Number Of Cumulus-oocyte Complexes Research Articles

Progestin primed ovarian stimulation using dydrogesterone from day 7 of the cycle onwards in oocyte donation cycles: a longitudinal study

Does a progestin-primed ovarian stimulation (PPOS) protocol with dydrogesterone from cycle day 7 yield similar outcomes compared with a gonadotrophin-releasing hormone (GnRH) antagonist protocol in the same oocyte donors? This retrospective longitudinal study included 128 cycles from 64 oocyte donors. All oocyte donors had the same type of gonadotrophin and daily dose in both stimulation cycles. The primary outcome was the number of cumulus-oocyte complexes (COC) retrieved. The number of COC retrieved (mean ± SD 19.7 ± 10.8 versus 19.2 ± 8.3; P = 0.5) and the number of metaphase II oocytes (15.5 ± 8.4 versus 16.2 ± 7.0; P = 0.19) were similar for the PPOS and GnRH antagonist protocols, respectively. The duration of stimulation (10.5 ± 1.5 days versus 10.8 ± 1.5 days; P = 0.14) and consumption of gonadotrophins (2271.9 ± 429.7 IU versus 2321.5 ± 403.4 IU; P = 0.2) were also comparable, without any cases of premature ovulation. Nevertheless, there was a significant difference in the total cost of medication per cycle: €898.3 ± 169.9 for the PPOS protocol versus €1196.4 ± 207.5 (P < 0.001) for the GnRH antagonist protocol. The number of oocytes retrieved and number of metaphase II oocytes were comparable in both stimulation protocols, with the advantage of significant cost reduction in favour of the PPOS protocol compared with the GnRH antagonist protocol. No cases of premature ovulation were observed, even when progestin was started later in the stimulation.

Phenotypic and Genetic Analyses of In Vitro Embryo Production Traits in Chinese Holstein Cattle.

Ovum pick up and in vitro embryo production (OPU-IVEP) is an essential technique in the dairy industry. The production efficiency of OPU-IVEP is significantly influenced by various factors, and phenotypic and genetic characteristics are highly variable in different populations. The objectives of this study were (1) to reveal the phenotypic characteristics, including population distribution, and impacts of donor age and month on in vitro embryo production and (2) to estimate genetic parameters for five in vitro embryo production traits in Chinese Holstein cattle. A total of 7311 OPU-IVEP records of 867 Holstein heifers from August 2021 to March 2023 were collected in this study. Five in vitro embryo production traits were defined, including the number of cumulus-oocyte complexes (NCOC), the number of cleaved embryos (NCLV), the number of grade I embryos (NGE), and the proportion of NCLV to NCOC (PCLV) and NGE to NCOC (PGE). A univariate repeatability animal model was employed to estimate heritability and repeatability, and a bivariate repeatability animal model was employed to estimate the genetic correlations among five in vitro embryo production traits. It was found that the in vitro embryo production traits were significantly influenced by season, as the NGE and PGE were significantly decreased from June to August. In addition, the production efficiency of OPU-IVEP was also influenced by donor age. On the observed scale, the estimates of heritability were 0.33 for NCOC, 0.24 for NCLV, 0.16 for NGE, 0.06 for PCLV, and 0.10 for PGE, respectively. On the log-transformed scale, the estimates of heritability of NCOC, NCLV, and NGE were 0.34, 0.18, and 0.13. The genetic correlations among NCOC, NCLV, and NGE ranged from 0.61 (NCLV and NGE) to 0.95 (NCOC and NCLV), considering both scales. However, there were low genetic correlations between NCOC and proportion traits (PCLV and PGE) on both the observed scale and the log-transformed scale. In the end, the variation in Chinese Holstein cattle was found to be considerable. The EBV value and average NCOC, NGE, and PGE for the top 10% donors presented extreme differences to those for the bottom 10% donors for NCOC (24.02 versus 2.60), NGE (3.42 versus 0.36), and PGE (30.54% versus 3.46%). Overall, the results of this study reveal that in vitro embryo production traits are heritable with low to high heritability, and the count traits (NCOC, NCLV, and NGE) and proportion traits (PCLV and PGE) reflect different aspects of in vitro embryo production and should be incorporated into genetic selection for improving the embryo production efficiency of dairy cattle.

O-133 Follicular flushing increases the number of cumulus oocyte complexes retrieved. A Randomized Controlled Trial

Abstract Study question Does follicular flushing increase the number of cumulus oocyte complexes (COCs) retrieved compared to single aspiration? Summary answer Follicular flushing increases the number of COCs retrieved compared to single aspiration. What is known already On the basis of published meta-analyses, follicular flushing does not seem to increase the number of COCs retrieved and the probability of pregnancy. However, eligible RCTs in these meta-analyses are characterized by significant heterogeneity regarding the population studied, the needle type and lumen diameter, the aspiration pressure and the number of flushing attempts performed. Moreover, most of the studies do not offer information regarding the flow rate of the aspiration system used. A constant flow rate has been employed in a single RCT, in which a higher number of COCs was retrieved after follicular flushing compared with single aspiration. Study design, size, duration This is a single-center, randomized controlled trial, performed between July and December 2022. One hundred and five patients undergoing oocyte retrieval for intracytoplasmic sperm injection, aged &amp;lt;43 years, with BMI 18-35kg/m2 were included in the study. Random allocation of each ovary to flushing or single aspiration was performed by a study nurse on the day of oocyte retrieval, using a computer generated randomization list. Patients could enter the study only once. Participants/materials, setting, methods All follicles ≥11 mm were aspirated with the same double-lumen needle, using the same aspiration pressure (190 mmHg) to achieve a flow rate of 0.42 ml/sec. If no COC was found in the initial aspirate, flushing was performed in the corresponding group until a COC was retrieved, or up to a maximum of five times. The primary outcome measure was the number of COCs retrieved per ovary randomized. Values are expressed as median (interquartile range). Main results and the role of chance Significantly more COCs were retrieved per ovary randomized in the follicular flushing versus the single aspiration group, in all patients [5 (7) versus 2 (3), p &amp;lt; 0.001, respectively], in patients with high [9 (3) versus 5 (4), p &amp;lt; 0.001, respectively], normal [5 (2) versus 2 (3), p &amp;lt; 0.001, respectively] and low [1 (1) versus 1 (1), p &amp;lt; 0.001, respectively] ovarian response. No COCs were retrieved in 2.9% of the ovaries in the flushing group versus 16.2% of the ovaries in the single aspiration group (p &amp;lt; 0.001). The oocyte retrieval rate, which was defined as the ratio of COCs retrieved to the number of aspirated follicles ≥11 mm, was significantly higher in the follicular flushing versus the single aspiration group, in all patients [88.9% (25) versus 45.5% (37.5), p &amp;lt; 0.001, respectively], as well as in patients with high [81.8% (15.1) versus 45.5% (25.0), p &amp;lt; 0.001, respectively], normal [85.7% (28.6) versus 40% (30.0), p &amp;lt; 0.001, respectively], and low [100% (0) versus 50% (100), p &amp;lt; 0.001, respectively] ovarian response. No significant differences were observed regarding maturation rate, fertilization rate and the proportion of good quality embryos on day 2 in all patients as well as in patients with high, normal and low ovarian response. Limitations, reasons for caution The current study design, by randomizing ovaries instead of patients, eliminates heterogeneity attributed to multiple confounders and allows a more accurate evaluation of the true effect of follicular flushing on the number of COCs retrieved. However, it does not allow the detection of its effect on the probability of pregnancy. Wider implications of the findings This is the first RCT to show that follicular flushing increases the number of COCs retrieved compared to single aspiration, independently of ovarian response. The data presented suggests that follicular flushing plays an important role in increasing the efficacy of oocyte retrieval. Trial registration number NCT 05473455

P-258 In-vitro oocyte maturation (IVM) in the ART clinic – What can we learn from our failed cycles?

Abstract Study question Why do some IVM cycles remain unsuccessful? Which factors contribute to failure in producing a usable embryo in IVM cycles? Summary answer Failure to obtain usable embryos after IVM was associated with oocyte dysfunction, inappropriate patient selection and unanticipated low oocyte yield. What is known already In-vitro maturation of oocytes retrieved from antral follicles has been offered as a mild-ART-approach to subfertile women with increased functional ovarian reserve, ovarian resistance to gonadotropins and in the context of fertility preservation. When using monophasic IVM culture systems, oocyte maturation, embryological development and pregnancy outcomes are generally lower compared to conventional ART (cART). Women who yield high numbers of cumulus oocyte complexes from a single egg retrieval procedure, such as those with polycystic ovary syndrome (PCOS), have been considered the best candidates for IVM, to compensate for the lower maturation rate and lower embryo yield from IVM cycles. Study design, size, duration This was a single-tertiary center, retrospective cohort study between May 2018 and February 2022 including 277 patients: 53 patients with failed IVM and 224 patients with a successful IVM cycle (inter-patient analysis). All ART cycles from patients with one failed IVM were investigated (intra-patient analysis). Failed IVM cycles were grouped: (i) ≤20% oocyte maturation; (ii) fertilization failure; (iii) embryo development failure. Patients with FSH resistance, fertility preservation or egg donation were excluded. Participants/materials, setting, methods Only patients with AMH &amp;gt;2.27 ng/ml were included. Oocytes retrieved in minimally stimulated non-hCG triggered IVM cycles were matured in a monophasic IVM system. Embryos were vitrified at cleavage stage and transferred in a hormonally substituted (HRT) cycle. Baseline patient characteristics, endocrinology and embryological data were analyzed. Intra-patient analysis was done using all ART cycles (failed IVM, IVM and cART) performed in one patient. Main results and the role of chance Inter-patient analysis revealed similar age, BMI and free testosterone, whereas AMH was lower in patients with a failed cycle (8.8 ± 3.5 vs 10.9 ± 6.4 ng/mL, p = 0.03). As expected, maturation, fertilization and useable embryos differed significantly in failed vs successful cycles: respectively 31% vs 48%; 40% vs 65%; 0% vs 50%; p &amp;lt; 0.0001. Out of 53 patients with a failed IVM cycle, 6 had oocyte maturation problems, 11 had failed fertilization and 36 had impaired embryological development. Intra-patient analysis revealed that in the failed oocyte maturation group, 2/6 patients had maturation problems that persisted in cART cycles, indicating oocyte dysfunction. Similarly, recurrent fertilization failure was noted in 3/11 patients, due to insufficient sperm quality (n = 2). In 8/36 patients, insufficient embryo quality was observed both after IVM and cART. Hence 13/53 (24.5%) failed IVM cycles were attributed to intrinsic patient characteristics. A subgroup of patients with a failed IVM cycle had normal maturation (3/6), fertilization (7/11) or good embryos (18/36) in other IVM or cART cycles. Factors associated with cycle failure included technical issues during egg retrieval resulting in unanticipated low oocyte yield (n = 14/40), and suboptimal inclusion criteria (no PCOS, n = 3/40). Limitations, reasons for caution This was a retrospective study and holds the possibility of unmeasured confounding factors. Wider implications of the findings Risk factors for IVM failure should be identified (e.g. non-PCOS, poor operator experience) and any underlying intrinsic patient conditions should be managed within a multi-disciplinary team. Optimal patient selection is key. Analysis of failed cycles helps clinicians to estimate the prognosis of future ART cycles in women with elevated AMH. Trial registration number not applicable

P-658 Clinical outcomes from ART in predicted hyperresponders: in-vitro maturation of oocytes versus conventional ovarian stimulation

Abstract Study question Do ongoing pregnancy rates (OPR) differ in predicted hyperresponders undergoing ART after in-vitro maturation of oocytes (IVM) compared with conventional ovarian stimulation (COS)? Summary answer One cycle of IVM is non-inferior to one cycle of COS in women with serum AMH levels &amp;gt;10ng/mL. What is known already Women with high antral follicle count (AFC) and elevated serum AMH levels, indicating an increased functional ovarian reserve, are prone to hyperresponse during ART treatment. To avoid iatrogenic complications of COS, IVM has been proposed as a mild-approach alternative treatment in predicted hyperresponders, including women with polycystic ovary syndrome (PCOS) who are eligible for ART. To date, inferior pregnancy rates from IVM compared to COS have hampered the uptake of IVM by ART clinics. However, it is unclear whether the efficiency gap between IVM and COS may differ depending on the extent of AMH elevation. Study design, size, duration This study is a retrospective cohort analysis of clinical and laboratory data from the first completed highly purified human menopausal gonadotropin (HP-hMG) primed, non-hCG triggered IVM or COS (FSH or HP-hMG stimulation in a GnRH antagonist protocol) cycle with ICSI in predicted hyperresponders ≤36 years of age at a tertiary referral university hospital. A total of 1707 cycles were included between January 2016 and June 2022. Participants/materials, setting, methods Predicted hyperresponse was defined as a serum AMH level &amp;gt;3.25ng/mL (Elecsys® AMH, Roche Diagnostics). The primary outcome was cumulative ongoing pregnancy rate (OPR) assessed 10-11 weeks after embryo transfer. The predefined non-inferiority limit was -10.0%. The analysis was adjusted for AMH strata. Time-to-pregnancy (TTP), defined by the time interval between egg retrieval and date of successful embryo transfer, was a secondary outcome. Statistical analysis was performed using a multivariable regression model controlling for potential confounders. Main results and the role of chance Data from 463 IVM cycles were compared with those from 1244 COS cycles. Women in the IVM group more often had a diagnosis of Rotterdam PCOS (434/463, 93.7%) compared to those undergoing COS (522/1193, 43.8%), were significantly younger (29.5 years vs 30.5 years, p ≤ 0.001), had a higher BMI (25.7kg/m² vs 25.1kg/m², p ≤ 0.01) and higher AMH (11.6ng/mL vs 5.3ng/mL, p ≤ 0.001). Although IVM cycles yielded more cumulus oocyte complexes (COCs) (25.8 COC vs 15.0 COC, p ≤ 0.001), both groups had similar numbers of mature oocytes (MII) (11.9 MII vs 10.6 MII, p = 0.9). In the entire cohort, non-adjusted cumulative OPR from IVM was significantly lower (198/463, 42.8%) compared to COS (794/1244, 63.8%), p ≤ 0.001. When analysing OPR across different serum AMH strata, cumulative OPR in both groups converged with increasing serum AMH, and OPR from IVM was non-inferior compared to COS from serum AMH levels &amp;gt;10ng/mL onwards (113/221, 51.1% [IVM]; 29/48, 60.4% [COS]). Time-To-Pregnancy was shorter in the IVM group compared to COS (11.3 weeks vs 21.0 weeks, p ≤ 0.001). Multivariable regression analysis adjusting for ART type, age, BMI, oocyte number and PCOS phenotype showed that the number of COCs was the only parameter associated with OPR in predicted hyperresponders with a serum AMH &amp;gt;10ng/mL. Limitations, reasons for caution These data should be interpreted with caution as the retrospective nature of the study holds the possibility of unmeasured confounding factors. Patients enrolled in a clinical IVM program received one-to-one care, in contrast with COS patients who received standard care, which may have influenced TTP. Wider implications of the findings Among subfertile women who are eligible for ART, IVM and COS resulted in comparable reproductive outcomes in a subset of women with a serum AMH &amp;gt;10ng/mL. These findings should be corroborated by an RCT comparing both treatments in selected patients with elevated AMH. Trial registration number Not applicable

O-294 Dual trigger versus hCG-only trigger in ICSI patients: an analysis of 8500 cycles

Abstract Study question Among patients undergoing ovarian stimulation for ICSI, does dual trigger with hCG and GnRH agonist offer any benefit over mono trigger with hCG alone? Summary answer Dual trigger is associated with a higher number of mature oocytes without any impact on the pregnancy rate. What is known already In ART cycles, final oocyte maturation and resumption of meiosis are generally triggered by the administration of hCG as a surrogate for the natural LH surge. Several studies have investigated the role of a combination of a bolus of GnRH agonists and hCG to mimic the natural peak of endogenous LH and FSH, without clear answers regarding clinical benefit. A meta-analysis showed highernumbers of good-quality embryos and increased ongoing pregnancy rate after dual trigger and a recent systematic review demonstrated significantly higher live birth rate (LBR) per cycle after administration of dual trigger compared to hCG-only trigger. Study design, size, duration The study was a retrospective, single-centre cohort study. In this study, 8,525 ICSI cycles were included between January 2017 and April 2022 at a tertiary referral University Hospital. We included all patients undergoing ICSI in a GnRH antagonist ovarian stimulation cycle. The mono trigger consisted of recombinant or highly purified urinary hCG. In the dual trigger group, a bolus of GnRH agonist was combined with the hCG trigger. Participants/materials, setting, methods Our cohort of 8,525 cycles was divided into two groups: Group A, the mono trigger group, (7,022 cycles), and Group B, the dual trigger group (1503 cycles). Patients who underwent IVF, pre-implantation genetic testing, oocyte donation, and fertility preservation were excluded. Patients with uterine anomalies and endocrine disorders were also excluded, as well as patients who received a triptorelin-only trigger because of hyperresponse. Main results and the role of chance There was no difference in the mean age between the dual vs mono trigger cohorts (35.93 ± 4.90 vs 35.52±4.85). The most common indication for ART was male factor infertility in both groups (21.36% in group B vs 27.11% in group A). Stimulation was shorter in the dual trigger cohort (10.41 vs 12.08 p = 0.04). The total number of cumulus oocyte complexes (8.26±5.32 vs 7.45±4.68, p &amp;lt; 0.001), mature oocytes (6.39±4.15 vs 5.99±3.80, p = 0.006) and fertilized oocytes (4.79±3.59 vs 4.33±3.27, p = &amp;lt;0.001) was higher in the dual trigger group compared to those in the mono trigger group. Day 5 embryo transfer was more prevalent in the dual trigger group (46.07% vs 33,84%, p &amp;lt; 0.001). Embryo utilization rate was higher in group A (61.37% ±31.98) than in group B (53.47% ±30.91 p = &amp;lt;0.001). Ongoing regnancy rate was similar in both groups (27.29% in group A vs 27.08% in group B). Pearson chi2, Mann Whitney and logistic regression tests were used for the analysis. Limitations, reasons for caution In spite of the large sample, this study is retrospective and holds the possibility of unmeasured confounders. Wider implications of the findings Although a dual trigger may increase the number of mature oocytes, this does not translate into a higher pregnancy nor live birth rate. Reduced oocyte competence of the “surplus” oocytes may underlie this observation. Further research should identify specific subgroups that may benefit from dual trigger across meaningful outcome parameters. Trial registration number not applicable

The Polymorphism Asn680Ser on the FSH Receptor and Abnormal Ovarian Response in Patients with Normal Values of AMH and AFC.

After the controlled ovarian stimulation (COS), the number of cumulus oocyte complexes collected is lower than predicted. The aim of this study is to understand if there is a possible reason for that deficient ovarian response. It was hypothesized that this is associated with the SNP (single-nucleotide polymorphism) of the FSH receptor (FSHr), specifically c.2039A > G, resulting in Asn680Ser. Two groups of patients were enrolled for this purpose: the normal (n = 36) and abnormal responses (n = 31). To predict the number of retrievable oocytes, according to the anti-Mũllerian hormone (AMH) and the antral follicle count (AFC), the following formula was applied in a log scale: the number of oocytes retrieved = 2.584 − 0.015 × (age) − 0.035 × (FSH) + 0.038 × (AMH) + 0.026 × (AFC). Then, when the number of oocytes collected was less than 50% of the calculated value, it was proposed that the patients result in an abnormal response. DNA sample blood was collected from the women, and then the genetic assessment for the Asn680Ser of the FSHr was evaluated in both groups. The differences between the two categories were statistically analyzed with an independent samples t test, a Mann−Whitney U test and a Chi-squared test. In a patient with an abnormal response, a significant prevalence of the amino acid serine at position 680 of the FSHr compared to the counterpart group (p < 0.05) was detected. In conclusion, according to the results, the genetic evaluation of the FSHr could represent an accurate and predictive feature for patients undergoing assisted reproductive technology treatment.

IVM: What are the RCTs Telling Us So Far?

Background: In Vitro Maturation (IVM) is a Assisted Reproduction Technology (ART) widely applied in livestock breeding, first attempted in human in the early nineties. While very successful in animals, the IVM technology has so-far not been widely adopted in human ART. The variation in IVM applications are mainly based on the use of gonadotrophins (FSH and/or HCG-trigger before oocyte collection) by the patient. The choice of the laboratory IVM protocols is dictated by the clinical preparation of the patient for oocyte pick-up. In this lecture the use of GV oocytes from fully stimulated cycles (“rescue IVM”) will not be considered. Aim: Summarize the learning from published clinical IVM studies in human from recent years, with a focus on the few randomized control trials (RCT). Method: Review by Pubmed search. Results: So far, the use of HCG administration before oocyte retrieval has been used by most ART centers, which adopted IVM (often in combination with few days of FSH, which makes it a ‘truncated IVF’). The largest series of latter kind (in 921 PCOS patients was reported by Ho et al. 2018) and yielded a 33.4% cumulative live birth rate over 12 months after 1 cycle. However available Cochrane review found no conclusive evidence for a positive role of HCG on number of retrieved or matured oocytes, nor on clinical outcome results. The role of FSH priming is proven in animal IVM studies, but clinically yet unproven in human IVM. The latest studies from Vuong L et al. (2020) applied as low as 150 IU HP-HMG for 2 days to aspirate a sufficient number of cumulus oocyte complexes (COC; 42 hours after the last FSH dose) to vitrify sufficient embryos of excellent quality for deferred transfer. A next RCTs in 351 PCOS patients was announced to evaluate the clinical requirement of FSH (Zheng X et al., 2020), however those results are still not published. Recent clinical data focused on the use of biphasic IVM culture systems in order to be capable of maturing COC from follicles smaller than 6 mm. After minimal FSH (300 IU total dose) and omission of HCG the large majority (&gt;80%) of compacted COC are aspirated from smaller than 6 mm follicles (Sanchez et al., 2019). Using the biphasic IVM culture principle (called ‘capacitation IVM’) several RCTs have been conducted to prove its superiority over ‘standard IVM’ in PCOS and ovarian tissue COC onco-fertility practice (Vuong L et al., 2020; Kirilova A et al., 2021). The CAPA-IVM culture method showed its potential compared to classical IVF standard of care in PCOM patients in a large non-inferiority RCT (546 patients): live birth rate after the first deferred transfer after Capa-IVM was 35% and conventional IVF 43%. After having observed low clinical pregnancies after non-HCG IVM (DeVos et al., 2011; Walls et al., 2015), additional small RCTs have confirmed the advantage of frozen vs fresh embryo transfer after CAPA-IVM (Vuong et al., 2021). Conclusion: Recent developments of IVM have been validated in few RCTs and IVM has been recently declared non-experimental after an extensive analysis of currently available data (ASRM Opinion paper published in 2021). The development of adequate culture media improves developmental capacity prior to meiotic maturation and brings the clinical results very close to the level of conventional IVF. The improved safety, the hugely simplified clinical approach and reduced cost structure could enhance access to ART across the globe for economically defavorised infertile couples.

P-238 Treatment options to overcome impaired fertilization and embryonic development caused by an oocyte-related deficiency in the Patl2-/- mouse model

Abstract Study question Can assisted oocyte activation (AOA) or spindle transfer (ST) overcome the lower fertilization and blastocyst rates observed in the Patl2-/- mouse model? Summary answer ST, but not AOA, has the potential to restore both normal fertilization and blastocyst rates in a mouse model with an oocyte-related deficiency. What is known already PATL2 is an RNA-transcriptional repressor involved in the regulation of maternal mRNAs during oocyte maturation. Mutations in PATL2 have been detected in patients with oocyte maturation arrest, reduced fertilization and embryo developmental arrest. Consequently, these patients are mostly referred to oocyte donation. Both AOA, inducing artificially calcium oscillations by calcium ionophores, and ST, replacing bad-quality oocyte cytoplasm with healthy cytoplasm, have been proposed to treat oocyte-related infertility. However, there is a lack of studies showing which oocyte-related infertility indications may benefit from which specific treatment. The Patl2-/- mouse model offers a unique opportunity to study the efficiency of these treatments. Study design, size, duration An experimental study using C57BL/6NTac-Patl2tm1a mice was conducted between April to December 2021. Breeding of heterozygous C57BL/6NTac-Patl2tm1a mice resulted in a total of 156 pups, from which 22 were homozygous C57BL/6NTac-Patl2tm1a ( Patl2-/-) females. Metaphase II (MII) oocytes were collected from Patl2-/- (test group) and Patl2+/+ (control group) females and used to evaluate different oocyte quality markers, as well as the efficiency of AOA and ST treatments to improve the oocyte-related subfertility observed in Patl2-/- mice. Participants/materials, setting, methods Four- to 12-week-old mice were subjected to ovarian hyperstimulation and oocyte collection, where oocyte maturation was assessed. Afterwards, MII oocytes were used to evaluate oocyte diameter, spindle abnormalities, activation rate (AR) and blastocyst rate (BR) after PIEZO-ICSI and calcium releasing capacity after SrCl2 exposure. Finally, Patl2-/- MII oocytes were treated with AOA, by direct exposure to SrCl2, or ST treatment, by transferring the Patl2-/- spindle to Patl2+/+ cytoplasm (previously enucleated) followed by SrCl2 exposure. Main results and the role of chance No difference in the number of cumulus-oocyte complexes (COCs) and mature MII oocytes was observed between 6 Patl2-/- mice (128 MII/159 COCs) and 5 Patl2+/+ mice (108 MII/132 COCs). However, Patl2-/- MII oocytes showed a significantly lower cytoplasm diameter (68,11 ± 2,25, n = 79) than Patl2+/+ oocytes (75,19 ± 2,09, n = 77, p-value&amp;lt;0,001), suggesting that Patl2 affects oocyte growth but does not seem to affect oocyte maturation. Nuclear maturity was not compromised either, as a similar percentage of oocytes with normal spindle was observed in Patl2-/- (48,4%, 15/31) and Patl2+/+ MII oocytes (50%, 18/36). Activation and blastocyst rates after PIEZO-ICSI were lower in Patl2-/- (AR = 45,5%, 5/11 and BR = 20,0%, 1/5) than in Patl2+/+(AR = 75,0%, 21/28 and BR = 52,4%, 11/21). In addition, total calcium released during oocyte activation after SrCl2 exposure was not different between Patl2-/- (AxF=6,88 AU, n = 30) and Patl2+/+ MII oocytes (AxF=5,04 AU, n = 28, p-value=0,087). In line with these results, AOA treatment did not improve activation and blastocyst rates in Patl2-/- oocytes (AR = 66,7%, 44/66 and BR = 45,5%, 20/44) compared to Patl2+/+ oocytes (AR = 96,5%, 110/114 and BR = 79,1%, 87/110, p-value&amp;lt;0,001). Nonetheless, when performing ST using Patl2-/- as spindle donors and Patl2+/+ as cytoplasm recipients, activation (8/8, 100,0%) and blastocyst rate (6/8, 75,0%) were restored to normal values. Limitations, reasons for caution This study describes preliminary results and is limited by its small sample size. In addition, we did not identify an increased number of immature oocytes and spindle abnormalities in Patl2-/- female mice in contrast to previously published reports. Wider implications of the findings ST could be offered to treat PATL2- related female-infertility caused by a cytoplasmic deficiency, to increase both fertilization and blastocyst rates. AOA would not be beneficial for these patients as calcium releasing capacity of Patl2-/- oocytes does not seem to be affected, suggesting a deficiency independent from calcium releasing machinery. Trial registration number Not applicable

O-127 Higher probability of live birth after testosterone pretreatment in women with poor ovarian response undergoing IVF: a systematic review and meta-analysis

Abstract Study question Does testosterone pretreatment increase the probability of pregnancy in poor responders undergoing ovarian stimulation with gonadotrophin-releasing hormone (GnRH) analogues and gonadotrophins for in-vitro fertilization (IVF)? Summary answer Testosterone pretreatment increases clinical pregnancy and live birth rates in poor responders undergoing ovarian stimulation for IVF. What is known already Androgens have been shown to stimulate early stages of follicular growth, increase the number of primary, pre-antral and antral follicles as well as increase ovarian sensitivity to follicle stimulating hormone (FSH). Although androgen supplementation has been evaluated in several randomized controlled trials (RCTs) and meta-analyzed in six systematic reviews until today currently no solid conclusions can be drawn regarding its effectiveness. Study design, size, duration A literature search was performed until September 2021 aiming to identify RCTs evaluating testosterone pretreatment in poor responders. Outcome measures included achievement of pregnancy, total dose of gonadotrophins required, duration of stimulation, estradiol levels, endometrial thickness and number of follicles ≥17 mm on the day of triggering final oocyte maturation, number of cumulus–oocyte complexes (COCs) retrieved, embryos transferred, metaphase II (MII) and 2-pronuclei oocytes (2pn) and the proportion of patients having an embryo transfer (ET). Participants/materials, setting, methods Eight RCTs published between 2006 and 2021 were analyzed, including 760 women. Pretreatment with transdermal testosterone gel was performed in all studies with a dose ranging from 10 to 12.5 mg/day for 10 to 56 days. In dichotomous data, estimates were expressed as risk ratio (RR) with 95% confidence intervals (CIs), using the fixed or random effects method. In continuous data, differences were pooled across resulting in a weighted mean difference (WMD) with 95% CI. Main results and the role of chance Testosterone pretreatment was associated with a significantly higher live birth (RR: 2.07, 95%CI: 1.09 to 3.92) and clinical pregnancy rate (RR: 2.25, 95%CI: 1.54) in women with POR undergoing IVF, while there was also a significant increase in the number of COCs retrieved (WMD: +0.88, 95% CI: +0.22 to + 1.54). Significantly less days to complete ovarian stimulation (WMD: -0.81 days, 95% CI: -1.46 to −0.16), a lower total dose of gonadotrophins (WMD: -368.8 IUs, 95% CI: −612.4 to -125.2), a thicker endometrium on the day of triggering final oocyte maturation (WMD: +0.83 mm, 95% CI: +0.13 to + 1.53) and a lower cancellation rate due to poor ovarian response (RR: 0.37, 95%CI: 0.20 to 0.71) were observed. No significant differences were observed in estradiol levels (WMD: -8.12 pg/mL, 95% CI: -118.2 to + 101.96), in the numbers of follicles ≥17 mm on the day of triggering final oocyte maturation (WMD: +0.82, 95%CI: -0.11 to + 1.74), of MII oocytes (WMD: +0.50, 95% CI: -0.17 to + 1.17), of 2pn oocytes (WMD: +0.49, 95% CI: -0.11 to + 1.10), of embryos transferred (WMD: +0.21, 95%CI: -0.07 to + 0.49) and in the proportion of patients with ET (RR: 1.00, 95% CI: 0.96 to 1.04). Limitations, reasons for caution The definition of poor ovarian response varied among studies and a considerable heterogeneity regarding the type, dose and duration of testosterone pretreatment was present. Although the present study is currently the largest meta-analysis evaluating testosterone pretreatment, the total number of patients is still not large enough to draw solid conclusions. Wider implications of the findings The current study suggests that the probability of pregnancy is increased in poor responders pretreated with transdermal testosterone. This increase, in the absence of other proven beneficial interventions in these patients, justifies the conduction of further relevant RCTs. Trial registration number CRD42021262098

Effects of PD-1 blockade on ovarian follicles in a prepubertal female mouse.

Immunotherapy has emerged at the forefront of cancer treatment. Checkpoint inhibitor pembrolizumab (KEYTRUDA), a chimeric antibody which targets programmed cell death protein 1 (PD-1), has been approved by the Food and Drug Administration (FDA) for use in pediatric patients with relapsed or refractory classical Hodgkin's lymphoma. However, there is currently no published data regarding the effects of pembrolizumab on the ovary of female pediatric patients. In this study, prepubertal immunocompetent and immunodeficient female mice were injected with pembrolizumab or anti-mouse PD-1 antibody. The number of primordial follicles significantly decreased post-injection of both pembrolizumab and anti-mouse PD-1 antibody in immunocompetent mice. However, no changes in follicle numbers were observed in immunodeficient nude mice. Superovulation test and vaginal opening experiments suggest that there is no difference in the number of cumulus-oocyte complexes (COCs) and the timing of puberty onset between the control and anti-mouse PD-1 antibody treatment groups, indicating that there is no effect on short-term fertility. Elevation of pro-inflammatory cytokine TNF-α following COX-2 upregulation was observed in the ovary. CD3+ T-cell infiltration was detected within some ovarian follicles and between stromal cells of the ovaries in mice following treatment with anti-mouse PD-1 antibody. Thus, PD-1 immune checkpoint blockade affects the ovarian reserve through a mechanism possibly involving inflammation following CD3+ T-cell infiltration.

Progesterone-induced blocking factor 1 and cytokine profile of follicular fluid of infertile women qualified to in vitro fertilization: The influence on fetus development and pregnancy outcome.

Introduction: Progesterone is essential for both the initiation and the maintenance of pregnancy. The immunological effects of progesterone are mediated by the progesterone-induced blocking factor (PIBF), which is an immunomodulatory factor with anti-abortive properties. The aim of the research was to establish the cytokine profile and PIBF1 concentration in follicular fluid (FF) of patients undergoing in vitro fertilization (IVF).Methods: Seventy-eight patients who qualified for IVF underwent a detailed medical interview, including the course of fertility treatment and physical, gynecological, and cytological examinations. The concentration of PIBF1, IL-18, IL-2, IL-4, IL-6, IL-10, interferon-γ (IFN-γ), IL-1α, IL-1β, IL-5, IL-8, and IL-15 in FF during ovarian puncture was measured using commercially available ELISA kits.Results: IL-1 beta concentration was lower in the FF of patients with successful IVF. IL-8 concentration in FF correlated with the number of cumulus-oocyte complexes (COC-1), metaphase II (MII), and top-quality embryos. PIBF1 concentration had a positive correlation with the number of MII and top-quality embryos. IL-2 and IL-6 concentrations were positively correlated with the number of COC-1 and MII. An important parameter in assessing the chances of successful IVF is the number of top-quality embryos achieved.Conclusion: Higher PIBF1 concentration in FF may indicate a greater possibility of successful IVF due to the higher number of top-quality embryos. IL-1 beta concentration was found to be lower in the FF of patients with successful IVF. Therefore, PIBF1 and IL-1 beta in FF could be candidates for a marker of successful IVF.

Contrasting effects of the Toll-like receptor 4 in determining ovarian follicle endowment and fertility in female adult mice

Toll-like receptor 4 (TLR4) is best known for its role in bacteria-produced lipopolysaccharide recognition. Regarding female reproduction, TLR4 is expressed by murine cumulus cells and participates in ovulation and in cumulus-oocyte complex (COC) expansion, maternal-fetal interaction and preterm labour. Despite these facts, the role of TLR4 in ovarian physiology is not fully understood. Therefore, the aim of the present study was to investigate the effects of TLR4 genetic ablation on mice folliculogenesis and female fertility, through analysis of reproductive crosses, ovarian responsiveness and follicular quantification in TLR4-/- (n = 94) and C57BL/6 mice [wild type (WT), n = 102]. TLR4-deficient pairs showed a reduced number of pups per litter (P = 0.037) compared with WT. TLR4-/- mice presented more primordial, primary, secondary and antral follicles (P < 0.001), however there was no difference in estrous cyclicity (P > 0.05). A lower (P = 0.006) number of COC was recovered from TLR4-/- mice oviducts after superovulation, and in heterozygous pairs, TLR4-/- females also showed a reduction in the pregnancy rate and in the number of fetuses per uterus (P = 0.007) when compared with WT. Altogether, these data suggest that TLR4 plays a role in the regulation of murine folliculogenesis and in determining ovarian endowment. TLR4 deficiency may affect ovulation and pregnancy rates, potentially decreasing fertility, therefore the potential side effects of its blockade have to be carefully investigated.

P–684 Impact of GnRH antagonist pretreatment on oocyte yield after ovarian stimulation: a retrospective analysis

Abstract Study question Does a 3-day pretreatment course with a GnRH antagonist in the early follicular phase increase the number of oocytes in a GnRH antagonist stimulation protocol? Summary answer The administration of 3 days of GnRH antagonist before starting ovarian stimulation in a GnRH antagonist protocol increases the number of COCs (Cumulus-Oocyte-Complexes). What is known already The GnRH antagonist protocol is characterized by higher gonadotropin and E2 serum levels at the start of ovarian stimulation (OS), compared with a long pituitary down regulation protocol. The unsuppressed FSH level at the start of a GnRH antagonist cycle allows the initial growth of follicles before addition of exogenous FSH, which may result in asynchrony of the follicular cohort. Menstrual administration of a GnRH antagonist can inhibit follicle growth and improve homogeneity of recruitable follicles. Previous studies showed a trend toward higher numbers of COCs and improved maturation and fertilization rates of retrieved oocytes. Study design, size, duration Retrospective single center crossover study, including consecutive women enrolled in an IVF program in a university hospital from January 2011 to December 2020. All women underwent one standard GnRH antagonist stimulation cycle (“standard cycle”) and one GnRH antagonist stimulation cycle preceded by early administration of GnRH antagonist for 3 days (“pretreatment cycle”). Women with basal progesterone levels &amp;gt;1.5ng/ml, and women undergoing oocyte freezing, oocyte donation or PGT were excluded. In total, 427 patients were included. Participants/materials, setting, methods Women were included when the pretreatment cycle occurred within a time interval of &amp;lt; 12 months following the start of stimulation in the standard cycle. The primary outcome was the total number of COCs. Main results and the role of chance The average female age was 35.1 ± 4.7 years. Indications for fertility treatment included unexplained infertility (34.3%), male-factor infertility (33.3%), age (16.9%), PCOS (8.2%) and endometriosis (2.6%). All cycles were divided into two groups: group 1 (standard, 427 cycles) and group 2 (pretreatment, 427 cycles). The mean duration of stimulation was similar in both groups (10.3 vs 10.3 days, p = 0.2). The starting dose of gonadotropin (196.8 vs 234.9IU, p &amp;lt; 0.001) and total amount of gonadotropin used (2000.7 vs 2415.2IU, p &amp;lt; 0.001) were higher in group 2. The total number of obtained COCs (6.2 vs 8.8 p &amp;lt; 0.001) and the number of mature oocytes (4.2 vs 6.4 p &amp;lt; 0.001) were significantly higher in group 2. The Generalized estimating equation (GEE) multivariate regression analysis showed that the pretreatment strategy had a significant positive effect on the number of COCs (coefficient 2.8, p value &amp;lt;0.001 after adjusting for the confounders age, indication of infertility, stimulation dose, type and total amount of gonatropins used). Limitations, reasons for caution Despite the large dataset, the presence of biases related to the retrospective study design cannot be excluded. Besides, the impact of GnRH pretreatment on pregnancy rate cannot be assessed because of the crossover design. Wider implications of the findings: A 3-day course of GnRH antagonist pretreatment increases the number of COCs obtained after OS. Furthermore, since the initiation of OS in a GnRH antagonist protocol relies on the occurrence of spontaneous menses, addition of three days of GnRH antagonist pretreatment may enhance scheduling flexibility without reducing efficacy. Trial registration number Not applicable

P–573 PGT-M for two or more disease carrier patients diagnosed after whole exome sequencing

Study questionCan whole exome sequencing (WES) before PGT-M identify previously unknown mutations for consanguineous couples having an increased risk of carrying more than one genetic disease?Summary answerWES has been successfully applied in combination with PGT-M by identifying new pathogenic mutations in addition to known gene mutations, extending the scope of PGT-M.What is known alreadyMost couples ignore their risk of being a carrier of an inherited genetic disease until they have an affected child. Rare, atypical, and undiagnosed autosomal-recessive disorders frequently occur in the offspring of consanguineous couples. Routine single gene diagnostic tests fail to detect any possible gene defects other than the clinically apparent one. Prospective WES or genetic carrier screening testing of consanguineous couples could identify couples who both are carriers of autosomal recessive diseases and thus encourage them to make informed reproductive decisions. Screening tests using NGS technology simultaneously sequence exons and exon-intron boundaries to determine disease carrier status.Study design, size, durationBetween January 2017 and October 2020, a total of 206 PGT-M couples applied to Istanbul Memorial Hospital ART Center. Of these couples, multigene PGT-M workups were carried for twelve couples who were carriers of more than one inherited disease. Eight couples were found to be carriers for two different diseases and four couples were carrying three diseases. All biopsies were performed at the blastocyst stage.Participants/materials, setting, methodsFor the 12 couples with multigene PGT-M workups the average female age was 31.0 ± 6.2. Nine of them initiated an ART cycle and the mean number of cumulus-oocyte complexes, metaphaseII oocytes, biopsied blastocysts and transferrable PGT-M embryos were 15 ± 6.9, 13.3 ± 6.3, 5.9 ± 2.0 and 2.9 ± 1.9, respectively. PGT-A was routinely performed for all couples with transferrable PGT-M tested embryos except one couple who refused PGT-A.Main results and the role of chanceA total of 28 different gene workups were completed for 26 genes. The inheritance mode of the 26 conditions was as follows: 20 autosomal recessive, four autosomal dominant and two X-linked recessive. Out of 12 couples, 9 of them initiated an ART cycle and transferrable embryos were found after PGT-M followed by PGT-A. Eight women had frozen embryo transfers resulting in five healthy babies (3 singletons and 1 twin), two pregnancies still ongoing and one biochemical miscarriage at the time of data collection. The couple who declined PGT-A testing prior to their frozen embryo transfer had anegative bhCG test. Three couples completed their workups but postponed their ART and PGT-M cycle due to Covid–19 pandemic.Limitations, reasons for cautionThe probability of finding at least one transferrable embryo after PGT-M is influenced by the inheritance mode of the disease. Late-onset diseases presumed to be caused by variants of unknown significance and polygenic diseases that are possibly influenced by environmental factors were not included in this study.Wider implications of the findings: With decreasing costs and improved availability of WES and genetic carrier screening panels, couples, especially consanguineous couples, who were previously shown to have one inherited disease may be offered to be screened for additional undiagnosed inherited diseases that may pose a threat for their offspring.Trial registration numberNot applicable

Clinical and Molecular Effects of GnRH Agonist and Antagonist on The Cumulus Cells in The In Vitro Fertilization Cycle.

Background:Gonadotropin-releasing hormone (GnRH) analogues have been extensively utilized in the ovarian stimulation cycle for suppression of endogenous rapid enhancement of luteinizing hormone (LH surge). Exclusive properties and functional mechanisms of GnRH analogues in in vitro fertilization (IVF) cycles are clearly described. This study was performed to evaluate clinical and molecular impacts of the GnRH agonist and antagonist protocols in IVF cycles. For This purpose, gene expression of cumulus cells (CCs) as well as clinical and embryological parameters were evaluated and compared between two groups (GnRH agonistand antagonist) during the IVF cycle. Materials and Methods:Twenty-one infertile individuals were enrolled in this study. Subjects were selected from two groups of GnRH agonist(n=10) treated patients and GnRH antagonist (n=11) treated individuals. The defined clinical embryological parameters were compared between the two groups. Expression of BAX, BCL-2, SURVIVIN, ALCAM, and VCAN genes were assessed in the CCs of the participants using the real-time polymerase chain reaction (PCR) technique.Results:The mean number of cumulus oocyte complex (COC), percentage of metaphase II (MII) oocytes, grade A embryo and clinical parameters did not show noticeable differences between the two groups. BAX gene expression in the CCs of the group treated with GnRH agonist was remarkably higher than those received GnRH antagonist treatment (P<0.001). The mRNA expression of BCL-2 and ALCM genes were considerably greater in the CCs of patients who underwent antagonist protocol in comparison to the group that received agonist protocol (P<0.001).Conclusion:Despite no considerable difference in the oocyte quality, embryo development, and clinical outcomes between the group treated with GnRH agonist and the one treated with antagonist protocol, the GnRH antagonist protocol was slightly more favorable. However, further clinical studies using molecular assessments are required to elucidate this controversial subject.

83 Response to follicle-stimulating hormone superstimulation in heifers with ovarian activity suppressed by active immunization against gonadotrophin-releasing hormone

In the present study, we evaluated the ovarian response to exogenous FSH stimulation in the absence of endogenous LH, using as experimental model heifers immunized against GnRH. Pubertal, cycling Nelore (Bos indicus) heifers were allocated into three experimental groups: (1) non-immunized, FSH stimulated (B−FSH+, n=5), (2) immunized, FSH stimulated (B+FSH+, n=5), and (3) immunized, nonstimulated (B+FSH−, n=5). Active immunization was obtained by 3 subcutaneous injections of 1.0mL anti-gonadotrophin-releasing hormone vaccine (Bopriva, Zoetis), given at 20-day intervals. Effective immunization was characterised by the absence of growing follicles &amp;gt;4mm or corpora lutea (CL) on the ovaries. Follicular wave emergence was synchronized in groups B+FSH+ and B+FSH− by follicle ablation, and in group B−FSH+ by using of a protocol consisting of an injection of 2mg of oestradiol benzoate and 0.5mg of sodium cloprostenol, and insertion of an intravaginal progesterone (P4) device (1g). Four days later (Day 0), groups B−FSH+ and B+FSH+ received 100mg of NIH-FSH-P1 (Folltropin-V, Vetoquinol), injected twice-a-day in 8 decreasing doses, and group B+FSH− received saline. Transvaginal ultrasonography (7.5MHz) was performed daily from Days 0 to 4 and the number and size of follicles were recorded. P4 devices of group B-FSH+ were removed at Day 3. All heifers underwent ovum pickup (OPU) at Day 4, and the cumulus–oocyte complexes (COC) recovered were graded for quality. Viable COC were used for invitro embryo production. The heifers were re-evaluated at Day 11 (7 days after OPU). The GLIMMIX procedure from SAS (SAS Institute Inc.) with repeated-measure statement was used to analyse the effects of group, day, and interactions; and the Chi-squared method was used to analyse binomial data. The results are shown as mean±s.e.m. A progressive increase in average follicle size was observed in groups B−FSH+ and B+FSH+ (P&amp;lt;0.0001), whereas no follicle growth was observed in group B+FSH− (P&amp;gt;0.05). Follicle growth rate was similar between groups B−FSH+ and B+FSH+, and both were greater than group B+FSH− (1.2±0.2 and 1.1±0.1 vs. 0.0±0.1 mm/d; P&amp;lt;0.0001). However, the smaller follicle size in group B+FSH+ at Day 0 resulted in smaller follicle size at Day 4, compared with group B−FSH+ (2.4±0.1 vs. 3.6±0.2 and 6.9±0.7 vs. 8.2±0.6mm, respectively; P&amp;lt;0.05). There was no (P&amp;gt;0.05) difference in the number of COC recovered among groups. The group B+FSH+ yielded fewer (P&amp;lt;0.01) COC of grades I and II and more (P&amp;lt;0.01) degenerated oocytes than groups B−FSH+ and B+FSH− (41.2% vs. 80.0% and 68.0%, and 34.0% vs. 19.8 and 7.0%, respectively). Nevertheless, blastocyst rates were similar (P&amp;gt;0.05) for B−FSH+, B+FSH+, and B+FSH− (57.1%, 45.9% and 44.2%, respectively). Residual follicles or luteal tissue were observed after OPU only in group B−FSH+, resulting in a significant difference in the size of ovaries between Days 0 and 11, compared with that of groups B+FSH+ and B+FSH− (3.7±1.4 vs. 0.2±0.2 and −0.2±0.2cm2, respectively; P&amp;lt;0.05). In summary, exogenous FSH supported follicle growth but did not improve oocyte quality in heifers immunized against GnRH. This research was funded by CAPES.

82 Invitro embryo production outcomes in adult goats is affected by season

In seasonal breeders, reproductive seasonality can have a substantial influence on the efficiency of assisted reproductive technologies. This study assessed seasonal effects on cleavage and blastocyst rates, as well as on quality of invitro-produced (IVP) goat embryos over 18 months. In total, 2348 (autumn: 811, spring: 404, summer: 639, and, winter: 494) cumulus–oocyte complexes (COC) were recovered from slaughterhouse ovaries during 49 replicates (autumn: 17, spring: 7, summer: 15, and, winter: 10) and matured in TCM-199 with 10ng mL−1 epidermal growth factor and 100µM cysteamine for 22h at 38.8°C (5% CO2 in air). Matured oocytes were fertilized with frozen/thawed semen in synthetic oviductal fluid (SOF) with 10% oestrus sheep serum. Sperm and oocytes were co-incubated for 16h at 38.8°C in a humidified atmosphere of 5% CO2 in air. Presumptive zygotes were cultured in SOF medium supplemented with bovine serum albumin (3mg mL−1) for 8 days at 38.8°C in a humidified atmosphere of 5% O2, 5% CO2 and 90% N2. At Day 2, 10% fetal calf serum was added to the culture droplets. The embryos produced were fixed and stained with Hoechst to count their total number of cells, under an epifluorescence microscope. The results of cleavage and blastocyst rates, including hatching rate, from each routine of IVEP were considered as replicates. These data were tested for normality by the Shapiro-Wilk test, before being subjected to ANOVA, followed by Tukey HSD test. The odds ratio (OR) among seasons (autumn: breeding; spring: anoestrus) were calculated. Values of P&amp;lt;0.05 were considered as significant, and the data reported are mean±s.e.m. Cleavage rate was lower (P&amp;lt;0.05) in spring (51±7.1%) than in either autumn (72±2.1%) or summer (71±2.0%) while winter (66±4.1%) had an intermediate value, being similar (P&amp;gt; 0.05) to all others. Indeed, greater possibility of cleavage was observed in autumn (OR: 2.43) and summer (OR: 2.39) compared with spring. The blastocyst rate from cleaved embryos was greater (P &amp;lt;0.05) in autumn (73±2.7%) than in spring (55±2.6%; OR: 2.18). As a result, the blastocyst formation rate from the initial number of COC entering IVM was greater (P&amp;lt;0.05) in autumn (52±2.5%) than in spring: 28±4.7%, summer: 45±2.3%, and winter: 42±2.1%; indeed, the spring season resulted in the lowest rate (P&amp;lt;0.05), compared with other seasons. Moreover, the OR in the blastocyst rate from initial number of COC was greater (P&amp;lt;0.05) in autumn compared with all other seasons and lower in spring compared with winter (OR: 0.54) and summer (OR: 0.48). There were no differences (P&amp;gt; 0.05) in the embryo hatching (mean: 66±2.0%) and blastocyst cell number (mean: 193±2.0 cells). In conclusion, the breeding season (autumn) leads to improved oocyte developmental competence, resulting in greater cleavage and blastocyst yield, whereas embryo quality remained similar throughout the year. Further studies at the molecular level might indicate the mechanisms involved and provide clues to alleviate the negative effect of season.

7 A record of 485 viable cumulus–oocyte complexes recovered and 165 viable embryos produced in a single ovum pickup session from a Senepol breed donor

The recovery of an exceptionally high number of cumulus–oocyte complexes (COC) in a single ovum pickup (OPU) session is rare, and thus seldom reported in the literature. In the present report, we describe one of these cases. The present data refers to an OPU session performed by author A. O. Resende in a commercial beef farm, located in the rural area of the city of Barretos, Brazil, on October 15, 2019. Breed heifers (N=12) underwent OPU at a random stage of oestrous cycle without any hormonal treatment. Follicle aspirations were performed following the standard operation procedures (SOP) established by ABS Pecplan OPU team, using a portable ultrasound (DP2200, Mindray do Brasil) equipped with a 7.5-MHz microconvex probe, a vacuum pump (WTA), and 18 gauge disposable Jelco catheter (Smith Medical). Invitro embryo production (IVP) was performed at Invitro Technologies laboratory, ABS Pecplan Uberaba, located 140km from the farm. On this day, 1312 oocytes were recovered from the 12 donors. However, a single heifer produced 485 viable COC, from over 500 total COC recovered. The COC from this donor were distributed in four groups, which underwent IVF with Y-sorted semen from four Senepol sires. The invitro embryo production was performed following the SOP established by invitro Technologies, ABS Pecplan. Embryo production was compared among sires by the chi-squared test. Of the 4 sires used during IVF, sire number 4 resulted in a higher blastocyst rate compared with the others (sire 1=25.4%, sire 2=30.6%, sire 3=33.9%, and sire 4=46.3%; P&amp;lt;0.01).The blastocyst rate differed (P&amp;lt;0.01) among sires: 25.4%a (31/122), 30.6%a (37/121), 33.9%a (41/121), and 46.3%b (56/121) for bulls A, B, C, and D, respectively. From the total of 165 viable embryos produced (34.0%), 120 were transferred fresh and the remaining 45 were vitrified using the SOP established by IVB Technologies, ABS. These results highlight the huge potential variation in antral follicle population and number of COC recovered from donors within the same population, breed, and reproductive status. It also shows the potential of IVP in the generation of offspring from high genetic indices.

A multilevel analysis identifies the different relationships between amino acids and the competence of oocytes matured individually or in groups

High-protein diets contribute to an increase in urea follicular concentrations associated with decreased fertility. Urea has been shown to interfere with the epidermal growth factor (EGF)/EGFR system, which has been shown to have a beneficial effect during in vitro maturation (IVM) of oocytes. Of note, the number of cumulus-oocyte complexes (COCs) in the maturation medium can change the maturation and the developmental competence of COCs. Therefore, it was hypothesized that, the presence of urea and EGF may have a differential effect on the depletion/appearance of AAs and competence of COCs matured individually (I-IVM system) or in groups (G-IVM system). In the G-IVM system, COCs increased consumption (depletion) of AAs compared with other groups in the presence of high-level urea (40 mg/dl) + EGF (10 ng/ml). In the I-IVM system, the non-cleaved COCs depleted more AAs than the cleaved COCs, in particular in the presence of urea. The combination of urea and EGF increased the depletion of AAs in the G-IVM system. However, the EGF abrogated the urea-induced depletion of AAs by the I-IVM COCs. The use of N-acetyl-l-cysteine as an EGFR inhibitor canceled urea-induced depletion of AAs. This shows the inhibiting effect of urea over the EGF/EGFR system. In the presence of urea + EGF, COCs had a lower degree of developmental competence than control in both I- and G-IVM systems. Arginine had the best predictive power to identify highly competent COCs in the G-IVM system, while glutamine was the best predictor of the cleavage in the I-IVM system. In conclusion, this multi-level study shows that COCs matured individually or in groups may have different association with AAs metabolism. These findings provide new insights into the relationships between AA metabolism and the subsequent developmental competence of COCs.