MII Rate Research Articles

Human oocyte zona pellucida abnormalities: evaluation of clinical impact for different zona pellucida abnormalities and role of using assisted hatching.

Zona pellucida (ZP) plays an important role in oocyte development and fertilisation, as well as in early embryo development. However, there is currently no exploration of systematic categorising and clinical treatment measures for patients with abnormal ZP, especially the fast and easy method that can be used clinically. In this study, 185 patients with abnormal ZP (ZPA) and 222 patients with normal ZP (ZPN) were enrolled and analysed. Patients with ZPA exhibited altered hormone levels and lower laboratory outcomes in in vitro fertilisation (IVF), such as reduced MII rate, cleavage rate, high-quality embryo rate, and blastocyst formation rate compared to ZPN group. ZPA patients were sub-categorised into ZPA-A/B/C/D group according to the status of oocyte polar body and the width of perivitelline space (PVS). There are also differences in the degree of sperm binding to ZP derived from different ZPA patients and the elasticity of the oocyte membrane. ZPA subgroup analysis revealed further disparities in various IVF parameters and pregnancy outcomes. In addition, by performing different treatments on oocytes derived from ZPA patients, we found that ZP assisted hatching before the first oocyte cleavage on D1 of embryonic development in the ZPA-A/B groups and before blastocyst expansion on D4 of embryonic development in the ZPA-C/D groups were beneficial to improve embryonic development. The degree of the impact on clinical outcomes is correlated with the types of ZPA, and laser-assisted hatching of the ZP helps to improve embryonic development in patients with ZPA.

Telomerase activity, telomere length, and the euploidy rate of human embryos

Background Telomeres maintain chromosome stability, while telomerase counteracts their progressive shortening. Telomere length varies between cell types, with leukocyte telomere length (LTL) decreasing with age. Reduced telomerase activity has been linked to reproductive issues in females, such as low pregnancy rates and premature ovarian failure, with recent studies indicating correlations between telomere length in granulosa cells and IVF outcomes. Objectives The study aims to explore the relationship between telomere length, telomerase activity, and euploid blastocyst rate in infertile women undergoing IVF/ICSI PGT-A cycles. Methods This prospective study involves 108 patients undergoing controlled ovarian stimulation and PGT-A. Telomere length and telomerase activity were measured in peripheral mononuclear cells and granulosa cells (GC), respectively. Results The telomere repeat copy number to single gene copy number ratio (T/S) results respectively 0.6 ± 0.8 in leukocytes and 0.7 ± 0.9 in GC. An inverse relationship was found between LTL and the patient’s age (p < .01). A higher aneuploid rate was noticed in patients with short LTL, with no differences in ovarian reserve markers (p = .15), number of oocytes retrieved (p = .33), and number of MII (p = 0.42). No significant association was noticed between telomere length in GC and patients’ age (p = 0.95), in ovarian reserve markers (p = 0.32), number of oocytes retrieved (p = .58), number of MII (p = .74) and aneuploidy rate (p = .65). Conclusion LTL shows a significant inverse correlation with patient age and higher aneuploidy rates. Telomere length in GCs does not correlate with patient age or reproductive outcomes, indicating differential telomere dynamics between leukocytes and granulosa cells.

Proximal tubal occlusion first or oocyte retrieval first for patients with hydrosalpinx?

Our study aimed to investigate the best time to manage hydrosalpinx to improve pregnancy outcomes during in vitro fertilization-embryo transfer (IVF-ET). Patients with hydrosalpinx who received IVF treatment were analyzed retrospectively. And two groups were included to compare the effects of different timing treatment of hydrosalpinx on IVF pregnancy outcomes, "Proximal Tubal Occlusion First Group" (Group Ligation-COH) and "Oocyte Retrieval First Group" (Group COH-Ligation). The main outcome measures included: ovarian response indexes, laboratory indexes and clinical pregnancy outcomes. Univariate and multivariate Logistic regression analysis was performed for outcome indicators, and the odds ratios (OR) and 95% confidence interval (CI) were used. A total of 1490 patients were included (n = 976 Ligation-COH and n = 514 COH-Ligation). The Gn starting dose and MII rate in group Ligation-COH were significantly higher than those in group COH-Ligation (203.33 ± 58.20 vs. 203.33 ± 58.20, 81.58% vs. 80.28%, P < 0.05). The number of oocytes obtained and the number of available D3 embryos in group COH-Ligation were higher than those in group Ligation-COH (15.10 ± 7.58 vs. 13.45 ± 6.42, 10.92 ± 5.81 vs. 9.94 ± 5.15, P < 0.05). Although the number of ET cycles per IVF cycle in group COH-Ligation was higher than that in group Ligation-COH (1.88 ± 1.00 vs. 1.48 ± 0.70, P < 0.05), the biochemical pregnancy rate, clinical pregnancy rate, multiple pregnancy rate, live birth rate and cumulative live birth rate in group Ligation-COH were significantly higher than those in group COH-Ligation (60.83% vs. 46.27% for biochemical pregnancy, 55.69% vs. 38.5% for clinical pregnancy, 26.18% vs. 17.74% for multiple pregnancy, 47.08% vs. 25.26% for live birth, 69.47% vs. 47.47% for cumulative live birth, P < 0.05), and the miscarriage rate in group Ligation-COH was lower than that in group COH-Ligation (10.47% vs. 17.20 for early abortion, 4.49% vs. 15.86% for late abortion, P < 0.05). In logistic regression analysis, after adjustment for age and multiple factors, the above results were still statistically significant differences (P < 0.001). For elderly patients, the clinical pregnancy rate, multiple birth rate and live birth rate in group Ligation-COH were also higher than those in group COH-Ligation (P < 0.001). No significant differences were detected for patients with diminished ovarian reserve. For the choice of ligation operation time, we recommend that patients choose tubal ligation first and then ovulation induction and oocyte retrieval treatment.

Lipid modulation during IVM increases the metabolism and improves the cryosurvival of cat oocytes

This study investigated the time course of lipid accumulation during IVM and assessed the role of lipid modulators added during IVM on lipid content, nuclear maturation, oxidative stress, mitochondrial activity, gene expression, and cryosurvival of cat oocytes. First, the lipid content of immature COCs was compared to those subjected to different IVM duration times (24, 28, and 32 h). Then, the lipid content was investigated after the use of different lipid modulators [conjugated linoleic acid (CLA), forskolin (FSK), l-carnitine (LC)]. Subsequently, both the CONTROL group and MIX 18 (CLA+FSK+LC) were compared regarding nuclear maturation, mitochondrial activity, reactive oxygen 19 species (ROS), and glutathione (GSH) levels, to the expression of SDHA, GDF9, BMP15, ZAR-1, 20 PRDX1, SIRT1, and SIRT3 genes (normalized by ACTB and YWHAZ genes); and to vitrification and 21 post-warming viability assessment. When not using any lipid modulator, an increase (P < 0.05) in lipid content could be observed after 28 h of IVM. The MIX group showed the greatest (P < 0.05) reduction in oocyte lipid content after 28 h of IVM. No difference (P > 0.05) was observed in the MII rate in the CONTROL (45%) and MIX (41%) groups and in mitochondrial activity ((1.00 ± 0.35 A U vs 1.19 ± 0.14 A U). Although ROS and GSH levels were higher (P < 0.05) in MIX than in CONTROL, the redox balance (ROS/GSH) was greater (P < 0.05) in the latter (C:1.00 ± 0.20b vs M:0.26 ± 0.06 a A.U). The GDF9, HSP70, PRDX1, and SIRT1 transcripts were downregulated (P < 0.05) in MIX-oocytes, compared to the CONTROL. After vitrification, MIX (74%) presented a higher (P < 0.05) viability compared to control (53%). In conclusion, MIX can reduce the total lipid content and improve viability after cryopreservation, however, it seems to affect the oocyte metabolism in a way that still needs to be better understood in the cat biological model.

Impact of letrozole co-treatment during ovarian stimulation on oocyte yield, embryo development, and live birth rate in women with normal ovarian reserve: secondary outcomes from the RIOT trial.

Impact of letrozole co-treatment during ovarian stimulation on oocyte yield, embryo development, and live birth rate in women with normal ovarian reserve: secondary outcomes from the RIOT trial.

Effect of chromosomal polymorphisms on the outcome of in vitro fertilization and embryo transfer.

The incidence of chromosomal polymorphisms (CP) is increased in infertile couples, but its impact on reproduction is uncertain, especially undergoing assisted reproductive technology treatment. The purpose of the present study was to investigate the effect of CP on the outcomes of in vitro fertilization/intracytoplasmic sperm injection-embryo transfer (IVF/ICSI-ET) treatment METHODS: A total of 1331 infertile couples undergoing IVF/ICSI treatment were involved in this retrospective case-control study. The participants were divided into 4 groups according to CP variations: (i) normal chromosomes (NC) group; (ii) CP group; (iii) both chromosomal polymorphisms (BCP) group; and (iv) double chromosomal polymorphisms (DCP) group. The CP group was further divided into five subgroups: qh+, D/G, inv(9), Yqh+ and Yqh-. The outcomes of IVF/ICSI-ET treatment were compared among the groups. There were no differences observed between the eight groups in terms of number of oocytes retrieved, MII rate, fertilization rate, cleaved embryo rate, and quality embryo rate for both females and males (p > 0.05). In both male and female, some of the CP subgroups experienced more oocyte retrieval operations and more embryo transfer operations to achieve pregnancy than the NC groups (p < 0.05). The rates of live births were significantly lower in some of the CP subgroups compared to the NC group (p < 0.05). In conclusion, the pregnancy outcomes of ET were affected by CP. It was speculated that this may be associated with the effect of chromosome polymorphism on embryo quality, although this could not be observed or determined by morphological evaluation.

P-686 Long photoperiod exposure results in glycometabolism disorder in the cumulus-oocyte complex in SD rats

Abstract Study question Does long photoperiod exposure cause glycometabolism changes in the cumulus-oocyte complex? Summary answer The levels of key enzymes and substrates involved in glycometabolism have changed in the cumulus-oocyte complex in SD rats due to circadian rhythm changes. What is known already Circadian rhythm is essential for maintaining the required reproductive activities in mammalians. In our previous research, long photoperiod exposure results in the reduction of ovulation number and the increase of ROS level in oocytes. In the process of oocyte maturation, the tricarboxylic acid cycle and energy metabolism gradually increase, while the ability of the oocyte to absorb glucose is low. It is mainly metabolized by cumulus granulosa cells into pyruvate and then transported to oocytes for subsequent metabolism. Metabolomics is a comprehensive analysis of small molecules, which provides key information about cellular metabolic states. Study design, size, duration Female SD rats, 6-8 weeks old, were randomized into two groups. The rats (n = 20) were exposed to the control photoperiod (12 hours light/12 hours dark) or long photoperiod (16 hours light/8 hours dark) for 12 weeks. Participants/materials, setting, methods Cumulus-oocyte complexes were collected after ovarian stimulation and numbers were counted. The parameters compared include MII rate, fertilization rate, and ovarian follicle number. We used mass spectrometry-based metabolomics to directly measure metabolite abundance. The glycometabolism pathway and its regulatory signaling molecules were analyzed by qPCR and/or Western blots. Main results and the role of chance Long photoperiod exposure significantly reduced the ovulation rate, MII rate and fertilization rate. Interestingly, we find secondary follicle and antral follicle were decreased (p &amp;lt; 0.01), while primordial follicle and primary follicle were increased (p &amp;lt; 0.01). We identified a total of 318 metabolites in COC. Among them, 59 metabolite levels have significant differences between the two groups (FDR &amp;lt; 0.05). The metabolical function analysis suggested that the phosphatidylinositol phosphate metabolism, pyruvate metabolism, phosphatidylcholine biosynthesis, and glycolysis pathways were influenced by circadian interference. In addition, hexokinase, glucose-6-phosphate isomerase, phosphofructokinase glyceraldehyde 3-phosphate expression level were down regulated. Limitations, reasons for caution More studies are required to address how clock gene regulating glycometabolism process. And Translating results in rat model to the human setting is a limitation of this study. Wider implications of the findings Our study findings are first to demonstrate that glycolmetabolism is regulated by long photoperiod exposure in follicle development. This knowledge could help to develop strategies to modulate specific recovery strategies based on metabolic regulator to improve human IVF. Trial registration number Not applicable

O-178 Conventional IVF results in better embryo development when compared with ICSI in patients without severe male factor

Abstract Study question Which fertilisation technique offers better embryo development results in patients without severe male factor? Summary answer Conventional IVF offers better results regarding embryo development than ICSI in patients without male factor when applying both techniques in the same cohort of oocytes. What is known already ICSI was originally indicated for severe male factor. Nevertheless, it is currently used for many other indications so that it has become the most used technique. One of the reasons for the use of ICSI is the fear of a fertilisation failure when using conventional IVF (cIVF). We have to consider that ICSI is more expensive and invasive. For this reason, it is important to know whether there is a justification to use it in these cases. Study design, size, duration It is a randomized prospective study where 68 IVF/ICSI cycles were analysed. The study comprised 812 oocytes and 469 embryos and was conducted from January to December 2022 in the Assisted Reproduction Unit of a tertiary hospital. In each cycle, we performed a mixed technique using cIVF and ICSI in the same cohort of oocytes. MII rate, fertilisation rate and embryo development were analysed. Participants/materials, setting, methods All consecutive cycles with at least 6 fresh oocytes and semen parameters suitable for cIVF were offered to participate. In each cycle, oocytes were divided into two groups in order of collection and the fertilisation technique (cIVF or ICSI) was randomly assigned for each group. All the embryos were cultured to blastocyst stage. Data of maturity, fertilisation and embryo development were individually registered for every oocyte. Data were statistically analysed by applying multilevel regression models. Main results and the role of chance The patients average age was 36.3±3.0 (28 to 40) and the number of oocytes per cycle was 11.9±5.1 (6 to 26). 396 oocytes (48.8%) were allocated to cIVF group and 416 (51.2%) to ICSI group. The percentage of mature oocytes was significantly higher in cIVF group than in ICSI group (88.9% vs 81.5%, p = 0.017), which is consistent with the later assessment of maturity in cIVF. There were no statistically significant differences between cIVF and ICSI groups in terms of fertilisation rate (59.6% vs 56.0%) and high-quality blastocyst rate (A+B) (9.3% vs 8.7%) when analysed per oocyte. We observed clinical but not statistical differences in blastocyst rate per oocyte (30.6% cIVF vs 23.6% ICSI, p = 0.09). However, blastocyst rate per fertilised oocyte showed statistically significant differences (51.3% cIVF vs 41.6% ICSI, p = 0.047). Additionally, we found statistically significant differences between cIVF and ICSI in the rate of usable blastocysts (transferred or cryopreserved) per oocyte (26.3% vs 18.3%, p &amp;lt; 0.01) and per fertilised oocyte (44.1% vs 32.2%, p &amp;lt; 0.01). In 57.8% of the cycles, the best blastocyst was obtained by cIVF and in 42.2% by ICSI. Fertilisation failure was observed in 3 patients with cIVF and 1 patient with ICSI. All of them had ≤4 mature eggs. Limitations, reasons for caution This study includes the 68 patients who met the recruitment requirements until this moment. It is a part of a wider work, which will analyse a higher number of oocytes and pregnancy and perinatal outcomes. For this reason, the sample size is a limitation to the interpretation of the results. Wider implications of the findings Our findings encourage the use of cIVF when there is no severe male factor. Considering that cIVF is cheaper and less invasive than ICSI, the exclusive use of ICSI in these patients does not seem to be justified according to our results. Trial registration number not applicable

P-791 Supplementation of mitochondria from endometrial mesenchymal stem cells (EnMSCs) improves oocyte quality in aged mice

Abstract Study question To improve the quality of aging oocytes by supplementing the mitochondria from endometrial mesenchymal stem cells (EnMSCs) via microinjection. Summary answer Supplementation with EnMSC-derived mitochondria can improve the quality of oocytes and promote embryo development in aging mice. What is known already Maternal aging is one of the major causes of reduced ovarian reserve and low oocyte quality in elderly women. Decreased oocyte quality is the main cause of age-related infertility. Mitochondria are multifunctional energy stations that determine the oocyte quality. The mitochondria in aging oocytes display functional impairments with mtDNA damage, which leads to reduced competence and developmental potential of oocytes. To improve oocyte quality, mitochondrial supplementation is carried out as a potential therapeutic approach. However, the selection of suitable cells as the source of mitochondria remains controversial. Study design, size, duration Study design: Eight-month-old ICR mice were purchased and then were further raised to ten months of age for use in the experiments. EnMSCs were stably cultivated and oocytes were extracted from ten-month-old ICR mice. Study size: About 300 GV oocytes and 134 MII oocytes were counted . 80 fertilized oocytes were transplanted into the fallopian tubes of surrogate mothers. Study duraton: About one half a year. Participants/materials, setting, methods Participants:ten-month-old ICR mice. Setting and methods: We cultivated EnMSCs from aged mice and extracted mitochondria from EnMSCs. And then GV oocytes were supplemented with mitochondria via microinjection. To further assess the effects of mitochondrial supplementation on embryo development, MII oocytes from aging mice were fertilized by ICSI combining EnMSCs mitochondria microinjection.To test whether mitochondrial supplementation could improve in vivo embryo development in aging mice, embryo transplantation was performed after mitochondrial microinjection combined with ICSI. Main results and the role of chance In this study, we found that the mitochondria derived from EnMSCs could significantly improve the quality of aging oocytes.Supplementation with EnMSC mitochondria significantly increased the blastocyst ratio of MII oocytes from aging mice after intracytoplasmic sperm injection (ICSI). We also found that the birth rate of mitochondria-injected aging oocytes was significantly increased after embryo transplantation. Limitations, reasons for caution Microinjection into oocytes was invasive and some oocytes died after microinjection. The ability of this method to improve oocyte quality was limited because the MII rate of oocytes after mitochondria supplementation was 35.65±1.75% while the rate of oocytes from 6–8 weeks of mice in a previous study was nearly 80%. Wider implications of the findings For clinical treatment, the EnMSCs can be obtained from the patient's own menstrual blood as the sources of mitochondrial supplementation. A noninvasive procedure for cell extraction ensures safety, and the findings of this work might provide a valuable strategy for patients with declining oocyte quality due to aging. Trial registration number Not applicable

Follicular homocysteine as a marker of oocyte quality in PCOS and the role of micronutrients

PurposeDoes follicular homocysteine predict the reproductive potential of oocytes following FSH stimulation in PCOS women? Can it be modulated by dietary interventions?MethodsThis was a prospective, randomized, interventional clinical study. Forty-eight PCOS women undergoing in vitro fertilization at a private fertility clinic were randomized for a dietary supplementation providing micronutrients involved in homocysteine clearance or no treatment. The supplement was assumed 2 months before stimulation until pick-up day. Monofollicular fluids were collected and frozen. After embryo transfer, the fluids from the follicles generating the transferred embryos were thawed and analyzed.ResultsFollicular homocysteine showed a negative correlation with clinical pregnancy both in the whole population (r = − 0.298; p = 0.041) and in controls (r = − 0.447, p = 0.053). The support achieved a non-significantly lower concentration of follicular homocysteine (median [IQR]–7.6 [13.2] vs 24.3 [22.9]). Supplemented patients required far less FSH for stimulation (1650 [325] vs 2250 [337], p = 0.00002) with no differences in the number of oocytes collected, MII rate, and fertilization rate. Supplemented patients enjoyed higher blastocyst rate (55% [20.5] vs 32% [16.5]; p = 0.0009) and a trend for improved implantation rate (64% vs 32%; p = 0.0606). Clinical pregnancy rates were 58% vs 33% in controls (p = ns).ConclusionFollicular homocysteine is a suitable reporter that might be investigated as a tool for oocyte-embryo selection. A diet enriched with methyl donors may be useful in PCOS and supplements may also help. These findings may be also true for non-PCOS women, which warrants investigation.The study was approved by the Acibadem University Research Ethics Committee (2017–3-42). Clinical trial retrospective registration number ISRCTN55983518.

Blastocyst production using a new equine FSH for in vitro maturation

Currently, the majority of the equine ART laboratories collect germinal vesicle (GV)-stage oocytes to perform ICSI, and the gonadotrophins used for in vitro maturation are mainly porcine, bovine, or human. In this study, the effect of two different concentrations of equine FSH (eFSH) in the maturation medium was evaluated. Cumulus-oocytes complexes (COCs) were collected via ultrasound-guided transvaginal follicle aspiration and randomly allotted to the groups. The control group was our standard maturation medium (BMM - TCM199-Earle's salts, 10% FBS, and additives) with human gonadotrophins added (Lazzari.et.al. Journal of Equine Veterinary Science. 2020; 89:103097); experimental groups were composed of BMM with eFSH added (0.01 and 0.05 IU/mL). After holding, COCs were transferred to maturation medium and cultured for 36h. ICSI was performed, and presumptive zygotes were cultured. The cleavage rate and number of cells were evaluated on D3, blastocyst formation and embryo quality, classified on A-B-C grade scale (adapted from Lewis.et.al. Reproduction, Fertility and Development. 2023; 35: 338-351), were evaluated from D6-10. Differences were analyzed by Fisher's Exact Test (significance level P<0.05). There was no difference (P>0.05) in degenerated, GV-stage, or MI oocytes after maturation for control, 0.01, and 0.05, respectively. The number of oocytes that reached the (meiosis) MII-stage was lower (P<0.05) for 0.01 U/mL (56.5%, 35/62)than the control (73.2%, 52/71), and also tended to be lower (P=0.0985) compared to 0.05 U/mL (71.2%, 47/66). There was no difference in cleavage or cell number among groups (Table 1). Group 0.01 U/mL had a higher (P<0.05) blastocyst rate (45.7%, 16/35) than control and 0.05 U/mL groups (28.8%,15/52; 25.5%, 12/47; respectively). No difference was found in embryo quality among groups. Although the 0.01 U/mL group showed a lower MII rate, it also showed numerically less 2-cell stage embryos, more 4-8 and 8-16 cells stage embryos, and more cleaved embryos. Additionally, the 0.01 U/mL group tended to have more high-quality embryos (grade A) than the control group and showed a higher total blastocyst rate compared to the other groups. Additional studies are required to establish an ideal maturation protocol using this new equine FSH.

Cadmium exposure impairs oocyte meiotic maturation by inducing endoplasmic reticulum stress in vitro maturation of porcine oocytes

Cadmium (Cd) is toxic metal that can induce various diseases, such as cardiovascular, nervous, and reproductive systems. This study investigated the effect of Cd exposure on porcine oocyte maturation and the underlying mechanism. Porcine cumulus-oocyte complexes were exposed various Cd concentration and tauroursodeoxycholic acid (TUDCA), an inhibitor of endoplasmic reticulum (ER) stress during in vitro maturation (IVM). After IVM, we evaluated meiotic maturation, ER stress, and oocyte quality by Cd exposure. Cd exposure inhibited cumulus cell expansion and meiotic maturation, increased oocyte degeneration, and induced ER stress. The levels of spliced XBP1 and ER stress-associated transcripts, markers of ER stress, were elevated in Cd-treated cumulus–oocyte complexes and denuded oocytes during IVM. Moreover, Cd-induced ER stress impaired oocyte quality by disrupting mitochondrial function and elevating intracellular reactive oxygen species levels while decreasing ER function. Interestingly, TUDCA supplementation significantly decreased the expression of ER stress-related genes and increased the quantity of ER compared with the Cd treatment. Additionally, TUDCA was also able to rescue excessive levels of ROS and restore normal mitochondrial function. Moreover, the addition of TUDCA under Cd exposure greatly ameliorated Cd-mediated detrimental effects on meiotic maturation and oocyte quality, including cumulus cell expansion and MII rate. These findings suggest that Cd exposure during IVM impairs the meiotic maturation of oocytes by inducing of ER stress.

Meiotic Development of Canine Oocytes from Poly-Ovular and Mono-Ovular Follicles after In Vitro Maturation

Poly-ovular follicles are defined as those with more than one oocyte present in single follicles. The occurrence frequency of this follicle type is higher in canines than that in other species. This study aimed to evaluate the in vitro meiotic maturation of dog oocytes from this follicle type in comparison to those from mono-ovular follicles of various sizes (small antral, medium antral, and large antral) considering different phases of the estrus cycle (anestrus, proestrus, estrus, and diestrus). Canine oocytes were obtained separately from the poly-ovular and mono-ovular antral follicles from the ovaries of adult females. In each experimental replicate, cumulus-oocyte complexes (COCs) from poly-ovular and mono-ovular follicles were incubated in supplemented TCM-199 at 38.5 °C and 5% CO2 for 72 h. After culturing, the meiotic development of each oocyte was evaluated using epifluorescence microscopy. Meiotic stages were classified into germinal vesicle (GV), germinal vesicle breakdown (GVBD), first metaphase (MI), and second metaphase (MII). Data were evaluated using an analysis of variance. Oocytes from poly-ovular follicles at all phases exhibited a higher (p < 0.05) percentage of oocytes arrested at the GV stage than those from mono-ovular follicles, showing the highest rate of GV in small antral follicles during anestrus. In contrast, there were no differences in MII rates (p < 0.05) in oocytes from mono-ovular and poly-ovular follicles during the estrus and diestrus phases in all sizes evaluated, with the highest MII rate in estrus. These results suggest that oocytes from poly-ovular follicles can resume meiosis at a slower rate than those from mono-ovular follicles; however, the maturation in vitro of such oocytes is possible. Furthermore, the relationship between the maturation capacity of oocytes from both poly-ovular and mono-ovular follicles depends on the ovarian cycle and follicular development.

Omega-3 fatty acids and L-carnitine prevent meiotic oocyte damage induced by follicular fluid from infertile women with endometriosis: an experimental study.

To assess whether follicular fluid (FF) from infertile women with endometriosis in advanced stages [moderate/severe (EIII/IV) without or with endometrioma (Endometrioma)] induce more oocyte damages than in early stages (minimal/mild: EI/II); and whether supplementation with L-carnitine (LC) and omega 3 (n3) can prevent these oocyte damages. Experimental study using bovine oocytes (obtained of ovaries from slaughterhouse), and human FF (samples were obtained during oocyte recovery for ICSI). Bovine oocytes were submitted to in vitro maturation (IVM) divided into 9 groups: no FF(No-FF), with 1% FF from infertile women without endometriosis (FFC), with EI/II, EIII/IV and Endometrioma, and with (or not) LC+n3 addition. After IVM, oocytes were fluorescently labelled and visualized by confocal microscopy to analyze chromosomes and spindle. FF from endometriosis decreased rate of normal MII (spindle assembly and chromosome alignment) compared to No-FF (87.2%) and FFC (87.2%). FFEIII/IV (80.7%) and FFEndometrioma (69.3%) decreased total MII rate compared to No-FF (91.9%) and FFC (89.2%), and FFEndometrioma had lower total MII rate compared to other groups. LC+n3 increased MII rate in the FFEIII/IV (80.7% vs. 90.8%) and the Endometrioma (69.3% vs. 86.4%), and it prevented damages in spindle and chromosomes in MII oocytes in the FFEI/II group (62.2% vs. 84.5%) and the FFEIII/IV group (70.2% vs. 84.1%). FF of endometriosis damaged the meiotic spindle of bovine MII oocytes. EIII/IV led to impaired nuclear maturation; FF from women with endometrioma had further negative impact in oocyte maturation. LC+n3 completely prevented the effects of FF from women with endometriosis on oocyte.

Metabolic enhancement of the one carbon metabolism (OCM) in bovine oocytes IVM increases the blastocyst rate: evidences for a OCM checkpoint

The one carbon metabolism (OCM) has a primary role in the process of oocyte maturation. In this study bovine oocytes were cultured for 24 h, up to MII stage, with standard medium supplemented or not with 8 metabolic enhancers of the OCM and the MII and blastocyst rate were compared. Additional analyses were performed on matured oocytes, cumulus cells, zygotes and blastocysts. The OCM supplementation increased the blastocyst rate derived from in vitro fertilization. The mitochondrial mass and DNMT3a protein expression were increased whereas DNA fragmentation decreased in matured oocytes. DNA methylation in female pronucleus of zygotes was increased. The supplementation did not directly affect the redox balance as ROS and GSH in matured oocytes and homocysteine in the spent medium were unchanged. The supplementation of the oocytes with metabolic enhancers of the OCM may increase the yield from the culture, likely due to improved DNA methylation and epigenetic programming. The lack of effects on MII rate with huge differences appearing at the blastocyst stage suggest the existence of a OCM metabolic check point that hampers oocytes progression to blastocyst post-fertilization, if they were not properly primed at the time of maturation.

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-674 Optimal aspiration pressure level of suction pump for oocyte retrieval in IVF-ET

Abstract Study question What is the appropriate suction pump pressure for oocyte retrieval in terms of oocytes and embryos quality? Summary answer The pressure of 150mmHg had a significantly higher number of oocytes retrieved compared with 120mmHg, but the oocyte quality and embryo quality were comparable. What is known already There is no conclusion on the optimal aspiration pressure level for oocyte retrieval in controlled ovarian stimulation (COS), and a variety of pressures between 100 and 200 mmHg are used, often based on manufacturer’s instructions. Study design, size, duration A retrospective analysis was performed on a total of 211 COS cycles of infertilie patients from September 2019 to December 2021 in a single tertiary center. 110 cycles were included in the 120 mmHg pressure group and 101 cycles were included in the 150 mmHg pressure group. Participants/materials, setting, methods Age, AMH, antral follicle count (AFC), the proportion of ICSI, the number of retrieved oocytes, the number of MII oocytes, MII rate, degeneration rate on day 1 of culture, and the number of embryos on day 3 of culture was compared and analyzed between the two groups. Variables such as Age, AMH, and AFC that may affect the results were corrected using analysis of covariance. Main results and the role of chance The number of retrieved oocytes was significantly higher in the 150 mmHg group compared to the 120 mmHg group (10.3 vs. 8.4, p = 0.016). Neither the number of MII oocytes nor the MII rate was significantly different between the two groups (6.1 vs. 5.5, p = 0.509 and 67.2% vs. 73.4%, p = 0.437 respectively). The degeneration rate on day 1 of culture and the number of embryos on day 3 of culture were also not significantly different between the two groups (6.2% vs. 4.4%, p = 0.282 and 5.7% vs. 4.7%, p = 0.109 respectively). Limitations, reasons for caution Since this study analyzed the appropriate pump pressure for oocyte retrieval in infertile patients, caution is needed in extending the study results to patients undergoing oocyte retrieval for the purpose of fertility preservation. Wider implications of the findings The results of this study revealed that oocyte collection could be efficiently performed without damaging the oocytes even with a higher aspiration pressure of 150 mmHg. Trial registration number not applicalbe

P-685 Optimal aspiration pressure level of suction pump for oocyte retrieval in IVF-ET

Abstract Study question What is the appropriate suction pump pressure for oocyte retrieval in terms of oocytes and embryos quality? Summary answer The aspiration pressure of 150mmHg had a significantly higher number of oocytes retrieved compared with 120mmHg, but the oocyte quality and embryo quality were comparable. What is known already There is no conclusion on the optimal aspiration pressure level for oocyte retrieval in controlled ovarian stimulation (COS), and a variety of pressures between 100 and 200 mmHg are used, often based on manufacturer’s instructions. Study design, size, duration A retrospective analysis was performed on a total of 211 COS cycles from September 2019 to December 2021 in a single tertiary center. The analysis was performed on patients who underwent COS for the purpose of infertility treatment. Participants/materials, setting, methods 110 cycles were included in the 120 mmHg pressure group and 101 cycles were included in the 150 mmHg pressure group. Age, AMH, antral follicle count (AFC), the proportion of ICSI, the number of retrieved oocytes, the number of MII oocytes, MII rate, degeneration rate on day 1, and the number of embryos on day 3 were compared between the two groups. Variables such as Age, AMH, and AFC were corrected using analysis of variance. Main results and the role of chance The number of retrieved oocytes was significantly higher in the 150 mmHg group compared to the 120 mmHg group (10.3 vs. 8.4, p = 0.016). Neither the number of MII oocytes nor the MII rate was significantly different between the two groups (6.1 vs. 5.5, p = 0.509 and 67.2% vs. 73.4%, p = 0.437 respectively). The degeneration rate on day 1 and the number of embryos on day 3 were also not significantly different between the two groups (6.2% vs. 4.4%, p = 0.282 and 5.7% vs. 4.7%, p = 0.109 respectively). Limitations, reasons for caution Since this study analyzed the appropriate pump pressure for oocyte retrieval in infertile patients, caution is needed in extending the study results to patients undergoing oocyte retrieval for the purpose of fertility preservation. Wider implications of the findings The results of this study revealed that oocyte collection could be efficiently performed without damaging the oocytes even with a higher aspiration pressure of 150 mmHg. Trial registration number Not applicable

The associations of serum metals concentrations with the intermediate and pregnancy outcomes in women undergoing in vitro fertilization (IVF)

BackgroundToxic and essential trace elements are reported to have impact on female fertility. However, studies on the potential synergistic or antagonistic effects of metal mixtures on IVF outcomes remain limited. ObjectiveTo evaluate whether serum concentrations of metals, individually and as mixtures, are associated with pregnancy outcomes in women undergoing IVF. MethodsIn a prospective birth cohort study about IVF from the First Affiliated Hospital of Anhui Medical University (n = 1184), we measured the concentrations of serum metals by ICP-MS according to a previously established method. Oocyte/embryo development indicators and follow-up results were also collected. The individual and joint effects of metals were estimated using logistic regressions and Bayesian kernel machine regressions (BKMR). ResultsAt embryonic stage, we found negative associations between the serum lead (Pb) (β = −0.14, 95%CI: −0.32, −0.04) and cadmium (Cd) (β = −0.24, 95%CI: −0.39, −0.09) concentrations and the high-quality embryos rate; and positive associations between the serum cobalt (Co) (β = 0.18, 95%CI: 0.05, 0.31) and selenium (Se) (β = 0.17, 95%CI: 0.06, 0.41) concentrations and the MII rate. Regarding to the pregnancy outcomes, the serum Pb was negatively related with successful implantation (OR=0.85, 95%CI: 0.77, 0.94) and clinical pregnancy (OR=0.95, 95%CI: 0.91, 0.99); and positively associated with spontaneous abortion (OR=1.39, 95% CI: 1.02, 1.91). The BKMR analysis showed linear or parabolic associations between the metal mixtures and pregnancy outcomes, with Pb showing the highest posterior inclusion probabilities. ConclusionsThe toxic (Pb, Cd) and essential (Co, Se) metals could be incorporated as simultaneous predictors of IVF outcomes including potential antagonistic effects, in which Pb exhibits major contributions.

Response of Lettuce to Silver Nanoparticles Under Drought Conditions

Nano-fertilization is an emerging technology to improve agricultural productivity under diverse ecosystems. Our study was conducted to investigate the effects of foliar fertilization of silver nanoparticles (AgNPs) on the growth, enzyme activities and water use efficiency (WUE) of lettuce (Lactuca sativa, L.) under drought conditions. A pot culture study with 4 AgNPs rates × 2 stress levels in a factorial combination of randomized complete block (RCB) design was conducted. The AgNPs levels were 0 (control), 25, 50, 75 and 100 mg/L) were imposed on lettuce seedlings under ambient and simulated drought (~50% field moisture capacity of soil) conditions. Lettuce plant height, total yields, leaf relative water content (RWC), chlorophyll content, membrane injury (MII), glutathione S-transferase (GST), glutathione reductase (GR), carboxylesterase (CaE), total phenolics and flavonoids and WUE were determined and/or calculated. The results showed that GST activity reduced in treatments of AgNPs + stress compared to stressed plants. In treatment of 100 mg L−1 AgNPs + stress, GR activity increased in treatments of 25, 50 and 75 mg L−1 AgNPs, but showed a significant decrease in 100 mg L−1 AgNPs + stress. CaE activity enhanced in 100 mg L−1 AgNPs + stress (about 1.55-fold) compared to stressed plants. Total flavonoid and phenolic contents were the highest in 50 mg L−1 AgNPs + stress. It was not obtained significant effects in the WUE rates. In 100 mg L−1 AgNPs, MII rates were the highest, and RWC rates were the lowest. Leaf width, plant height and total yield decreased at doses of AgNPs.