular signal-regulated kinase 1 and 2 (ERK1/2) signaling pathway, induced by these EGF-like factors. In addition, we found that the upregulation of PTX3 expression triggered by the EGF-like factors was completely reversed by either pretreatment with the epidermal growth factor receptor (EGFR) inhibitor, AG1478, or knockdown of EGFR, suggesting that EGFR is crucial for activating the ERK1/2 signaling pathway in hGL cells. Overall, our findings indicate that AREG, BTC, and EREG may modulate human cumulus expansion during the periovulatory stage through the upregulation of PTX3.

Acrosin activity negatively influences the cumulative live birth rate in patients undergoing IVF treatment

Research questionIs acrosin activity related to cumulative live birth rate (CLBR) over 1 year after IVF, intracytoplasmic sperm injection (ICSI) treatment or both? DesignRetrospective monocentric cohort study of 5704 couples who started IVF/ICSI treatments between 2016 and 2021. Acrosin activity was determined by a modified Kennedy method using a commercial kit. Patients were divided into two groups according to their acrosin activity: below 25 μIU/106 spermatozoa; and an acrosin activity 25 μIU/106 spermatozoa or above. Primary outcome was the CLBR, defined as an ongoing pregnancy leading to live birth that had arisen from all embryo transfers carried out within 1 year after the first ovum retrieval. Both conservative and optimistic methods were used for estimating CLBRs. ResultsThe CLBRs of patients with an acrosin activity below 25 μIU/106 spermatozoa were found to be significantly lower than those of patients with an acrosin activity 25 μIU/106 spermatozoa or above by conservative (48.5% versus 55.4%, P = 0.02) and optimistic (63.7% versus 70.3%, P = 0.047) methods after adjusting for confounders. When acrosin activity was regarded as a continuous variable, significant negative relationships between acrosin activity and CLBR were identified in subgroups: young couples (men and women aged younger than 30 years) and couples from whom no more than 10 eggs were retrieved. ConclusionLow acrosin activity levels were correlated with decreasing CLBRs over 1 year. These findings suggest that acrosin activity can be used as a predictor for CLBRs before starting IVF/ICSI treatment to enhance the effectiveness of counselling.

Intra-ovarian inflammatory states and their associations with embryo quality in normal-BMI PCOS patients undergoing IVF treatment

BackgroundPolycystic ovary syndrome (PCOS) is the main cause of anovulatory infertility in women of reproductive age, and low-grade chronic inflammation plays a key role in the occurrence and development of PCOS. However, obesity, as a likely confounding factor, can affect the inflammatory state of PCOS patients.ObjectiveThe aim of this study was to comprehensively investigate intra-ovarian inflammatory states and their impact on embryo quality in PCOS patients with a normal BMI undergoing IVF treatment.MethodsDIA-mass spectrometry-based proteomics and bioinformatic analysis were combined to comprehensively profile the protein expression of granulosa cells (GCs) from 5 normal-BMI PCOS patients and 5 controls. Thirty-four cytokines were further systematically detected in follicular fluid (FF) from 32 age- and BMI-matched normal-BMI patients using Luminex liquid chip suspension technology. Next, the differentially expressed cytokines were evaluated by enzyme-linked immunosorbent assay (ELISA) in 24 newly recruited subjects, and the relationship between these cytokines and embryo quality in PCOS patients was analysed. Finally, these cytokine levels were compared and evaluated in PCOS patients with different androgen levels.ResultsProteomic analysis showed that the suppression of substance metabolism and steroid biosynthesis, more interestingly, resulted in an enhanced immune and inflammatory response in the GCs of normal-BMI PCOS patients and prompted the involvement of cytokines in this process. Luminex analysis further showed that FF macrophage inflammatory protein-1 beta (MIP-1β) and stromal cell-derived factor-1 alpha (SDF-1α) levels were significantly increased in normal-BMI PCOS patients compared to controls (P = 0.005; P = 0.035, respectively), and the ELISA results were consistent with these findings. Besides, FF MIP-1β showed an inverse correlation with the number of D3 good-quality embryos and the good-quality blastocyst rate in patients with PCOS (P = 0.006; P = 0.003, respectively), which remained significant after correction for multiple comparisons. Moreover, SDF-1α levels had no relationship with embryo development in PCOS patients. Additionally, SDF-1α levels were significantly lower in PCOS patients with high androgen levels than in controls (P = 0.031).ConclusionsLocal ovarian inflammation was present in normal-BMI PCOS patients, affecting follicular development, and FF MIP-1β may be a potential biomarker associated with embryo quality in normal-BMI PCOS patients.Graphical abstract

Role of thyroid stimulating hormone in the maintenance and functioning of the human corpus luteum.

To evaluate the impact of high thyroid stimulating hormone (TSH) levels on human granulosa-luteal (hGL) cells. hGL cells were isolated from follicular aspirates derived from patients undergoing IVF treatment without any thyroid disorder (serum TSH 0.5-2mU/L). Cells were cultured at 37°C in DMEM, supplemented with 5% FBS. The cells were treated with 1nM LH and increasing concentrations of TSH. At the end of culture, conditioned medium and cells were collected to analyze progesterone production, cell viability, and mRNA levels of genes involved in the steroidogenesis process. Human ovarian tissues were analyzed for TSH receptor (TSHR) expression by IHC. The expression of TSHR was detected in human corpus luteum by IHC and in hGL by RT-PCR. In hGL cells, TSH treatment did not modulate progesterone production nor the expression of steroidogenic genes, such as p450scc and HSD3b 1/2. However, TSH induced a dose-dependent increase in cell death. Finally, TSH did not affect LH-induced p450scc and HSD3b1/2 expression while LH partially reverted TSH negative effect on cell death in hGL. Elevated TSH levels in hypothyroid women may be associated with impaired CL functioning and maintenance. These findings open a new line of research for the importance of the treatment of women with thyroid dysfunction that could contribute to the onset of infertility.

The Role of Infertility Panel Testing with Next Generation Sequencing in a Cohort of Infertile Patients undergoing IVF Treatment

The Role of Infertility Panel Testing with Next Generation Sequencing in a Cohort of Infertile Patients undergoing IVF Treatment

Human granulosa cells of poor ovarian responder patients display telomeres shortening.

We aimed to compare the telomere length in granulosa cells of the young normal and poor ovarian responder patients and elderly patients undergoing ovarian stimulation for IVF. The main outcome measures granulosa cells telomere Length in the 3 study groups of patients undergoing IVF treatment in our center. 1) young normal responder patients (< 35years); 2) young (< 35years) poor ovarian responder patients; and 3) Elderly patients (40-45years). Granulosa cells were obtained at the time of oocyte retrieval. Granulosa cells telomere length was assessed by absolute human telomere length quantification qPCR Assay. The telomere length of the young normal responder was significantly longer as compared to young poor ovarian responder (15.5 vs 9.6KB, p < 0.001) and the elderly patients (15.5 vs 10.66KB, p < 0.002). No significant difference was observed in the telomere length between the young poor ovarian responder and the elderly patients. Granulosa cells telomere length of the young normal responder was found to be significantly longer than young poor ovarian responder or elderly patients, highlighting the role of telomere length as a predictor, or contributor to poor oocyte yield following IVF treatment.

A retrospective observational study to see the effect of dual trigger on IVF outcomes in poor ovarian responders

Background: Poor Responders Patients undergoing IVF treatment were given Dual Trigger for final oocyte maturation. Poor responders are the patients in whom, less number of egg were retrieved. Typically, they were with advanced maternal age and poor ovarian reserve (AFC <5-7 follicle or AMH < 1.2ng/ml) or with a history of previous Poor ovarian reserve (<3 oocytes) with Controlled ovarian stimulation. This study aimed to examine the effectiveness of dual trigger for final oocyte maturation in poor responder patients based on Bologna criteria (2011).Methods: A total of 30 IVF cycles of poor responder patients were retrospectively analyzed. The study group (15 patients) was given dual triggers (HCG and GnRH agonist) for final oocyte maturation whereas in control group final oocyte maturation was performed with HCG only. GnRH antagonist Protocol was used in both the groups.Baseline characteristics, number of oocyte retrieved, the number of M-2 and the quality of embryo of both groups were compared.Results: Both the groups were comparable in baseline characteristics. In our study, there was not much difference in number of oocyte retrieved, but there was higher number of M2 and top quality embryo, compared to control group.Conclusion: Dual trigger might be a superior option for final oocyte maturation as compared to hCG trigger alone in terms of IVF cycles outcomes in poor responders and further large scale randomized prospective studies needed to validate our results.

Cigarette smoke is associated with up-regulation of inducible NOS and COX-2 protein expression and activity in granulosa cells of women undergoing in vitro fertilization

Cigarette smoke exposure represents a well-established ovotoxic exogenous stress, but the molecular mechanisms underlying of this effect are still unclear. Cigarette smoke upregulates inflammatory genes in the female reproductive organs, therefore an abnormal inflammation response may contribute to the impairment of female fertility. In this study we investigated for the first time the effect of cigarette smoke exposure on NOS and COX expression and activity and on their transcription factors (CREB and NF-kB) in human GCs and on the release of NO and PGE2 in the FF in smoking and non-smoking patients undergoing IVF treatment. In addition, correlation analysis between AMH serum levels, an index of ovarian reserve, and smoking exposure or iNOS and COX-2 protein expression levels were performed using a Pearson correlation method. Cigarette smoke exposure resulted in a significant increase of iNOS and COX-2 protein expression together with an increase of iNOS activity and PGE2 levels. pNF-kB and pCREB protein expression were upregulated in the GCs of smokers compared to non-smokers. The habit of smoking was negatively correlated with serum AMH levels, and positively correlated with iNOS and COX-2 protein expression levels. The data presented in the current study revealed a novel molecular mechanism underlying the toxic effects of cigarette smoke on fertility. Additional pathways mediating the effects of cigarette smoke exposure in human GCs cannot be excluded and should be investigated in future studies.

Letrozole cotreatment with progestin-primed ovarian stimulation in women with polycystic ovary syndrome undergoing IVF treatment.

Background: Progestin is an alternative to gonadotropin-releasing hormone (GnRH) analogues in the follicular phase to suppress the premature luteinizing hormone (LH) surge in women with polycystic ovary syndrome (PCOS). However, progestin-primed ovarian stimulation (PPOS) is always accompanied by increased pituitary suppression and gonadotropin consumption. Previous studies suggested that letrozole appeared to have the potential to reduce the total gonadotropin dose required for ovarian stimulation. A retrospective cohort study was performed to evaluate the efficacy of PPOS with or without letrozole in infertile women with PCOS. Methods: This retrospective cohort study included 448 women with PCOS who underwent controlled ovarian stimulation (COS) with human menopausal gonadotropin (hMG) and medroxyprogesterone acetate (MPA) (n = 224) or hMG and MPA cotreatment with LE (n = 224) from January 2018 to March 2021 after propensity-score matching. The primary outcome measure was the hMG dose. The secondary outcomes were the durations of ovarian stimulation, the implantation rate, the number of oocytes retrieved and viable embryos, oocyte maturity and fertilization rates, the percentage of women with profound pituitary suppression (luteinizing hormone [LH] <1.0 IU/L on the trigger day). Results: The hMG doses (1949.89 ± 725.03 IU vs 2017.41 ± 653.32 IU, p > 0.05) and durations of ovarian stimulation (9.03 ± 1.79 days vs 9.21 ± 2.18 days, p > 0.05) were similar between the two groups. The implantation rate was significantly higher in the study group (MPA + hMG + LE) than in the control group (MPA + hMG) (42.22 vs 34.69%, p < 0.05). The numbers of oocytes and embryos retrieved were similar between the two groups. Interestingly, letrozole cotreatment was associated with decreased oocyte maturity and fertilization rates in comparison with standard PPOS protocols even though mature and fertilized oocyte yields were comparable. Compared with those in the control group, the LH values on the trigger day were significantly higher in the study group, together with significantly reduced pituitary suppression. Conclusion: Letrozole combined with PPOS cannot reduce hMG consumption in PCOS patients undergoing IVF treatment and shows no beneficial effect on cycle characteristics of COS. However, letrozole supplementation manifests as a superior implantation rate to that of the standard PPOS protocol in women with PCOS.

P-667 Follitropin delta for ovarian stimulation in daily practice in a Dutch IVF centre

Abstract Study question This study reports individualized follitropin delta dosing in a real life setting in patients undergoing IVF treatment. Summary answer The use of follitropin delta, in real-world clinical setting with a broad and varied patient population, shows results aligned with the ESTHER-1 registration trial (NCT01956110). What is known already Individualized follitropin delta dosing based on anti-Müllerian Hormone (AMH) and body weight (BW) is as effective as treatment with Chinese hamster ovary (CHO) derived follitropin alfa, and leads to a more predictable ovarian response and improved safety. Results of the ESTHER-1 trial show: similar pregnancy rates and less frequent excessive ovarian response compared to women treated with follitropin alfa. Moreover, the incidence of ovarian hyperstimulation syndrome (OHSS) was reduced, fewer poor responses occurred in women with AMH &amp;lt;15 pmol/L and fewer excessive responses in women with AMH ≥ 15 pmol/L. Study design, size, duration Descriptive analysis from real-world data obtained in the Erasmus University Medical Centre. Since its introduction in 2017, 2369 treatment cycles have been performed with follitropin delta in our center. In 499 patients, with a regular ovulatory menstrual cycle, follitropin delta was used in a GnRH-antagonist protocol in their first IVF cycle in 2017. Subsequent fresh embryo transfers were done on day 5. EMC patients were compared to patients in the ESTHER-1 trial using follitropin delta. Participants/materials, setting, methods Women are prescribed follitropin delta according to the dose algorithm based on the patient’s BW and serum AMH levels as measured by the automated Elecsys® AMH plus immunoassay (Roche Diagnostics). Results of the treatment were collected from the clinical records. The outcomes of the first IVF cycles with follitropin delta are reported. Data were expressed as mean (SD) or percentages and statistically analyzed using t-test and chi-square test. Main results and the role of chance EMC patients were significantly older (33.9 (4.5) vs. 33.4 (3.9) years (p &amp;lt; 0.01) and had higher AMH serum levels (21.5 (13.2) vs. 19.4 (14.6) pmol/l (p &amp;lt; 0.001) compared to those in the ESTHER-1 trial. Less patients started IVF in a naïve cycle (75.4% vs. 100% (p &amp;lt; 0.001) and BW was higher (68.6 (11.1) vs. 64.7 (10.7) kg (p &amp;lt; 0.01). Retrieved number of oocytes (10.6 (5.7) vs. 10.0 (5.6) (p &amp;lt; 0.05), number of cleavage stage embryos (6.1 (3.6) vs. 5.4 (3.7) (p &amp;lt; 0.001), number of cryopreserved embryos (2.3 (2.3) vs. 1.9 (2.4) (p &amp;lt; 0.001) and proportion of women with embryos cryopreserved (72.4% vs. 63.4% (p &amp;lt; 0.01) were all significantly higher in the EMC patients. Proportion of women with 8-14 oocytes was similar (47.3% vs. 43.3% of patients (NS). Women with AMH &amp;lt;15 pmol/L had more oocytes retrieved (8.9 (4.5) vs. 8.0 (4.3) (p &amp;lt; 0.01). Women with AMH ≥15 pmol//L had similar numbers of oocytes retrieved (11.4 (6.0) vs. 11.6 (5.9) (NS). More cycles were cancelled with an insufficient number of follicles in the overall population (7.4% vs. 3.8% (p &amp;lt; 0.01). Hospitalization due to OHSS was similar (0.6% vs. 0.3% (NS). Positive hCG test (40.7% vs. 38.6% (NS) and ongoing pregnancy rate per started cycle (32% vs. 30.7% (NS) were similar. Limitations, reasons for caution This is a retrospective analysis of the use of follitropin delta in patients in daily clinical practice. As this population was drawn from daily clinical practice, it is a heterogeneous population in which no strict in- or exclusion criteria were applied. Wider implications of the findings Individualized follitropin delta treatment of a broad patient population as presented here shows clinically similar results in ovarian response compared to the previously reported ESTHER-1 trial. Moreover, pregnancy rates were also similar even though patient characteristics were slightly different. Trial registration number not applicable

P-595 Heterogeneity of preovulatory follicle cell types between normo- and hyporesponders

Abstract Study question Is there a difference in the proportions of preovulatory follicle cell types between normo- and hyporesponders? Summary answer Human preovulatory follicles consist of 14 distinct cell types, 3 of them are significantly different between normo- and hyporesponder patients. What is known already Human ovarian follicles are a diverse dynamic environment for oocytes to mature and ovulate. Follicles consist of multiple cell types in layers and altogether they are responsible for a repertoire of biological processes. Dysregulations in follicular cells could result in hyporesponsiveness to ovarian stimulation. Hyporesponse might lead to the failure of IVF, due to a low number of retrieved oocytes. Its pathophysiological mechanisms have not been fully elucidated so far. This condition affects up to 25% of patients and investigating their follicular cell populations in detail is highly informative for increasing their prognosis for the live birth outcome. Study design, size, duration Study groups consisted of IVF patients with normoresponse (n = 10) and hyporesponse (n = 9) to rFSH stimulation according to antagonist protocol undergoing in vitro fertilization treatment at Nova Vita Clinic, Estonia. Hyporesponse to treatment was diagnosed if ≥ 200 IU of rFSH was administered to retrieve an oocyte. All study participants were ≤40 years of age with matched BMI and normal ovarian reserve. Women with polycystic ovary morphology and ovarian abnormalities observed by ultrasound examination were excluded. Participants/materials, setting, methods Oocyte pick-up was performed for patients after 36 hours of hCG trigger. Cumulus-oocyte complex was removed from the follicular fluid for IVF and the remaining cells were collected by centrifugation and treated with hyaluronidase and DNase. Genome-wide single-cell RNA-seq was performed for &amp;gt;6000 individual cells of 3 normoresponder patients each. RNA-seq was performed for pooled follicular cells of each participant. CIBERTSORTx software was used to deconvolute the proportion of cell types from bulk RNA-seq data. Main results and the role of chance By analyzing a total number of 24 213 single cells from preovulatory follicles, we identified 4 immune cell lineages (CD45+), including neutrophils, T cells, M1 and M2 macrophages along with 10 non-immune cell lineages (CD45-): epithelial cells and theca cells, cumulus cells and 7 subtypes of granulosa cells with different functional transcriptomic profiles (p &amp;lt; 0.05). We identified granulosa cell subclusters that were highly active in progesterone or estrogen production, extracellular matrix remodeling, ovulational process, cell migration, and apoptosis. Comparing the proportion of cell populations between normo- and hyporesponders from deconvoluted RNA-seq data (adjusted to age), we identified that 3 cell clusters varied with statistical significance (p &amp;lt; 0.05). These detected clusters with different proportions between the studied patient groups suggest their role in contributing to hyporesponse in a preovulatory follicle regarding patients undergoing IVF treatment. Limitations, reasons for caution The small sample size limits the study. Wider implications of the findings Our work advances understanding the heterogeneity of cell types in human preovulatory follicles. The differences in the proportions of cell types of preovulatory follicles of hyporesponders could provide valuable insight for assisting IVF treatment by introducing potential therapeutic targets to improve their live birth outcome. Trial registration number not applicable

P-570 Prokineticin-1 follicular fluid levels are strongly associated with diminished ovarian reserve and poor ovarian response: A prospective observational study

Abstract Study question Are Prokineticin-1 (PROK1) follicular fluid (FF) levels associated with poor ovarian response (POR) as well as with other well-known biomarkers associated with ovarian reserve status? Summary answer Poor responders present with increased PROK1 FF levels. Also, PROK1 FF levels are negatively correlated with anti-müllerian hormone (AMH) levels and number of oocytes retrieved. What is known already Prokineticin-1 is an angiogenic factor with pleiotropic properties. Considering its role in reproduction, PROK1 mediates significant angiogenic functions in the fetal-maternal interface. Thus, alterations in PROK1 expression and/or function lead to placenta-derived pregnancy complications. Recently published studies also indicate that PROK1 is associated with ovarian function and oocyte competence. However, limited data associating PROK1 with POR are published. This study uniquely investigates possible associations between PROK1 FF levels in POR cases, with stimulation outcome, as well as with FF levels of other factors related to ovarian functionality, namely VEGF, bone morphogenetic protein 15 (BMP-15) and Pigment Epithelium Derived Factor (PEDF). Study design, size, duration This prospective-observational study was collaboratively conducted between November 2020 and July 2021 at Genesis Athens Clinic and at the University of Athens Medical School. A total of 64 patients undergoing IVF treatment were enrolled. The study group comprised of 32 POR patients defined according to the Bologna criteria. The control group consisted of 32 normal responder women undergoing IVF due to tubal factor and/or mild male factor infertility. Patients with other infertility aetiologies were excluded. Participants/materials, setting, methods Participants in both groups received the standard short GnRH-antagonist protocol. Prior to ovarian stimulation, participants were subjected to basic infertility investigation, including antral follicle count and AMH levels evaluation. The FF samples were collected as part of the oocyte retrieval process, then centrifuged and stored at -80 °C till analysis. Follicular fluid levels of PROK1, VEGF, BMP-15 and PEDF were evaluated via ELISA employing commercially available kits. Statistical analysis was performed employing R Programming Language. Main results and the role of chance Significantly higher PROK1 (3229.63 ± 2372.66 vs 1945.95 ± 1408.01 pg/ml; P-value&amp;lt;0.0001), VEGF (2309.63 ± 412.49 vs 2013.22 ± 330.16 pg/ml; P-value=0.006) and lower BMP-15 (434.13 ± 106.38 vs 532.5 ± 108.26 pg/ml; P-value=0.001) levels were recorded in the POR group. No difference was observed regarding PEDF levels (7.43 ± 1.95 vs 8.23 ± 2.54 ng/ml; P-value=0.2). However, the POR group presented with lower PEDF/VEGF ratio (3.33 ± 1.08 vs 4.18 ± 1.41; P-value=0.02), indicating a reduced antioxidant capacity. PROK1 levels were negatively correlated with AMH (P-value=0.04), number of oocytes retrieved (P-value=0.001) and number of MII oocytes (P-value=0.005). BMP-15 was positively correlated with number of oocytes retrieved (P-value&amp;lt;0.001), number of MII oocytes (P-value=0.002), number of normally fertilized zygotes (P-value=0.007), number of cleavage stage embryos (P-value=0.03) and number of blastocysts (P-value=0.04). VEGF was negatively correlated with AMH (P-value=0.004) and number of oocytes retrieved (P-value=0.03). These correlations remained significant when adjusting for ovarian response status. PROK1 levels, with a cut-off value of 2854.25 pg/ml, were able to predict ovarian response status with an area under the curve at 0.64. Sensitivity was 0.55, specificity was 0.88, and accuracy was 0.71. The positive and negative predictive values were 81.82% and 66.67%, respectively. Limitations, reasons for caution Limitations of our study refer to the limited size of the studied population, as well as to the lack of data considering pregnancy outcomes. Moreover, molecular data with regards to the possible role of PROK1 on POR pathogenesis are required. Future studies are needed to verify the results presented herein. Wider implications of the findings Data presented herein indicate that PROK1 FF levels are strongly associated with diminished ovarian reserve and POR. Considering that FF-PROK1 presents with a similar profile with FF-VEGF, we can form the hypothesis that the compromised angiogenesis observed in POR patients leads to PROK1 and VEGF increase via a negative-feedback-loop. Trial registration number Not applicable

O-173 Artificial intelligence blastocyst ploidy distinction through morphokinetics data

Abstract Study question Is it possible to estimate blastocyst ploidy with an artificial intelligence (AI) algorithm built using embryo morphokinetics data? Summary answer AI was able to estimate blastocysts status in euploid or aneuploid with an accuracy of 0.99 (training) and 0.71 and 0.70 AUC (blind-test), respectively. What is known already Morphokinetic parameters are well associated with implantation rates, thus ensuring time-lapse as a useful tool to enhance embryo ranking and selection. However, regarding embryo ploidy status, clinics still rely exclusively on molecular/genetic testing. Our previous studies has indicated that euploid blastocysts are faster at time of pronucleous fading (tPNf) and time to blastulation (tB). Considering the amount of data derived from the morphokinetic annotations and the promising results that AI approach is bringing to the field, this study aims to verify AI accuracy to distinguish euploid and aneuploidy blastocysts using 17 morphokinetics parameters and prospectively compared to embryo biopsy results. Study design, size, duration This is a prospective cohort study including 402 embryos (cultured in a time-lapse incubator, EmbryoscopePlus, Vitrolife) from 140 patients undergoing IVF treatment with preimplantation genetic testing for aneuploidy (NGS platform) after inform consent form signature between July 2019 and September 2021. Morphokinetics annotations were analyzed by AI in an association of the technique of artificial neural networks (ANN) and genetic algorithms (GA) for ploidy assessment. Participants/materials, setting, methods Morphokinetic parameters were manually annotated up to full-expanded blastocyst time. Time intervals and time ratios were calculated, resulting in 17 variables for the IA analysis. Of the 402 embryos data, 252 were randomly divided into training, validating and testing (70%, 15% and 15%, respectively) of the ANN. The remaining 150 data were used for the blind test. The area under the curve (AUC) of the receiver operating characteristic curve was measured to obtain predictive power. Main results and the role of chance From 402 blastocysts biopsied, 185 were euploid and 217 aneuploid. The AI algorithm was trained with 176 embryos (AUC for both euploid and aneuploid= 0.99), tested with 38 embryos (AUC for euploid=0.62 and aneuploid=0.61), validated with 38 embryos (AUC for euploid=0.82 and aneuploid=0.83). For the blind-test 150 embryos were used (AUC for euploid=0.70 and aneuploid=0.71). Blind-test database was checked only after AI algorithm was tested. Limitations, reasons for caution The development of this AI algorithm was built from a single IVF center database. The absence of an extent dataset for the blind-test does not allow us to transpose this algorithm for clinical use at this moment. Wider implications of the findings The use of artificial intelligence for embryo assessment is a promising tool in IVF laboratories. In our model, using only morphokinetics, a considerable predictive power to evaluate euploid (0.71 AUC) and aneuploid (0.70 AUC) embryos was achieved, indicating a potential use as a non-invasive approach for embryo ranking and selection. Trial registration number Not applicable

P-571 The newly developed highly sensitive reagent GL13 indicates extensively increased cellular senescence in the follicular fluid of poor responder patients: A prospective observational study

Abstract Study question Do women of diminished ovarian reserve and poor ovarian response (POR) present with increased lipofuscin follicular fluid (FF) levels, indicating increased ovarian cellular senescence? Summary answer Poor responders present with a six-fold increased lipofuscin FF levels indicating extensive senescence in POR ovaries. Lipofuscin may be a new sensitive biomarker for POR. What is known already Lipofuscin is a nondegradable substrate of metabolism accumulating in cells due to impaired mitochondrial/lysosome/proteasome function, upon stress or damage. It is well-documented that lipofuscin constitutes a highly sensitive biomarker of cellular aging and senescence. Recently, a novel reagent (GL13) coupled by a hybrid histochemical–immunohistochemical method were developed for detecting and measuring soluble lipofuscin levels in biological fluids, ascertaining high sensitivity and specificity. Data indicate that lipofuscin levels are associated with oocyte competence and age-related infertility. However, hitherto no data has been published indicating the value of lipofuscin FF levels as a biomarker towards accurately predicting ovarian reserve and response. Study design, size, duration This prospective observational study was collaboratively conducted between November 2020 and September 2021 at the Athens University Medical School and at Genesis Athens Clinic. A total of 32 patients undergoing IVF treatment were enrolled. The study group comprised of 16 POR patients defined according to the Bologna criteria. The control group consisted of 16 normal responder women undergoing IVF due to tubal factor or/and mild male factor infertility. Patients with other infertility aetiologies were excluded. Participants/materials, setting, methods Participants in both groups received the standard short GnRH-antagonist protocol. The FF samples were collected as part of the oocyte retrieval process. Lipid and protein parts of lipofuscin were isolated from FF samples and stained with GL13. The complex of lipofuscin-GL13 was labeled with an anti-biotin HRP conjugated antibody and detected employing a chemiluminescence reaction. Luminescence was measured and signal intensity was corresponding to lipofuscin concentration. Statistical analysis was performed employing R Programming Language. Main results and the role of chance Poor responders presented with a statistically significant six-fold higher lipofuscin FF levels in comparison to the normal responder group (869.21 ± 501.87 vs 146.6 ± 107.64 RLU; P-value &amp;lt;0.0001). Lipofuscin levels were negatively correlated with AFC (Spearman’s Rho: -0.68; P-value &amp;lt;0.0001), AMH levels (Rho: -0.61; P-value =0.0002), estradiol levels on triggering day (Rho: -0.68; P-value &amp;lt;0.0001), number of oocytes retrieved (Rho: -0.54; P-value =0.001), number of mature metaphase II (MII) oocytes obtained (Rho: -0.58; P-value =0.0008), number of normally fertilized (2PN) zygotes (Rho: -0.51; P-value =0.003), number of cleavage stage embryos (Rho: -0.48; P-value =0.005) and number of blastocyst stage embryos (Rho: -0.41; P-value =0.02). These correlations remained statistically significant when adjusting for ovarian stimulation response status. No association was established between Lipofuscin levels and embryo quality neither on Day 3 nor on Day 5. Lipofuscin levels, with a cut-off value at 294, were able to predict ovarian stimulation response status with an area under the curve (AUC) at 0.96. The sensitivity was 0.875, the specificity was 0.938 and the accuracy was 0.906. The positive predictive value was 88.24% and the negative predictive value was 93.33%. Limitations, reasons for caution Limitations of our study refer to the limited size of the studied population, as well as to the lack of data referring to pregnancy outcomes. Moreover, molecular data with regards to the possible mechanisms leading to the observed increased senescence are required. Future studies are needed to verify these findings. Wider implications of the findings Data presented herein indicates, for the first time in literature, that lipofuscin FF levels measured via the GL13 method may be a promising and sensitive tool for predicting POR and stimulation outcome. Lipofuscin could further serve as a valuable novel biomarker indicating ovarian senescence, ovarian reserve status and oocyte competence. Trial registration number Not Applicable

Depression and suicidality amongst infertile women: a hidden pandemic?

IntroductionThe psychosocial impact of infertility has been well researched and documented. However very little research has been conducted to assess the causative relationship between infertility and serious psychiatric illness such as suicide.We conducted a cross-sectional study to investigate suicidal risk ( suicidal ideation/ suicide attempts) amongst 100 infertile women undergoing infertility treatment at an IVF Centre based in Kolkata, India. Allied parameters such as depression, anxiety, quality of life were also studied.ObjectivesThe goal of the presence study was to assess the suicidal risk ( suicidal ideation / attempted suicide ) amongst infertile women undergoing infertility/ ART treatment. And to examine the possible etiological factors behind increased levels of suicidal risk amongst infertile women.MethodsMini International Neuropsychiatric Interview was administered to 100 female patient’s undergoing IVF treatment and 100 control patient’s , visiting an IVF Centre based in Kolkata. Chi square test, independent t test and Z test used for statistical analysis.ResultsAccording to the results obtained by assessment of MINI scale , the prevalence of major depressive disorder (50 % ) followed by Dysthymia (25%) was documented among infertile group while suicidality at 15% were significantly higher than other disorder (Z = 3.80, p&gt; .001). No such cases of suicidality or Dysthymia was found among patients of control group. (fertile group).ConclusionsRoutine screening of suicidal risk and depression should be conducted for all patients undergoing IVF treatment. Infertility specialists should recognize psychiatric morbidity amongst infertile patients for subsequent referral and treatment.DisclosureNo significant relationships.

The BNT162b2 mRNA Covid-19 vaccine does not impair sperm parameters

Research questionDoes the BNT162b2 COVID-19 vaccine affect sperm parameters of patients with a normal or an abnormal semen analysis? DesignData were collected from male patients undergoing IVF treatment after completing vaccination between February 2021 and June 2021 (post-vaccine). For comparison, records of the same patients were reviewed before the vaccination (pre-vaccine) back to January 2017. Patients with azoospermia were excluded. Sperm parameters were compared between pre- and post-vaccine groups. Each patient served as self-control. ResultsSeventy-two patients were included in the study (median interquartile range [IQR] age 35.7 [33.0–43.0] years), of whom 57 had a normal semen analysis. The time between the first vaccine and the post-vaccine sperm analysis was 71.0 (40.5–104.8) days. The sperm parameters before and after the vaccination were as follows: sperm volume before 3.0 (2.0–4.0) and after 3.0 (1.6–3.9) ml, P = 0.02; sperm concentration before 26.5 (14.0–64.7) and after 31.0 (14.2–80.0) 106/ml, P = 0.35; and total motile sperm count before 33.7 (9.0–66.0) and after 29 (6.0–97.5)106, P = 0.96. Sub-group analyses were conducted for patients with male infertility and patients with a normal semen analysis. Neither of the sub-groups showed significant changes after vaccination. ConclusionSperm parameters showed no significant changes after vaccination among men with a normal and abnormal semen analysis. Therefore, the BNT162b2 vaccine does not seem to affect sperm parameters. The preliminary results are reassuring for the entire global population, currently undergoing intense vaccination campaigns against COVID-19.