Fertilization Rate Of Oocytes Research Articles

Implementation and Evaluation of Preimplantation Genetic Testing at Vilnius University Hospital Santaros Klinikos.

The most effective treatment of infertility is in vitro fertilization (IVF). IVF with Preimplantation Genetic Testing (PGT) allows to identify embryos with a genetic abnormality associated with a specific medical disorder and to select the most optimal embryos for the transfer. PGT is divided into structural rearrangement testing (PGT-SR), monogenetic disorder testing (PGT-M), and aneuploidy testing (PGT-A). This study mostly analyzes PGT-SR, also describes a few cases of PGT-M. The aim of this study was to implement PGT procedure at Vilnius University Hospital Santaros Klinikos (VUHSK) Santaros Fertility Centre (SFC) and to perform retrospective analysis of PGT procedures after the implementation. A single-center retrospective analysis was carried out. The study population included infertile couples who underwent PGT at SFC, VUHSK from January 01st, 2017 to December 31st, 2020. Ion PGM platform (Life Technologies, USA) and Ion ReproSeq PGS View Kit (Life Technologies, USA) were used for the whole genome amplification. Results were assessed using descriptive statistics. PGT was successfully implemented in VUHSK in 2017. During the analyzed time period, thirty-four PGT procedures were performed for 26 couples. Two procedures were performed in 2017, 7 procedures - in 2018, 13 - in 2019, and 12 - in 2020. In comparison with all IVF procedures, 2.5% procedures were IVF with PGT, a highest percentage was in 2020 (3.8% of all procedures). The main indication for PGT was balanced chromosomal rearrangements (in 85.3% cases). In all 34 cases 515 oocytes were aspirated in total, 309 oocytes were fertilized, oocytes fertilization rate exceeded 60%. A normal diploid karyotype was found in 46 (16.8%) biopsied embryos. Out of all PGT procedures, 9 (26.5%) resulted in a clinical pregnancy. Six (66.7%) pregnancies were confirmed in 2019, and 3 (33.3%) - in 2020. Three (33.3%) pregnancies resulted in spontaneous abortion, 6 (66.7%) - in delivery. The implementation of PGT in VUHSK was successful. The most common indication for PGT was a reciprocal translocation. Oocytes fertilization rate exceeded 60%, a normal karyotype was found less than in one-fifth of biopsied embryos. A highest clinical pregnancy rate was achieved in 2019 when almost half of women conceived, which is probably related to the experience gained by the multidisciplinary team. This is the first study analyzing IVF with PGT in Lithuania, however, the results should be interpreted with caution due to a low number of total procedures performed.

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-148 Denudation employing human recombinant hyaluronidase enzyme does not affect fertilization and blastocyst formation rates in sibling oocytes from patients undergoing ART: A prospective randomized study

Abstract Study question Does human recombinant hyaluronidase (Cumulase®) improve fertilization and blastocyst formation rates in sibling human oocytes compared to bovine-derived hyaluronidase? Summary answer The use of Cumulase® for oocyte denudation does not appear to affect fertilization or usable blastocyst formation rate in patients undergoing intracytoplasmic sperm injection (ICSI). What is known already Bovine-derived hyaluronidase has been widely employed to denude the cumulus-oocyte complexes (COCs) prior to ICSI or oocyte vitrification. Nonetheless, the recent concern for its safety, due to its animal origin, has led to the development of alternatives ascertaining the enzyme’s purity. It has been reported that human recombinant hyaluronidase is not equally effective as its bovine-derived analogue, leading to a longer time required for complete denudation coupled by the subsequent toxicity risk for oocytes. This may result to lower fertilization and blastocyst formation rates. Although there are some studies on IVF outcomes with recombinant hyaluronidase, its use remains rather limited. Study design, size, duration This randomized double-blinded clinical trial was conducted at Citmer Reproductive Medicine, in Puebla and Monterrey, Mexico from February to December 2021. The impact of denudation on sibling oocytes from 103 patients employing bovine-derived hyaluronidase (InVitroCare®) or human recombinant hyaluronidase (ICSI Cumulase® (rHuPH20), CooperSurgical®) was evaluated. A total of 1237 oocytes were assigned as follows: The Hyaluronidase Group included 626 oocytes, while the Cumulase® Group included 611 oocytes. Preimplantation Genetic Testing was performed in 18 cycles. Participants/materials, setting, methods After retrieval, COCs were incubated for 2-4 h and placed in microdropets with 10 IU/mL bovine-derived hyaluronidase or Cumulase® in culture media GlobalTotal® (LifeGlobal®) for granulosa cells removal. COCs were gently pipetted up and down employing 20 μL and 150 μm tips. Metaphase II oocytes were injected and incubated for 17-20 hours in 8% CO2, 20% O2, 37°C. Normally fertilized zygotes were cultured to blastocyst stage and embryo development was evaluated. Seventy-nine blastocysts were biopsied. Main results and the role of chance The mean age of the patients was 32.12±1.11 years old. A total of 506 and 493 metaphase II oocytes from Groups 1 and 2 respectively were subjected to ICSI. Normal fertilization rates for oocytes treated with Hyaluronidase and Cumulase® were 71.34% (n = 361) and 72.41% (n = 357) respectively, with no statistically significant differences between the two groups (p = 0.35). Usable blastocysts rate (selected for embryo transfer or blastocyst cryopreservation) did not differ with any statistical difference between the bovine-derived hyaluronidase (n = 170, 47.09%) and the Cumulase® treated oocytes (n = 158, 44.25%), (p = 0.44). Preimplantation genetic testing for aneuploidies (PGT-A) was performed in 18 cycles. Seventy-nine blastocysts were biopsied and tested. Fourty-seven embryos from the bovine-derived hyaluronidase group were biopsied, presenting with a euploidy rate of 51.06% (n = 24), compared to 32 embryos from the recombinant hyaluronidase group with a euploidy rate of 50% (n = 16). Results indicated no statistically significant difference (p = 0.92). These data support that human recombinant hyaluronidase presents with similar efficacy to bovine-derived hyaluronidase. in terms of normal fertilization, embryo development as well as euploidy rates. Limitations, reasons for caution The small sample size regarding the euploidy outcome stands as a limitation of the study. Further studies should focus on the effect of recombinant hyaluronidase on genetic anomalies on blastocysts. Wider implications of the findings Fertilization, usable blastocyst and euploidy rates did not differ when employing bovine-derived or human recombinant hyaluronidase, rendering the latter a safe and efficient alternative. However, it is important to assess long-term effects, as only a limited number of studies report on perinatal and neonatal outcomes Trial registration number 25-12-21MTYPUECIT

P-192 ATP5G3 and VEGFC expression in cumulus cells are non-invasive markers of fertilization rate

Abstract Study question Could expression of aging- and hypoxia-related genes in cumulus cells be used as non-invasive markers of fertilization rate in ART? Summary answer The expression levels of specific aging- (ATP5G3) and hypoxia-related genes (VEGFC) in human cumulus cells correlate with oocyte ability to be fertilized. What is known already Throughout a woman reproductive life, cumulus cells (CC) protect the dormant oocyte from damage, act as sensors of the follicular microenvironment and act as gatekeeper for the oocyte developmental potential. One such mechanism relays on the hypoxia-dependent response, which controls the cell metabolic and oxidative state. With age, hypoxia tolerance decreases in the whole organism, including the ovary. Recently, alterations in CC response to the follicular microenvironment have been described in advanced maternal age (AMA) women. We investigate whether expression levels of hypoxia-responsive genes in CC could be used as non-invasive marker of fertilization rate in human oocytes. Study design, size, duration Prospective study of 94 women (41 oocyte donors and 53 patients) recruited from 2018 to 2021. The overall age was 32±8.0 (range 18-46); 25±4.3 (range 18-34) for oocyte donors and 38±4.6 (range 26-46) for patients. The mean antral follicular count (AFC) was 18±10.9 (range 1-48) and the mean MII rate at ovum pick-up (OPU) was 75±17.8% (range 20-100%). Participants/materials, setting, methods Pooled CC were collected from each woman after OPU; total RNA was extracted, reverse transcribed to cDNA using random hexamers and expression of aging- (LYZ, FGF2, ATP5G3, AMH, ANXA5) and hypoxia-related genes (HIF-1A, NOS2, NOS3, HMOX1, TXNIP, CLU, FABP3, TGFBR3, VEGFC) was detected by qPCR and normalized against TBP. Expression levels were plotted against woman age, AFC, MII rate at OPU (MR), and fertilization rates (FR) and non-parametric Spearman’s correlation was applied. Main results and the role of chance Overall, MII and FR rates did not decrease with increasing woman’s age (rs= -0.2, p = 0.06 and rs= -0.1, p = 0.39, respectively). When analyzing expression levels of aging markers (LYZ, FGF2, ATP5G3, AMH, ANXA5), none of them correlate with age, AFC, or MR (rs&amp;lt;│0.1│, p &amp;gt; 0.05) in CCs. Moreover, although abundant, the expression of HIF1A, the main hypoxia-related gene, was not correlated with age, AFC, MR or FR. However, the expression of several genes regulated by HIF1A did show correlation with age, MR or FR. CLU (a secreted chaperone involved in cell death), TXNIP (a tumor suppressor gene involved in redox regulation) and NOS2 (an inducible and calcium-independent isoform of nitric oxide synthase) correlated positively with age (rs=0.25, p = 0.014; rs=0.31, p = 0.0027; and rs=0.24, p = 0.03, respectively). We further analyzed CC gene expression against FR and we found that ATP5G3 and VEGFC levels decreased with increasing FR, independently of age (rs=-0.26, p = 0.044 and rs=-0.31, p = 0.014; respectively). Limitations, reasons for caution The analysis of pooled CC allows for cohort interpretation of results, and they cannot be extrapolated at this time to single oocyte’s reproductive outcome prediction. Wider implications of the findings We found that the fertilization potential of a cohort of oocytes is related to the ability of CC to respond to oxidative stress and hypoxia, pointing at ATP5G3 and VEGFC expression as markers for fertilization success. Further research is needed to assess their association with embryo quality and pregnancy outcomes. Trial registration number NA

P-119 Oxidative stress in the native semen versus strict sperm morphology as predictors of fertilization in couples undergoing IVF

Abstract Study question Is oxidative stress in the native semen a better predictor of fertilization that strict sperm morphology in couples undergoing IVF? Summary answer Compared to strict sperm morphology, oxidative stress in the native semen is a better predictor of fertilization than strict sperm morphology in couples undergoing IVF What is known already Oxidative stress (OS) in native semen was studied before by measuring oxidative-reductive potential (ORP) and was found to be a good predictor of fertilization in-vitro with a cut-off point of 1.34 mV/106 sperm/mL below which the fertilizing capacity of the sperm is diminished. Similarly, strict sperm morphology (SM) was shown to be a good predictor of fertilization with a cut-off point around 4%. However, the superiority of either of these parameters as a better predictor of fertilization needs to be determined and one of the best methods to test this theory is in the IVF model (not the ICSI model). Study design, size, duration This prospective cohort study was conducted between September 2017 and December 2018. Couples with unexplained infertility were treated for one cycle of combined IVF/ICSI if 12 oocytes or more were retrieved. If good embryos resulted from IVF, 2 were transferred. If no fertilization occurred from IVF, 2 embryos resulting from ICSI were transferred. All remaining embryos were frozen. A total of 108 couples were enrolled but only 25 fulfilled the criteria and completed the study. Participants/materials, setting, methods A total of 575 oocytes were retrieved from the 25 patients (mean ± SD = 20.5 ± 5.6 oocyte/cycle) but only 3 to 5 oocytes per patient were inseminated by conventional IVF and the rest with ICSI. OS was determined in native semen by measuring oxidative reduction potential (ORP) using the MiOXYS system and the results correlated with the fertilization rate (FR) in the IVF-fertilized oocytes. Strict morphology of the same sample was also determined. Main results and the role of chance Out of the 108 oocytes inseminated with conventional IVF, 36 reached the 2PN stage (FR = 33.3%). The mean (± SD) ORP in the native semen in couples with = &amp;gt;50% IVF fertilization was 1.02 ± 0.1 mV/106 sperm/mL which is significantly lower than in couples with &amp;lt;50% fertilization (2.05 ± 0.7 mV/106 sperm/mL) (P &amp;lt; 0.02). The sensitivity, specificity, PPV, NPV, +LR and –LR of the ORP assay in predicting = &amp;gt;50% fertilization were 70.0%, 60.0%, 53.8%, 75.0%, 0.80 and 0.20 respectively. On the contrary, the mean (± SD) SM in the native semen in couples with = &amp;gt;50% IVF fertilization was 9.0 ± 4.74 which is slightly (but insignificantly) higher than in couples with &amp;lt;50% fertilization (8.5333 ± 5.25) (P &amp;gt; 0.5). The sensitivity, specificity, PPV, NPV, +LR and –LR of SM in predicting = &amp;gt;50% IVF fertilization were 40.0%, 80.0%, 57.1%, 66.7%, 2.00 and 0.75 respectively. The AUC for ORP was equal to 0.854167 with a cut-off point at 1.5692 mV/106 sperm/ml. On the contrary, the AUC for SM was equal to 0.5367 with a cut-off point at 8, denoting that ORP in the native semen is a better predictor of fertilization in IVF compared to SM. Limitations, reasons for caution The small sample size of this study can be considered a limitation but these early results can be further confirmed by larger prospective studies involving couples with various causes of infertility, although the criteria required for the recruitment of these couples are not easy to fulfill. Wider implications of the findings Measurement of ORP in semen should be added as a parameter to evaluate the fertilizing capacity of the sperm in routine sperm analysis. This can help clinicians treating couples with unexplained or male factor infertility determine whether anti-oxidant therapy, IUI, IVF or ICSI is the best management option. Trial registration number No applicable

Autologous ovarian platelet rich plasma treatment improves oocyte and embryo quality: a before-after prospective study

Platelet-rich plasma (PRP) is used for successful regeneration of female reproductive tissues. However, little is known about the effect of ovarian PRP treatment on oocyte and embryo quality. The objective of our study was to assess the role of autologous ovarian PRP treatment on ovarian reserve and number and quality of oocytes and embryos in women with poor ovarian response (POR) undergoing in-vitro fertilization cycles. A total of 66 women with POR were treated with ovarian PRP injection in two subsequent menstrual cycles. The antral follicle counts, serum anti-Mullerian hormone, follicle-stimulating hormone levels, fertilization rate, number and quality of oocytes, and embryos were assessed and compared between the cycle before and after PRP treatment. Ovarian PRP treatment resulted in insignificantly lower follicle-stimulating hormone levels, significantly higher antral follicle count, anti-Mullerian hormone, number of retrieved oocytes, and insignificantly higher fertilization rate. However, the mean number of Day 5 embryos (2.19 ± 1.45 vs. 1.58 ± 1.30, p = 0.01), the percentage of high-quality oocytes (45.29% ± 42.40% vs. 15.21% ± 30.24%, p < 0.01) and the percentage of grade-I blastocysts (52.10% ± 37.94% vs. 12.86% ± 22.97%, p < 0.01) were significantly higher after PRP treatment in comparison to the pretreatment period. Moreover, the mean MII oocyte quality (1.60 ± 0.54 vs. 2.31 ± 0.63, p < 0.01) and mean blastocyst quality (1.53 ± 0.45 vs. 2.42 ± 0.63, p < 0.01) were significantly improved in the post-treatment period. In conclusion, the applied autologous ovarian PRP treatment in poor responders may significantly improve oocyte and embryo quality.

P-449 Storage of vitrified oocytes up to 8 years does not affect pregnancy and live birth rates: analysis of 5,362 cycles

Abstract Study question Does long-term storage of vitrified oocytes affect laboratory and reproductive outcomes after ICSI? Summary answer The time spent by vitrified oocytes in liquid nitrogen banks does not affect oocyte survival, fertilization rate nor reproductive outcomes up to live birth. What is known already Vitrification is the most efficient approach to oocyte cryopreservation and is commonly applied in oocyte donation programs. The survival rate can be &amp;gt;90% and vitrified-warmed oocytes provide comparable reproductive results to fresh oocytes. However, even with modern cryobanking technology, there are potential temperature fluctuations caused by access to cryopreserved material, transportation, stocktaking events, and regular maintenance. While some studies indicate that a multiyear storage of vitrified oocytes does not affect pregnancy rates, others found decreased reproductive outcomes after as little as 6 months, and further investigations in large cohorts are needed to confirm the safety of long-term oocyte storage. Study design, size, duration Retrospective cohort study of 5,362 cycles with vitrified oocytes from oocyte donors carried out between 2013 and 2021. All cycles included fresh embryo transfer (ET). Oocyte vitrification and warming were carried using the Cryotop® protocol (Kitazato). We analyzed the effect of storage time of vitrified oocytes in liquid nitrogen vapors on laboratory outcomes (oocyte survival, fertilization rate (FR), percentage of viable embryos) and reproductive outcomes (pregnancy -biochemical and clinical- and live birth (LB) rates). Participants/materials, setting, methods Eight categories of storage time were established: 0-0.25 years (reference group, ≤3 months), 0.25-0.5, 0.5-1, 1-1.5, 1.5-2, 2-3, 3-4, and &amp;gt;4 years. The effect of oocyte storage time on laboratory and reproductive outcomes was analyzed by linear and logistic regression, respectively. These multivariate analyses were adjusted by multiple factors: sperm origin (partner vs donor), sperm parameters, number of oocytes inseminated, number of embryos transferred and day of ET (2-3 vs 5), among others. Main results and the role of chance Mean recipient age was 42.0±4.6, while oocyte donors age was 26.1±4.6. The mean number of thawed oocytes was 8.0±2.5, all surviving oocytes were inseminated by ICSI with partner (84.9%) or donor sperm (15.1%). The oocyte storage time ranged from 3 days to 8.2 years (mean: 0.7 ± 0.9 years). We did not find significant differences in oocyte storage time between positive and negative pregnancy and LB, nor between different survival rates (&amp;lt;100 vs = 100%), FR (&amp;lt;70 vs ≥ 70%) and percentages of viable embryos (&amp;lt;50 vs ≥ 50%) at univariate analysis (p &amp;gt; 0.05 in all cases). The mean oocyte survival rate was 90.2%±14.7% and, after adjusting for confounders, did not decrease with longer storage time (i.e. 88.9% for time &amp;gt;4 years, p = 0.963). Similarly, the linear regression model did not show a significant effect of storage time on FR, which was close to 70% in all categories (range: 67.6-70.6, p &amp;gt; 0.05), nor a decrease in the percentage of viable embryos. Finally, reproductive outcomes were similar across storage times (p &amp;gt; 0.05 for all categories when compared to the reference 0-0.25); specifically, long-term oocyte storage (&amp;gt;4 years) did not affect the chances of clinical pregnancy (OR: 0.657 [0.395-1.092], p = 0.194) and LB (OR: 0.666 [0.393-1.128], p = 0.231). Limitations, reasons for caution These results cannot be extended to cycles using the patient’s own oocytes or involving a severe male factor (testicular spermatozoa or criptozoospermia), which were excluded. Due to the retrospective nature of the study, some uncontrolled variables could affect the results. Wider implications of the findings This is the first study evaluating the effect of long-term oocyte storage on a large cohort of patients undergoing oocyte donation, thus partially discarding the effect of the female factor. Reassuringly, these results indicate that long-term storage of oocytes is a safe option for young patients and oocyte banks. Trial registration number Not applicable

P-150 clinical pregnancy rates of oocyte donor cycles are similar when universal media and/or oocyte-specific media are used for oocyte vitrification and warming

Abstract Study question Did the use of universal vitrification/warming media have an impact on the clinical pregnancy rates in oocyte donation cycles compared with oocyte-specific vitrification/warming media? Summary answer Using universal media for oocyte vitrification and warming yields similar clinical pregnancy rates but higher oocyte survival rates than oocyte-specific media. What is known already Vitrification is now the gold standard for oocyte and embryo cryopreservation. Several commercial kits are available, some are designed for specific cell types (e.g., oocytes, zygotes, cleavage-stage embryos or blastocysts) and others are suitable for several stages, therefore termed “universal”. While the composition and the exposition protocol of stage specific media are optimized for specific cell types, “universal” media display a single composition, therefore exposition protocols need to be adapted to each specific developmental stage to ensure optimal cell survival rates. The potential use of the universal media allows to optimize the management of media in ART laboratories. Study design, size, duration This is a retrospective monocentric study. We compared the results obtained in our center for oocyte vitrification in donor cycles with the successive use of oocyte-specific vitrification/warming media (RapidVit/Warm™ Oocyte, Vitrolife) and afterwards universal vitrification/warming media (RapidVit/Warm™ Omni, Vitrolife). We analyzed 111 oocyte recipient ICSI cycles performed between March 2016 and July 2020 (86 recipient couples) and their 81 corresponding oocyte donation cycles (79 donors) with these media. Participants/materials, setting, methods Oocyte recipient ICSI cycles were divided in three groups depending on the combination of vitrification and warming media used for the oocyte vitrification and warming procedures: “specific/specific” (S/S), “specific/universal” (S/U) and “universal/universal” (U/U). The primary outcome was the clinical pregnancy rate per embryo transfer. Secondary outcomes were the oocyte survival rates, fertilization rates, cleavage and blastocyst rates, live birth and miscarriage rates. Main results and the role of chance The age of the donors (31.7+/-3.6 yo) and the recipients (35.1+/-4.5) was similar for the three groups (p &amp;gt; 0.05). Total FSH dose was similar for the 3 groups (1814.2+/-505UI) (p &amp;gt; 0.05) Clinical pregnancy rates were similar when universal vitrification/warming media were used (25.6% for S/U; 25.8% for U/U) compared with oocyte-specific vitrification/warming media (12.2% for S/S, p = 0.25). We observed higher oocyte survival rates when universal media were used (93.7% for U/U, 85.5% for S/U) compared with the use of the oocyte-specific media (75.6% for S/S, p &amp;lt; 0.0001). Fertilization (74.9% for U/U; 68% for S/U; 68.1% for S/S), cleavage (97.9% for U/U; 94.6% for S/U; 89.3% for S/S), and blastulation rates (46.6% for U/U; 50.3% for S/U; 41.7% for S/S) were not different between the three groups (p &amp;gt; 0.05). There was no difference in miscarriage (6.5% for U/U; 10.2% for S/U; 4.9% for S/S) and live birth rates (19.4% for U/U; 15.4% for S/U; 7.3% for S/S) between the 3 groups (p &amp;gt; 0.05). Limitations, reasons for caution The preliminary results of this retrospective study need to be confirmed by a larger prospective study. Wider implications of the findings The use of vitrification/warming universal media, which improves the management of media used for vitrification of oocytes and embryos in ART laboratory, allows to obtain similar clinical pregnancy rates for oocyte recipient donors. Trial registration number not applicable

P-155 Follicular fluid antibodies associated with quality of oocytes and fertilization rate in IVF-ICSI cycles

Abstract Study question Which follicular fluid (FF) autoantibodies influence oocyte quality and its fertilization potential in the settings of IVF-ICSI? Summary answer The measured follicular fluid levels of anti-thyroperoxidase and anti-aromatase antibodies are associated with lower number of mature oocytes and fertilization rate in IVF-ICSI cycles. What is known already Nowadays it is known that there is clear correlation between serum and follicular fluid antibody levels indicating their ability to pass freely through blood-follicle barrier. It is believed that anti-thyroid antibodies may exert direct toxic effect on the growing follicle and adversely affect the quality of future embryo due to the direct interaction with zona pellucida antigens, disordering its functional role and, consequently, altering oocyte fertilization and developmental potential. However, at this point no cases of anti-aromatase antibody detection in the follicular fluid of women undergoing fertility treatment have been reported. Study design, size, duration A prospective cohort study including 90 patients undergoing IVF-ICSI was performed at the Department of ART, The D. O. Ott Research Institute of Obstetrics and Gynecology from September 2018 to March 2020. During oocyte retrieval only the first aspiration including cumulus-oocyte complexes (COC) were used for the study. After COC removal, the pre-ovulatory FF was centrifuged at 2000×g for 10 min. The cell-free supernatants were divided into aliquots and stored at 80 oC until assay. Participants/materials, setting, methods FF investigation included anti-thyroperoxidase (AT-ТРО, BlueGene, Сhina, E01T0531) and anti-aromatase (anti-ARO, Cloud-Clone Corp., USA, AEC319Hu) antibodies detection using commercial ELISA kits. The threshold value for each antibody was calculated as 3*SD from the mean values of patients investigated. Thus, the study group included 52 women with FF autoantibodies ≥ (M + 3*SD) (if at least one marker positive), the comparison group comprised 38 women with FF autoantibodies &amp;lt; (M + 3*SD). Main results and the role of chance In the study group, the number of mature oocytes was notably lower than in the comparison group (6.5 (3.5; 9.5) vs 9 (8; 11); P &amp;lt; 0,01). A significant inverse correlation between follicular fluid AT-TPO (rs =-0.5; P &amp;lt; 0.05) and anti-ARO (rs =-0.5; P &amp;lt; 0.05) antibody levels and the number of mature oocytes was revealed in all investigated samples. In addition, despite no difference in the rate of mature oocytes between the groups (84% vs 88%, P &amp;gt; 0.05) it negatively correlated with anti-ARO FF (rs =-0.4; P &amp;lt; 0.05) among all the participants. Evaluation of oocyte fertilization rate using a linear regression analysis showed its significant dependence on the follicular fluid anti-aromatase antibody levels (R = 0,33; P &amp;lt; 0,01). Limitations, reasons for caution Small sample size Wider implications of the findings Further studies with larger sample size evaluating anti-ARO antibodies are needed in order to understand its origin and effect on early steps of human reproduction. Trial registration number not applicable

Comparing the IVM laboratory outcomes between stimulated IVF with unstimulated natural cycles.

Recently, more attention has been raised towards fertility preservation in women with cancer. One option is in vitro maturation (IVM) of the immature oocytes as there is not enough time for induction of an ovarian stimulation protocol. The aim was to compare the IVM laboratory outcomes between stimulated and unstimulated (natural) in vitro fertilization (IVF) cycles. In total, 234 immature oocytes collected from 15 cancer patients who underwent an IVM programme (natural IVM) and 23 IVF cycles with a controlled ovarian hyperstimulation protocol (stimulated IVM) were analyzed. The oocyte morphology, zona pellucida (ZP), and meiotic spindle presence were measured using PolScope technology. Also, the rates of oocyte maturation and fertilization were assessed in both groups. The IVM rate was higher in the stimulated cycle (P < 0.05), but the fertilization rate was insignificant in comparison with unstimulated cycles. There were no significant differences in the spindle visualization and ZP birefringence scoring between the groups (P > 0.05). The oocyte normal morphology was better in the stimulated cycle compared with the natural cycle (P < 0.05). In conclusion, IVM can be recommended for cancer patients as an alternative treatment when there is insufficient time for conventional IVF before chemotherapy initiation.

Pre-IVF treatment with a GnRH antagonist in women with endometriosis (PREGNANT): study protocol for a prospective, double-blind, placebo-controlled trial

IntroductionInfertility is a common complication of endometriosis. While in vitro fertilisation-embryo transfer (IVF) successfully treats endometriosis-associated infertility, there is some evidence that pregnancy rates may be diminished in women seeing...

Effects of IGF-1 on the Three-Dimensional Culture of Ovarian Preantral Follicles and Superovulation Rates in Mice.

Simple SummaryIn this experiment, mouse secondary follicles were cultured in the three-dimensional culture system of alginate gel with different concentrations of IGF-1. According to the results of follicle growth, oocyte maturation, the levels of hormone (17β-estradiol, progesterone and AMH) and the expressions of genes related to hormone secretion, oocyte-secreted factors, apoptosis and gonadotropin receptor, the optimal concentration of IGF-1 was determined to be 10 ng/mL in the culture medium. Moreover, the intraperitoneal injection of IGF-1 before superovulation in mice could increase the number of ovulated oocytes and reduce their degraded rates.Insulin-like growth factor-1 (IGF-1) plays a crucial role during folliculogenesis, which has been demonstrated by previous research. However, the optimal IGF-1 dosage in the three-dimensional (3D) culture system is unknown. Mouse secondary follicles (140–150 µm) were cultured for 6 days within an alginate bead in a medium supplemented with 0 (G0), 5 ng/mL (G5), 10 ng/mL (G10), or 50 ng/mL IGF-1 (G50). Secretions of 17β-estradiol and progesterone were significantly increased in G10 and G50 (p < 0.05). However, G50 significantly inhibited follicular growth (p < 0.05), while G10 showed a higher oocyte maturation rate. Thus, the 10 ng/mL IGF-1 was used in subsequent experiments. IGF-1 enhanced the function of granulosa cells (GCs) by upregulating expressions of Star, Cyp19a1, Hsd3b1, Fshr, and Lhcgr. Oocyte secretory function was promoted by upregulating expressions of Bmp-15, Gdf-9, and Fgf-8. Addition of IGF-1 showed anti-apoptotic effect. However, G10 did not improve fertilization rate of MII oocytes compared to G0. In an intraperitoneal injection experiment in mice, IGF-1 significantly increased the number of ovulated oocytes (p < 0.05). In conclusion, 10 ng/mL IGF-1 can promote the production of mature oocytes in the 3D culture medium and injection of IGF-1 before superovulation increases the number of ovulated oocytes.

High efficiency of homemade culture medium supplemented with GDF9-β in human oocytes for rescue in vitro maturation

ObjectiveOptimizing culture media for the incubation of immature oocytes is a vital strategy to increase the oocyte maturation rate during in vitro maturation (IVM) programs. This study evaluated the IVM and fertilization rates of human germinal vesicle (GV) and metaphase I (MI) oocytes using two different maturation media (commercial and homemade) with or without growth differentiation factor 9-β (GDF9-β). supplementation.MethodsImmature oocytes from intracytoplasmic sperm injection (ICSI) cycles were collected and assigned to one of two IVM culture media (commercial or homemade; cleavage-stage base). After maturation, MII oocytes were examined under an inverted microscope for the presence of the polar body, zona pellucida (ZP) birefringence, and meiotic spindle (MS) visualization after maturation in four conditions (commercial or homemade medium, with or without GDF9-β. ICSI was done for matured oocytes, and fertilization was confirmed by the visualization of two distinct pronuclei and two polar bodies.ResultsNo significant differences were found between the two culture media in terms of the time and rate of oocyte maturation or the rate of fertilization (p>0.05). Growth factor supplementation increased the 24-hour maturation rate for both GV and MI oocytes only in homemade medium. The maturation rate after 24 hours was higher for MI oocytes (p<0.05). Similar results were observed for MS visualization and ZP structure in both types of media (p>0.05).ConclusionHigher rates of oocyte maturation and fertilization were observed after application of homemade medium supplemented with GDF9-β. Therefore, this combination may be recommended as an alternative for clinical IVM programs.

Bovine salpingitis: Histopathology, bacteriology, cytology and transcriptomic approaches and its impact on the oocyte competence

The present study was performed to examine the histopathology, cytology, bacteriology and expression pattern of a targeted set of genes of cytokines in the oviduct of cows with inflammation (Experiment 1). In addition, the effects of oviductal fluid from cows with salpingitis on the oocyte maturation and fertilization in vitro were examined (Experiment 2). The most frequent bacterial co-infection was Escherichia coli and Fusobacterium necrophorum, which was always associated with severe histopathologic salpingitis. Out of 15 cows with histologically healthy uterus, only one cow (6.7%) displayed the histologic signs of mild salpingitis, whereas from 50 cows with endometritis, 48 cows (96%) showed histologically different grades of salpingitis. The mRNA expression of IL1β, CD14, IL8 and CASP3 was significantly different among all groups of salpingitis (P < 0.05) with the highest level of mRNA expression in the sever grade of salpingitis. Results of experiment 2 showed a significant decline in the oocytes with peripheral free mitochondria and fertilization rate in the salpingitis group than the no- salpingitis group (P < 0.05). In conclusion, our results showed that histologically detected salpingitis is in most cases associated with histologic and cytologic endometritis. The pattern of the gene expression of chemokines and cytokines was altered in association with different grades of salpingitis. Further, we observed a decline in the peripherally located mitochondria and lower fertilization rate in oocytes following addition of oviductal fluid collected from the cows with sapingitis to the maturation media.

Effects of nicotinamide on follicular development and the quality of oocytes

BackgroundNicotinamide (NAM) is an important antioxidant, which is closely related to female fertility, but its role has not been clearly elucidated. The purpose of the present study was to investigate the effects of NAM on follicular development at different stages and the quality of oocytes.MethodsThe concentration of NAM in follicular fluid (FF) of 236 women undergoing in vitro fertilization (IVF) was ascertained by enzyme-linked immunosorbent assay (ELISA), and the correlation between NAM and clinical indexes was analyzed. During the in vitro maturation (IVM) of mice cumulus-oocyte complexes (COCs), different concentrations of NAM were added to check the maturation rate and fertilization rate. The reactive oxygen species (ROS) levels in the oocytes treated with different hydrogen peroxide (H2O2) and NAM were assessed. Immunofluorescence staining was performed to measure the proportion of abnormal spindles.ResultsThe level of NAM in large follicles was significantly higher than that in small follicles. In mature FF, the NAM concentration was positively correlated with the rates of oocyte maturation and fertilization. Five mM NAM treatment during IVM increased maturation rate and fertilization rate in the oxidative stress model, and significantly reduced the increase of ROS levels induced by H2O2 in mice oocytes.ConclusionsHigher levels of NAM in FF are associated with larger follicle development. The supplement of 5 mM NAM during IVM may improve mice oocyte quality, reducing damage caused by oxidative stress.

Women with polycystic ovary syndrome (PCOS) have reduced melatonin concentrations in their follicles and have mild sleep disturbances

BackgroundPolycystic ovary syndrome (PCOS) is a common gynecologic disorder related to abnormal circadian rhythm. Therefore, we aimed to find whether the level of melatonin, a rhythm regulating hormone changed in the ovarian microenvironment in this disease.MethodsThe melatonin concentrations in follicular fluid (FF) were measured in 35 PCOS and 36 non-PCOS women undergoing in vitro fertilization (IVF) treatment.ResultsThe FF melatonin concentration was significantly lower in PCOS women than non-PCOS women (p = 0.045) and it was found positively correlated with serum basal FSH level (r = 0.308, p = 0.013). In IVF procedures, there was no significant difference in the fertilization rate of oocytes between the two groups, but the high-quality embryogenesis rate on the third day of the PCOS group was significantly lower than that of the control group (p = 0.042), which showed a weak positive correlation with the FF melatonin concentration (rs = 0.240, p = 0.044). Furthermore, there was no significant difference in overall pregnancy outcome. The PSQI questionnaire showed that sleep disorders were more likely to exist in the PCOS group, though there was no significant difference.ConclusionThe obtained results suggested PCOS women had lower melatonin concentrations in the ovarian microenvironment.

Oncostatin M and its receptor in women with polycystic ovary syndrome and association with assisted reproductive technology outcomes

The role of adipokines in ovarian-related disorders such as polycystic ovary syndrome (PCOS) has been reported. However, the involvement of Oncostatin M (OSM), a recently identified adipokine, in ovarian function is unknown. Therefore, we investigated the association of the OSM signaling pathway with ovarian functions and PCOS pathogenesis. This case-control study enrolled 30 PCOS and 30 healthy women who underwent the intracytoplasmic sperm injection procedure. OSM and OSM receptor (OSMR) levels were evaluated in the follicular fluid (FF). Moreover, the expression of insulin receptor substrates (IRS1 and IRS2), OSM, OSMR, suppressor of cytokine signaling 3 (SOCS3), and androgen receptor (AR) genes were analyzed in the isolated cumulus cells (CCs). For the in-vitro experiment, the effect of recombinant OSM on the expression of related genes in isolated CCs was analyzed. Follicular concentrations of OSM and OSMR were significantly lower in PCOS (123.91±48.58 pg/ml and 0.93±0.35ng/ml, respectively) compared to control women (283.53±96.62pg/ml and 1.45±0.18ng/ml, respectively; p<0.001) and were positively correlated with the oocyte maturation (r=0.611 and r=0.611, respectively) and fertilization (r=0.592 and r=0.627, respectively) rates in the PCOS group. Furthermore, the SOCS3 expression was upregulated about eight times in PCOS patients compared to the controls (p<0.05). The treatment of cells with recombinant OSM significantly increased SOCS3, OSMR, IRS-1, and -2 expression and decreased AR expression. The decreased levels of OSM and its receptor in PCOS patients, possibly mediated by SOCS3, could negatively affect oocyte maturation and fertilization rates.

What is the optimum number of follicular flushes in mono-follicular in-vitro fertilization cycles in a poor responder population?

Objective:Assessment of the optimal number of follicular flushes on retrieval rate and quality of oocytes in mono-follicular in-vitro fertilization (IVF) cycles.Material and Methods:A retrospective analysis of 246 oocyte pick-up procedures in mono-follicular IVF cycles of 226 poor responder women was performed. The primary endpoint was oocyte retrieval rate in the initial aspirate versus subsequent flushing episodes. The secondary endpoints were oocyte maturity, fertilization rates and embryo cleavage.Results:The procedure was successful in 187 cycles (76%), of which 160 metaphase-II oocytes were retrieved. Retrieval rates were similar for natural and modified natural cycles (p=0.595). The initial aspirate provided 54% of the total yield and the rest was obtained from up to four episodes of flushing. Follicular flushing increased oocyte recovery rate from 41.1% to 76%. None of the oocytes retrieved after three flushes fertilized. Oocyte maturity, fertilization and embryo cleavage rates were comparable for oocytes from the initial aspirate and one or two episodes of flushing. Oocytes obtained after the third flushing episode developed into poor quality embryos.Conclusion:Flushing confers a benefit for oocyte recover rates in mono-follicular IVF cycles in poor responder women. However, more than three attempts at flushing were not associated with good outcome.

Effect of GnRH agonist alone or combined with different low-dose hCG on cumulative live birth rate for high responders in GnRH antagonist cycles: a retrospective study

BackgroundThere is insufficient evidence regarding the impact of dual trigger on oocyte maturity and reproductive outcomes in high responders. Thus, we aimed to explore the effect of gonadotropin-releasing hormone agonist (GnRHa) trigger alone or combined with different low-dose human chorionic gonadotropin (hCG) regimens on rates of oocyte maturation and cumulative live birth in high responders who underwent a freeze-all strategy in GnRH antagonist cycles.MethodsA total of 1343 cycles were divided into three groups according to different trigger protocols: group A received GnRHa 0.2 mg (n = 577), group B received GnRHa 0.2 mg and hCG 1000 IU (n = 403), and group C received GnRHa 0.2 mg and hCG 2000 IU (n = 363).ResultsThere were no significant differences in age, body mass index, and rates of oocyte maturation, fertilization, available embryo, and top-quality embryo among the groups. However, the incidence of moderate to severe ovarian hyperstimulation syndrome (OHSS) was significantly different among the three groups (0% in group A, 1.49% in group B, and 1.38% in group C). For the first frozen embryo transfer (FET) cycle, there were no significant differences in the number of transferred embryos and rates of implantation, clinical pregnancy, live birth, and early miscarriage among the three groups. Additionally, the cumulative ongoing pregnancy rate and cumulative live birth rate were not significantly different among the three groups. Similarly, there were no significant differences in gestational age, birth weight, birth height, and the proportion of low birth weight among subgroups stratified by singleton or twin.ConclusionsGnRHa trigger combined with low-dose hCG (1000 IU or 2000 IU) did not improve oocyte maturity and embryo quality and was still associated with an increased risk of moderate to severe OHSS. Therefore, for high responders treated with the freeze-all strategy, the single GnRHa trigger is recommended for final oocyte maturation, which can prevent the occurrence of moderate to severe OHSS and obtain satisfactory pregnancy and neonatal outcomes in subsequent FET cycles.

IVF under COVID-19: treatment outcomes of fresh ART cycles.

Study QuestionDoes prior SARS-CoV-2 infection in women undergoing fertility treatments affect the outcomes of fresh ART cycles?Summary AnswerSARS-CoV-2 infection does not affect fresh ART treatment outcomes, except for a possible long term negative effect on oocyte yield (>180 days post infection).What Is Known AlreadyA single previous study suggested no evidence that a history of asymptomatic or mild SARS-CoV-2 infection in females caused impairment of fresh ART treatment outcomes.Study Design, Size, DurationRetrospective cohort study, including all SARS-CoV-2 infected women who underwent fresh ART cycles within a year from infection (the first cycle post infection), between October 2020 and June 2021, matched to non-diagnosed controls.Participants/Materials, Setting, MethodsPatients from two large IVF units in Israel who were infected with SARS-CoV-2 and later underwent fresh ART cycles were matched by age to non-diagnosed, non-vaccinated controls. Demographics, cycle characteristics and cycle outcomes, including oocyte yield, maturation rate, fertilization rate, number of frozen embryos per cycle, and clinical pregnancy rates, were compared between groups.Main Results And The Role Of ChanceOne hundred and twenty-one infected patients and 121 controls who underwent fresh ART cycles were included. Oocyte yield (12.50 versus 11.29; p = 0.169) and mature oocyte rate (78% versus 82%; p = 0.144) in all fresh cycles were similar between groups, as were fertilization rates, number of frozen embryos per cycle and clinical pregnancy rates (43% versus 40%; p = 0.737) in fresh cycles with an embryo transfer. In a logistic regression model, SARS-CoV-2 infection more than 180 days prior to retrieval had a negative effect on oocyte yield (p = 0.018, Slope=-4.08, 95% CI -7.41 – -0.75), although the sample size was small.Limitations, Reasons For CautionA retrospective study with data that was not uniformly generated under a study protocol, no antibody testing for the control group.Wider Implications Of The FindingsThe study findings suggest that SARS-CoV-2 infection does not affect treatment outcomes, including oocyte yield, fertilization and maturation rate, number of good quality embryos, and clinical pregnancy rates, in fresh ART cycles, except for a possible long term negative effect on oocyte yield when retrieval occurs > 180 days post SARS-CoV-2 infection. Further studies are warranted to support these findings.Study funding/competing interest(s)None.Trial registration number0010-21-HMC, 0094-21-ASF