Total Oocytes Research Articles

174 Use of mesenchymal stem cell treatment to improve oocyte yield and invitro embryo production in cattle

Cumulative tissue damage and chronic inflammation associated with frequent ovum pickup (OPU) may lead to a progressive reduction in the number and quality of the oocytes recovered, particularly in donors with a high antral follicle count. The aim of this study was to evaluate the effect of an intraovarian treatment with mesenchymal stem cells (MSC) on oocyte yield, quality, and development potential during invitro embryo production (IVEP) in cattle donors undergoing repeated OPU. Mesenchymal stem cells were previously isolated from adipose tissue, cultured in Dulbecco's modified Eagle medium until reaching 80% confluence, isolated with trypsin, and frozen in liquid N2 until use. Characterisation of MSC was carried out according to the guidelines of the International Society for Cellular Therapy. Nelore (Bos indicus) cows (n=5) were used in this study, with the ovaries as replicates. The cows underwent eight OPU sessions at 15-day intervals, and the oocytes recovered were graded and used for IVEP with the semen of a single sire and batch under similar invitro culture conditions. To ensure a high inflammatory response, immediately after the fourth OPU session all ovaries received 30 additional punctures, performed with a 16-gauge Jelco needle. Six hours later, the left ovary of each cow was injected with 500µL of Dulbecco's modified phosphate buffered saline (control ovary) and the right ovary received 500µL of Dulbecco's modified phosphate buffered saline with 2.5×106 allogenic MSC (treated ovary). Oocyte yield and embryo production before and after treatment were recorded for each ovary and donor. Grade I blastocysts produced from control and treated ovaries were used for gene expression evaluation. Data was analysed using the repeated-measures procedure of SAS (SAS Institute Inc.) to account for the effects of treatment, time, and interactions. There was no difference (P>0.05) in any endpoint before treatment (sessions 1-4) between the right and left ovaries. Thus, differences between ovaries observed in OPU sessions 5-8 were assumed to be due to the treatment. After the injection of MSC, more total and viable oocytes were collected from the right ovaries compared with the left ovaries (15.3±2.2 vs. 8.7±1.2 (P<0.02) and 13.6±2.1 vs. 7.1±1.0 (P<0.01), respectively), resulting in more embryos produced invitro (7.6±1.2 vs. 3.6±0.6, respectively; P<0.01) as well as more initial and expanded blastocysts (1.4±0.3 vs. 0.4±0.1 and 4.4±0.9 vs. 2.1±0.4, respectively; P<0.04). The proportion of viable oocytes recovered from the right ovary after treatment was greater than that from the left ovary (89.1% vs. 81.5%; P<0.05). However, blastocyst rates did not differ between ovaries before or after treatment (50.4% vs. 55.5%: P>0.05). In the blastocysts produced from treated ovaries, SLC2A3 was overexpressed (P<0.04), whereas there was no difference for the expression of KRT8, PLAC8, SLC2A1, CASP3, PRDX3, or SOD2 (P>0.05), suggesting potential differences in glucose uptake and metabolism. In conclusion, intraovarian treatment with MSC improved oocyte yield and quality and may be an alternative to increase IVEP from donors under intensive OPU schedules. This research was supported by CNPq, CAPES, and Fazenda Grupo Esplanada.

200 Maturation method affects lipid accumulation in bovine oocytes

Invitro maturation is a key step in invitro embryo production, since its success will depend on the availability of good quality oocytes. Previous studies have shown that it is during this stage that the greatest accumulation of lipid droplets occurs, which is reflected in the amount of lipid present in embryos produced invitro. However, this is not observed when maturation is performed invivo. Therefore, we hypothesised that lipid accumulation would be avoided if oocyte maturation were carried out in ovarian follicles following intrafollicular transfer of immature oocytes (IFIOT). We compared lipid accumulation in oocytes matured invitro, invivo, and by IFIOT. A total of 90 Nellore heifers were distributed in 3 experimental groups: donors of immature oocytes (D-IMA), ovulators of IFIOT oocytes (D-OV), and superstimulated donors of invivo-matured oocytes (D-FSH). All animals rotated through all groups during the experiment. To obtain immature oocytes, the D-IMA were submitted to ovum pickup (OPU), in which aspiration medium was supplemented with 500μM 3-isobutyl-1-methylxanthine (IBMX), and, after selection, part of the oocytes were cultured invitro for 22h (MatF) and part were used for IFIOT (MatT). To perform MatT, the D-OV had their ovulation synchronized by a progesterone and benzoate oestradiol protocol, in which 30h after the implant removal, the IFIOT was performed on the dominant follicle. The D-FSH oocytes were stimulated with 80mg of FSH (Folltropin; Vetoquinol) over 4 days, every 12h, in decreasing doses. At the same time that the immature oocytes were placed in MatF and IFIOT, ovulation was induced with the gonadotrophin releasing hormone (GnRH) analogue (50µg of lecirelin) in D-OV and D-FSH groups. After 22h, matured oocytes were either removed from culture (MatF) or recovered from follicles by OPU (MatT and MatS). From the recovered oocytes of all groups, only those with a polar body were used for lipid droplet evaluation. To quantify lipid accumulation, denuded oocytes were fixed and stained with boron-dipyrromethene (Bodipy) 493/503 (20 µgmL−1) and evaluated by confocal microscopy. Captured images were evaluated in the ImageJ program (National Institutes of Health), and lipid content was determined by calculating the ratio of the area of the lipid droplets to total oocyte area. Data were analysed by ANOVA with statistical significance set at P<0.05. A total of 95 oocytes were evaluated: 25 immature (CT), 24 invitro (MatF), 30 invivo (MatS), and 16 invivo (MatT). The mean area containing lipid droplets in immature oocytes (14%±0.9) was similar (P>0.05) to that observed in both invivo maturation systems (MatS=17.26%±0.8 and MatT=14.11%±0.9). However, in the MatF oocytes, lipid content (24.34%±1) increased during maturation and was higher than in the other groups (P<0.05). We showed for the first time that oocytes matured by IFIOT are similar to those invivo matured with regard to lipid content, which may imply their superior quality over those matured invitro. This new maturation method opens new possibilities for biotechnologies that need to use mature oocytes, such invitro embryo production, oocyte and embryo cryopreservation, cloning, and transgenesis. This study was supported by FAP-DF and Capes.

180 Glucose versus fatty acids: Different energy supplies for the bovine oocyte

Studies show that optimal concentrations of fatty acids (FA) and glucose are crucial for proper oocyte development, maturation, and further embryo quality support (Sutton McDowall et al. 2010 Reproduction 139, 685-95, https://doi.org/10.1530/REP-09-0345; Sutton McDowall et al. 2014 Theriogenology 82, 95-103, https://doi.org/10.1016/j.theriogenology.2014.03.011). A balance between metabolism of FA and glucose in oocytes needs to be maintained; however, it is not clear what the preferable pathway of energy production is and how it affects the oocyte. The aim of the experiment was to selectively block pathways of either glucose or FA metabolism during IVM of bovine oocytes to reveal changes within oocyte lipid droplets (LD) under crucial changes in energy metabolism. Cumulus-oocyte complexes were matured under standard conditions (Warzych et al. 2007Mol. Reprod. Dev. 74, 280-289; https://doi.org/10.1002/mrd.20610) without FA and glucose supplementation. The experimental groups were (1) control (IVM in basic medium), (2) group with inhibited glucose metabolism [supplementation with 1.5 µM iodoacetate (IO, inhibitor of glycolysis) and 150 µM dehydroepiandrosterone (DHEA, inhibitor of pentose phosphate pathway)], and (3) group with inhibited FA metabolism (150 µM etomoxir supplementation, ETO). Oocytes after 24h of IVM were stained with boron-dipyrromethene (BODIPY) 493/503 dye (lipid droplets) and 4′,6-diamidino-2-phenylindole (DAPI; chromatin) and analysed using a confocal microscope (LSM 880 AiryScan FAST; Zeiss). Obtained data were analysed using the Kruskal-Wallis test. The MII rate decreased to 60.5% for IO+DHEA (P<0.05) and to 78% for the ETO group compared with the control (83.8%). The average LD area (% of total oocyte area) significantly decreased in both experimental groups (IO+DHEA 6.72±2.5, ETO 6.28±3.2; P<0.01) compared with control (8.7±3.6). Total lipid content (intensity of the fluorescence), was significantly lower in experimental groups (IO+DHEA: 1.17×106±3×105, ETO: 7.1×104±2×104 vs. control: 1.68×106±4.6×105; P<0.01). With regard to the total number of LD, only in the IO+DHEA group were significantly fewer LD noted (987±343; P<0.05), whereas the ETO group did not differ significantly (1146±414) compared with control (1148±357). The ETO group had significantly lower total lipid content (P<0.01) and higher total LD number (P<0.05) compared with IO+DHEA. The area of lipid droplets did not differ between the experimental groups. These results show that blocking glucose metabolism strongly affects the nuclear maturation process of the oocyte, limiting the number of oocytes that reach the MII stage. A significant decrease in total lipid content in the ETO group may suggest strong utilisation of lipids during the maturation process, when de novo synthesis of lipids is blocked. Accumulation of lipids in LD before maturation allows this process to be passed even under the applied FA metabolism inhibitory conditions. In contrast, inhibition of glucose metabolism negatively affects oocyte development strictly after inhibition conditions, indicating the greater significance of glucose for proper oocyte maturation. Further studies are being conducted to investigate the effect of inhibitory systems on lipids in bovine cumulus cells and further embryos. Funding for this study was provided by National Science Centre, Poland (project no 2017/27/B/NZ9/00904).

Age, body weight and ovarian function affect oocyte size and morphology in non-PCOS patients undergoing intracytoplasmic sperm injection (ICSI)

The size of oocytes was previously reported to be smaller in obese women with polycystic ovary syndrome (PCOS). In the present prospective cohort study, we sought to determine whether oocyte size and morphology are associated with patient characteristics in non-PCOS women. Oocyte and oolemmal diameter were measured, enlarged perivitelline space (PVS) and ooplasmic granulation were assessed in 308 MII oocytes from 77 IVF/ICSI couples. Statistical analysis was undertaken using SAS version 9.4 (SAS institute Inc., USA). Continuous values are presented as mean ± SD and compared using a two-sample t-test or Mann-Whitney U test as appropriate. Categorical parameters are presented as proportions and compared using a Fisher exact test. Logistic and linear regression models were used to control for the effect of age for categorical and continuous variables respectively. P-value < 0.05 was considered statistically significant. Patients presented with a mean age of 40.3±5.0 years, had a BMI of 25.1±6.1 kg/m2, median AMH levels of 0.6 ng/ml and produced a median of 4 oocytes. Mean total oocyte diameter was 163.2±7.4 μm (range 145.8–182.1 μm), while oolemmal diameter was 109.4±4.1 μm (range 98.5–122.3 μm). After adjusting for age and ovarian reserve increasing BMI was associated with decreased total oocyte diameter (p<0.05). Total oocyte diameter was also inversely associated with AMH levels (p = 0.03) and oocyte yield (p = 0.04). In contrast to total oocyte diameter, oolemmal diameter was not related to patient characteristics. Younger women and those with large oocyte yields demonstrated fewer oocytes with ooplasmic granulation (p<0.05 and p = 0.01). After adjustments for age, ooplasmic granulation was also less frequently observed in oocytes from women with higher AMH (p = 0.03) and increasing BMI (p<0.01). Fertilization was more likely in oocytes with larger oolemmal diameter (p = 0.008). Embryos from oocytes with larger total and ooplasmic diameters were more likely to be transferred or frozen (p = 0.004 and p = 0.01). In non-PCOS infertile women, BMI and ovarian function relate to total oocyte diameter. These results expand on previously observed associations between oocyte size and BMI in women with PCOS. They indicate the importance of detailed oocyte assessments, which may aid the currently used criteria for embryo selection and help to better understand how oocyte status is associated with later embryo development.

Shorter telomere length of white blood cells is associated with higher rates of aneuploidy in women undergoing in vitro fertilization

Telomeres are tandem repeats of the sequence TTAGGG located at the ends of chromosomes. Telomere shortening is a key mechanism of cell senescence and aging. Telomere shortening has been associated with decreased oocyte quality through disruption of chromosome alignment and spindle structure. In this study, we sought to evaluate whether the telomere length of cumulus cells (CC) or white blood cells (WBC) in an infertile population is associated with reproductive aging. Prospective cohort study. Women undergoing IVF between July 2017 and December 2018 were recruited for the study under Institutional Review Board approval. Blood and CC were collected at the time of oocyte retrieval. Genomic DNA was isolated and stored at -80°C. Telomere DNA length was measured by quantitative real-time PCR and normalized to AluYa5 sequence as an endogenous control for each sample. Linear regression was applied to determine if telomere length (TL) of WBC or CC was associated with patient age, number of oocytes retrieved, number of mature (M2) oocytes retrieved, blastulation rate, aneuploidy rate, serum anti-mullerian hormone (AMH), and serum estrogen (E2) level on day of hCG or GnRHa administration. TL data was available for WBC samples from 156 individuals and for CC samples from 142 patients. Data was available for both tissue types in 139 patients (age range 25.7 to 45.0 years, mean 35.0 ± 4.0 years). As expected, WBC telomere length declined with increasing age (p=0.006). In contrast, CC TL was equivalent in patients of all ages and failed to show anticipated age-related shortening. As such, CC TL was unrelated to any index of ovarian performance including number of oocytes retrieved (p=0.95), M2 oocytes retrieved (p=0.81), blastulation rate (p=0.98), aneuploidy rate (p=0.30), AMH level (p=0.32), or mid-cycle E2 level (p=0.77). Consistent with these data, CC TL was not associated with patient age (p=0.99). While WBC TL declined with increasing maternal age, it was a poor predictor of quantitative ovarian performance [total oocytes retrieved (p=0.47), number of M2 oocytes retrieved (p=0.30), AMH level (p=0.13), E2 level (p=0.36), and blastulation rate (p=0.48)]. However, TL of WBC samples was associated with embryonic competence as evidenced by aneuploidy rate (p=0.02), with shorter TL associated with higher aneuploidy. The TL of CC was not associated with patient age or any index of ovarian or embryonic performance. Declining WBC TL was associated with increasing maternal age and increasing rates of embryonic aneuploidy. Further studies are necessary to determine if changes in peripheral somatic cell TL are truly prognostic for aneuploidy rate within a given maternal age group or if this finding is simply reflective of a simultaneous change which occurs with age.

Comparison between AMH levels with total oocyte, total embryo, embryo quality, and pregnancy on in vitro fertilization patient at Aster fertility clinic Hasan Sadikin hospital in 2015-2016

Comparison between AMH levels with total oocyte, total embryo, embryo quality, and pregnancy on in vitro fertilization patient at Aster fertility clinic Hasan Sadikin hospital in 2015-2016

Evaluation of in Vitro Fertilization Index by Caudal Spermatozoa Capacitation with Different Heparin Concentration in Iraqi Sheep

Sperm capacitation refers to the physiological changes in which spermatozoa must undergo in order to have its ability to penetrate and fertilize a mature oocyte. Recognition of the phenomenon was quite important to early in vitro fertilization (IVF) experiments as well as to the fields of embryology and reproductive biology. This initial study is designed to study the process of capacitation of ram sperm with three heparin levels (50,100,150 IU). 140 pairs of abattoir testicular samples (70 pairs each season), with 70 ewe genitalia specimens (35 each season). Spermatozoa sample were capacitated with heparin in CO2 incubator for 45 minutes, and evaluated by its total motility and its ability to penetrate oocytes zona pellucida for fertilization. Different heparin level for spermatozoa capacitation in relation to seasonal influences revealed that; in regarding to capacitation index there was an increased spermatozoa activity and motility significantly for the three levels within season, but capacitation index showed significant variations between the three heparin levels out of breeding season in which the highest heparin level gave the best capacitation index than the other two. Results of oocytes collection in relation to seasonal influences revealed to; total oocytes number was 2035 oocytes on both season in which 1039 oocytes were obtained within season while a number of 1006 oocytes was collected out of season. Oocytes maturity index within season time is about 40% for the oocytes grade V.good and good grade, fair and poor grade is 25%.Oocytes collected out of breeding time showed range between 25% for the v. good and good grade to 10% for the other. Fertilizable index for embryos development after capacitated spermatozoa adding to the medium containing matured oocytes is range from 35% for the v. good and good graded oocytes within a season to 10% out of season. In conclusion; heparin can induced spermatozoa capacitation in any levels through the breeding season while the high level of heparin gave better results, fertilizable index and embryo production are both gave best result via breeding season.

7. GENETIC TESTING FOR COPY NUMBER VARIATIONS IN FIRST POLAR BODIES AND OOCYTES BY SINGLE-CELL NEXT-GENERATION SEQUENCING

Introduction Female meiotic errors are the main cause of whole chromosomal aneuploidy in human embryos and significantly contribute to early embryo loss. Meiotic chromosomal malsegregation occurs during divisions of the maturing oocyte in oogenesis concurrently with extrusion of two polar bodies: polar body first (PB1) and polar body second (PB2). Based on meiosis mechanism, genome of a single oocyte can be deduced by analyzing its sibling polar bodies. PB genetic testing can identify abnormalities of maternal meiotic origin and may be the only form of preimplantation genetic testing for aneuploidies (PGT-A) accepted by patients who object embryo biopsy for ideological reasons. In the majority of previous studies, array comparative genomic hybridization (aCGH) was a method of choice for comprehensive chromosome testing in polar bodies. Here, we test the use of single-cell next-generation sequencing (NGS) method in polar body testing for detecting copy number variations (CNVs) in PB1 genome and validate methods for whole genome amplification (WGA) of PB1. Material and methods In this study, single-cell NGS technology was used to asses ploidy status of oocytes and their sibling PB1. A total of 120 single cells (60 first polar bodies and 60 oocytes) were collected from in vitro fertilization (IVF) couples and subjected for WGA using four different kits based on three distinct amplification systems: (i) polymerase chain reaction (PCR-based WGA), (ii) multiple displacement amplification (MDA) or (iii) multiple annealing and looping-based amplification cycles (MALBAC). WGA products were processed and analyzed with VeriSeq PGS MiSeq kit on MiSeq sequencing platform (Illumina) and random samples were validated with the aCGH method. Amplification uniformity and accuracy for CNV detection of the four tested kits were evaluated. Results Ploidy status was determined for 112 of 120 (93,3%) samples (84/112 based on direct analysis and 28/112 based on indirect analysis of the well-amplified sibling PB1 or oocyte). Estimated aneuploidy rate for the studied group which resembles the total oocyte abnormality resulting from meiosis I errors rate was 33% (37/112 samples). In the group of well-amplified PB1-oocyte pairs (33/60) a 97% concordance between the chromosomal status of PB1 and the corresponding oocyte was observed. Both whole chromosome nondisjunction (6 of 37 aneuploid samples) and abnormal chromatid pre-division and segregation were detected (31 of 37 aneuploid samples). The result is concordant with previous studies - abnormal chromatid segregation was the dominant aneuploidy-causing mechanism. Differences in WGA kits performance including amplification uniformity, aneuploidy calling potential and a number of interpretable NGS results were evaluated and the most optimal kit for PB1 genome WGA was pre-selected. Results from aCGH validation of the randomly selected samples showed full consistency with the NGS results. Conclusions The NGS-based method used for PB1 genome analysis showed a high predictive potential of PB1 in deducing ploidy status of the corresponding oocyte and is a promising method for PGT-A of oocytes. The study also contributes to a better understanding of the chromosome segregation patterns and maternal meiosis errors. Supported by: POIR.02.03.02-14- 0092/17

Clinical pregnancy (CP) and live birth (LB) increase significantly with each additional fertilized oocyte up to nine, and CP and LB decline after that: an analysis of 15,803 first fresh in vitro fertilization cycles from the Society for Assisted Reproductive Technology registry

To study the association between the total number of fertilized oocytes available and pregnancy outcomes in first fresh IVF cycles with a single blastocyst transfer. Retrospective cohort study. Not applicable. A total of 15,803 patients from SART reporting fertility clinics. None. Primary outcomes were clinical pregnancy (CP), live birth (LB), and miscarriage rates. Logistic regression was used to investigate the association between the number of fertilized eggs and each outcome. The median number of total oocytes was 15, and the median number of fertilized oocytes was nine. The odds of a clinical pregnancy were 8% higher for each additional fertilized oocyte up to nine (odds ratio [OR] 1.08; 95% confidence interval [CI] 1.07-1.10) and declined by 9% for every additional fertilized oocyte after nine (OR 0.91; 95% CI 0.89-0.94). Similarly, the odds of an LB was 8% higher for every additional fertilized oocyte up to none (OR 1.08; 95% CI 1.06-1.10) and declined by 8% for every additional fertilized oocyte over nine (OR 0.92; 95% CI 0.90-0.94). Odds of pregnancy outcomes (CP, LB) increase significantly with every additional fertilized oocyte up to nine, and CP and LB decline after that in first fresh autologous cycles with a single blastocyst transfer.

Meiotic Chromosomes, Synaptonemal Complexes in a Female Viviparous Lizard (Zootoca vivipara) in Prophase I of Meiosis

In the females of the viviparous lizard Zootoca vivipara (Lichtenstein, 1823) (family Lacertidae) from Northwest Russia (2n = 35: 32A (acrocentric autosomes) + Z1Z2W sex chromosomes), the ovarian lumen germinal vesicles (oocytes), as well as germinal lamina cells, were examined. Chromosome preparations were obtained using the direct method and the method of total oocyte nuclei spreading developed by Dresser and Moses. Chromosome preparations were stained with Giemsa; for visualization of synaptonemal complexes (SCs), total preparations of oocyte nuclei were stained with silver nitrate and DAPI. It was demonstrated that, during oogenesis in the female, primary follicles enter the early stages of the meiotic prophase I (stages from leptotene to diplotene, lampbrush chromosomes are formed). Here, for the first time, total oocyte spreads were obtained and studied. On the basis of light microscopic analysis of the oocyte SCs and taking into account their length in a female with 2n = 35, the SC karyotype is presented, consisting of 19 SC elements, among which 16 SC autosomal bivalents are distinguished. The remaining three SC elements, according to the authors, can be univalents of Z1Z2W sex chromosomes or one WZ1 bivalent and Z2 and B chromosome univalents. As in the SC karyotype of Z. vivipara males, in a female, specific features in the morphology of SC elements of chromosomes 5 and 6 were observed.

Is corifollitropin alfa effective in controlled ovarian stimulation among all poor ovarian responders? A retrospective comparative study

Several studies have compared the effectiveness of corifollitropin alfa versus daily gonadotropins in poor ovarian responders (PORs) undergoing controlled ovarian stimulation (COS), showing conflicting results in terms of IVF outcomes. Given the heterogeneity of patients included in the classification of POR according to ‘Bologna criteria’, the aim of this study was to evaluate the impact of corifollitropin alfa in two different categories of POR distinguished according to patients’ antral follicle count (AFC). We retrospectively evaluated 104 infertile POR, split into two groups according to AFC (Group A ≤ 5; Group B > 5) and subgroups according to the ovarian stimulation regimen (corifollitropin alfa plus daily gonadotropins (Subgroup 1) versus daily gonadotropins alone (Subgroup 2)). Outcome measures were total oocytes, MII oocytes, total embryos, follicular output rate (FORT), implantation rate (IR), clinical pregnancy rate (CPR), miscarriage rate (MR), and live birth rate (LBR). Subgroup A1 experienced a lower number of total oocytes, MII oocytes, total embryos, and FORT (p < .05) in comparison to Subgroup A2, while no difference was found when comparing Subgroups B1 and B2. No difference was found between subgroups even in terms of IR, CPR, MR, and LBR. In conclusion, corifollitropin alfa may be as effective as daily gonadotropins in POR with AFC > 5 undergoing COS, while it might be inferior to daily gonadotropins in POR with AFC ≤ 5.

Testosterone therapy for women with poor ovarian response undergoing IVF: a meta-analysis of randomized controlled trials.

The aim of the present systematic review and meta-analysis was to summarize evidence on the effectiveness of testosterone supplementation for poor ovarian responders (POR) on IVF outcomes. The primary outcome was live birth rate (LBR); secondary outcomes were clinical pregnancy rate (CPR), miscarriage rate (MR), total and MII oocytes, and total embryos. This meta-analysis of randomized controlled trials (RCTs) evaluates the effects of testosterone administration before/during COS compared with a control group in patients defined as POR. The primary outcome was live birth rate (LBR); secondary outcomes were clinical pregnancy rate (CPR), miscarriage rate (MR), total and MII oocytes, and total embryos. Pooled results were expressed as risk ratio (RR) or mean differences (MD) with 95% confidence interval (95% CI). Sources of heterogeneity were investigated through sensitivity and subgroup analysis. All analyses were performed by using the random-effects model. Women receiving testosterone showed higher LBR (RR 2.29, 95% CI 1.31-4.01, p = 0.004), CPR (RR 2.32, 95% CI 1.47-3.64, p = 0.0003), total oocytes (MD = 1.28 [95% CI 0.83, 1.73], p < 0.00001), MII oocytes (MD = 0.96 [95% CI 0.28, 1.65], p = 0.006), and total embryos (MD = 1.17 [95% CI 0.67, 1.67], p < 0.00001) in comparison to controls, with no difference in MR (p = ns). Sensitivity and subgroup analysis did not provide statistical changes to the pooled results. Testosterone therapy seems promising to improve the success at IVF in POR patients. Further RCTs with rigorous methodology and inclusion criteria are still mandatory.

Is embryogenesis and ART outcome different in polycystic ovary syndrome?

Introduction: As oogenesis is a continuum of months, oocyte quality will be dictated by events that occur during both the growth and maturation stages of development. Morphological variation of the oocyte and embryo result from intrinsic factors such as age and genetic defects or extrinsic factors such as stimulation protocol, culture condition, and nutrition. Controlled ovarian stimulation (COH) interferes with the balance of forces within the ovary and overrides their endogenous pattern of control to optimize normal selection process. Aim: 1. To compare the embryogenesis and outcome of assisted reproductive technology (ART) in polycystic ovary syndrome (PCOS) and non-PCOS women. 2. To determine whether embryo quality, implantation rate (IR) and clinical pregnancy rate (CPR) are related to estradiol (E2) or progesterone (P4) levels on the day of human chorionic gonadotropin (hCG). Materials and Methods: This was a retrospective case–control study of 425 women at a Tertiary Care Center from January 2017 to December 2018. Of these 183 were PCOS and 242 non-PCOS matched for body mass index and age. We compared the follicle stimulating hormone, antral follicle count (AFC), anti-Mullerian hormone, thyroid-stimulating hormone, total gonadotropin utilized, total days of stimulation, estradiol (E2), and progesterone (P4) on the day of hCG trigger between both groups. Before entering the in vitro fertilization program, patients were classified into PCOS and non-PCOS depending on the AFC at the baseline scan done on day 2/3 of the menstrual cycle. We calculated the fertilization, cleavage, blastocyst formation rate, and utilization rate of the embryos from the total oocytes retrieved and fertilized in four different groups classified according to the E2 levels on the day of the hCG (≤1000, 1001–2000, 2001–3000, >3000 pg/ml) in both PCOS and non-PCOS women. We also looked at the IR and CPR in the above four groups in fresh cycle and included the frozen embryo transfer cycles to calculate the utilization rate. Statistical comparison was done using the Mann–Whitney U test. Results: There was no statistically significant difference in the fertilization rate (P = 0.803), cleavage rate was higher in PCOS group (P = 0.001) and blastocyst formation rate was higher in non-PCOS group (P 3000 pg/ml. The IR was higher in the PCOS group as compared to non-PCOS in women with E2 between 2000 and 3000 (P = 0.020). No difference in the CPR across the E2 groups. The utilization rate of the embryos per egg retrieved and per 2PN was statistically higher in patients with an E2 Conclusion: As embryogenesis and endometrial receptivity can be affected by high E2 and P4 levels it is highly recommended to closely monitor COS cycles by measuring serum E2 and P4 levels on D2 and at the time of hCG trigger. The dose of gonadotropins dose should be chosen in such a way that the number of oocytes retrieved should not be more than 10 to 12. When E2 and P4 are elevated over a threshold value, one should cryopreserve the embryo and transfer them in the subsequent cycle to decrease the incidence of ovarian hyperstimulation syndrome and improve the IR.

Establishing an Anti-Müllerian Hormone Cutoff for Diagnosis of Polycystic Ovarian Syndrome in Women of Reproductive Age-Bearing Indian Ethnicity Using the Automated Anti-Müllerian Hormone Assay.

Context:Polycystic ovary syndrome diagnosed by Rotterdam criteria, is the most common cause of anovulatory infertility. The criteria of polycystic ovarian morphology (PCOM) are subject to operator variability and technological advances. Serum anti-Müllerian hormone (AMH) level has been proposed as a more reliable alternative to antral follicle count. There is a paucity of data on use of AMH for diagnosis of PCOS in Indian women.Aim and Objectives:The aim of this study is to determine a cutoff level for AMH that could facilitate diagnosis of PCOS and its phenotypes in women of Indian origin using the automated (Roche) assay and to compare the competence of oocytes in PCOS and non-PCOS women undergoing in vitro fertilization-intracytoplasmic sperm injection (IVF-ICSI).Materials and Methodology:A total of 367 women undergoing treatment at our fertility center between February 2017 and August 2017 were prospectively enrolled in this study. Of these, 133 were diagnosed with PCOS, 69 had isolated PCOM, and 165 (controls) had normal ovaries on ultrasound examination. Serum AMH levels were assessed using the fully automated Roche Elecsys® immunoassay. Gonadotropin-releasing hormone antagonist protocol was used for IVF-ICSI in all patients.Statistical Analysis Used:Quantitative variables were compared using the Mann–Whitney test. Qualitative variables were correlated using the Chi-square test. P < 0.05 was considered to be statistically significant.Results:Mean AMH concentrations in women with PCOS was higher (7.56 ± 4.36 ng/mL) in comparison to PCOM and controls. Serum AMH concentration >5.03 ng/mL could facilitate diagnosis of PCOS (area under the curve = 0.826); sensitivity –70.68%, specificity of 79.91%. There was no difference in the ratio of mature to total oocytes retrieved in the three groups (P > 0.05). Mean number of mature oocytes was lower in controls than PCOS and PCOM (P < 0.001).Conclusions:Serum AMH concentration >5.03 ng/mL could be used as cutoff value for the diagnosis of PCOS in women of Indian origin.

The UV filter benzophenone 3, alters early follicular assembly in rat whole ovary cultures

Our goal was to study the effect of BP3 (benzophenone 3) in the follicular assembly and the potential involvement of Foxl2 pathway using whole ovary cultures. Ovaries were collected from Wistar rats at birth, treated in vitro with vehicle (0.01% DMSO), BP3 (5.8 nM, 276 nM, 576 nM and 876 nM) or ESR2 inhibitor (0.1 nM), and cultured for 7 days. Nest breakdown, follicular assembly and the expression of several regulators of these processes (p27, Foxl2, Sox9, Bmp2, Cyp19 and Fst) were evaluated. In vitro exposure to BP3 (5.8 nM) decreased the population of total oocytes, the number of nests per ovary and early primary follicles population. In addition, BP3 (5.8 nM) induced overexpression of Foxl2 mRNA levels through ESR2 but increased Fst mRNA levels independently from ESR2 or Foxl2. We also observed that the number of p27-positive oocytes was decreased after BP3 (5.8 nM). On the other hand, exposure to BP3-276 increased total oocytes, the number of nests per ovary and decreased primary follicles. In addition, BP3-276 induced no changes of Foxl2 mRNA levels through ESR2 but increased Fst mRNA levels independently from ESR2 or Foxl2. In conclusion, our study clearly shows that exposure to BP3 is to perturb the early events of germ cell development as showed here in whole ovary cultures.

Razmnožavanje brgljuna, Engraulis encrasicolus (Linnaeus, 1758) u Bokokotorskom zaljevu (Crna Gora, južni Jadran)

The annual alteration of gonad morphology in anchovy, Engraulis encrasicolus (Linnaeus,1758), caught in the period from July 2006 to June 2007 using beach seines in the region of Boka Kotorska Bay (Montenegro, southern Adriatic) was studied. Samples were taken each month. Total length of individuals ranged from 6.6 to 12.2 cm, while weight ranged from 1.58 to 11.27 g, Their ovaries were extracted and weighed, and a piece of female gonad tissue was sampled for histological analysis. The length–weight relationship of all anchovy specimens was described by the equation: \(W = 0.0032\) \(L_T^3.2537; (r^2 = 0.9528)\). The lowest GSI values were found in November and remained at low levels until April, which corresponded to the gonad rest stage. The GSI increased gradually during April and May, which coincides with the begining of spawning period. Although primary oocytes (stage I) were present during all months, their percentage increased from October and was the highest during the winter months (November-March 100%). Mature cells, stage IV, were present during April – October period, with highest value in May when they comprised 12.3% of total oocyte number. An increase in the percentage of oocytes in the yolk vesicle (II) and yolk (III) stages occurred in April, remaining almost unchanged until October. Smallest average oocyte size was noticed in period November – February with an average of 59.69 μm, while greatest size was recorded in April 2007, 185.84 μm.

Lower follicular n-3 polyunsaturated fatty acid levels are associated with a better response to ovarian stimulation.

To analyze in detail the fatty acid (FA) composition of follicular fluid (FF) from two-sized follicles at oocyte retrieval and to determine associations of the FAs from large follicles with the woman's age and the response to ovarian stimulation. Observational study. University and fertility clinic. Sixty-four women (age 19-46), consisting of unfertile patients and oocyte donors, undergoing controlled ovarian stimulation. None. FF from small (< 12mm) and large (≥ 18mm) follicles was collected at oocyte retrieval. FAs by gas chromatography-flame ionization detection. Thirty-two FAs with chain lengths ranging from 14 to 25 carbons were identified. There was a readjustment in FA distribution as follicle size increased, raising very long-chain saturated FAs, nervonic (24:1n-9), arachidonic (20:4n-6), and n-3 polyunsaturated FAs (PUFA, P < 0.001), the latter mainly due to an increase in docosahexaenoic acid (22:6n-3, DHA). In large follicles, double bond and peroxidizability indices and total n-3 PUFA, particularly DHA, correlated positively with the woman's age and negatively with the number of total and mature oocytes, total and top-quality embryos, and fertilization rate. We have described 32 FAs in ovarian FF, of which 16 changed their distribution with follicle size. The results also indicate that lower n-3 PUFA levels in large follicles, which are associated with younger women, predict a better response to ovarian stimulation based on the recovery of total and mature oocytes, total and top-quality embryos, and fertilization rate per cycle. The fatty acid profile of ovarian FF changes as the follicle grows and lower n-3 PUFA levels in large follicles, associated with younger women, predict a better response to ovarian stimulation.

133 Test of minimum-intervention protocols for optimizing in vitro embryo production in bison

The study was done to determine whether minimal handling protocols for ovarian synchronization and ovarian superstimulation may be used to increase in vitro embryo production in bison. Ultrasound-guided cumulus-oocyte complex (COC) collection was done in a group of bison (n=23; random start) during the anovulatory season to synchronize new follicular wave emergence. The COC were classified morphologically (compact-good and -regular, expanded, denuded, degenerate) but not processed further. At the time of COC collection (Day 0), bison were assigned randomly to 3 groups and given 5mL of saline IM (non-superstimulated controls; n=11), 10 Armour units of pFSH (Antrin R10, Kyoritsu Seiyaku Corp., Tokyo, Japan) in 5mL of saline IM once per day from Day 0 to 2 (regular FSH; n=5), or 30 armour units of a sustained-release form of pFSH (Antrin R10Al, Kyoritsu Seiyaku Corp.) in 5mL of saline SC on Day 0 (long-acting FSH; n=7). On Day 4, a second COC collection was performed. Only compact COC were processed. The COC were matured in vitro for 25 to 28h at 38.8°C, fertilized (2×106 sperm mL−1) and co-incubated at 38.8°C in 5% CO2 for 18h. Presumptive zygotes were denuded and cultured at 38.8°C in 5% O2, 5% CO2 and 90% N2. Nominal data were compared by t-test and analysis of variance. Binomial data were compared among groups by chi-squared. There was no difference between the first (random) COC collection (n=23) and second collection (n=11 non-superstimulated controls) in the total number of follicles detected, but the distribution among size categories (3-4, 4-8, and &amp;gt;8mm) differed, i.e. fewer in the 3 to 4mm category at the time of the second COC collection (12.2±1.0v. 8.1±1.4; P&amp;lt;0.05). In the nonstimulated control group, there were no differences between the first and second COC collections in the number of follicles aspirated (12.7±1.0v. 10.4±1.5), number of COC collected (7.7±0.9v. 5.3±1.3), or in the categorical distribution of COC. At the second COC collection, the number of follicles in the &amp;gt;8mm category was greater in the regular FSH group than in the control or long-acting FSH groups (2.8±0.5v. 1.1±0.3, and 1.9±0.4, respectively; P&amp;lt;0.05), but no differences were detected in the number of follicles aspirated, COC collected, or in the categorical distribution of COC. The cleavage rate (of total oocytes submitted to in vitro maturation), recorded 2 days after IVF, was higher in the control group than in either the regular FSH or long-acting FSH groups [25/35 (71.4%), 7/28 (25.0%), 8/35 (22.8%); P&amp;lt;0.0001]. The freezable embryo production rate, recorded 7 days after IVF, was greater in the control group than in the regular FSH or long-acting FSH groups [19/35 (54.3%), 5/28 (17.9%), 5/35 (14.3%); P&amp;lt;0.01]. In conclusion, minimal-handling interventions used in the present study to increase embryo production in bison were not effective, likely as a result of the timing, frequency, and duration of superstimulation. A random start resulted in greater COC collection than collection 4 days after ovarian synchronization, and embryo production rates were greater in non-superstimulated bison. This work was supported by Parks Canada and Saskatchewan ADF. Antrin products donated by Kyoritsu Seiyaku Corp., Japan.

168 Melatonin slightly alleviates the effect of heat shock on nuclear maturation of the bovine oocyte

Exposing bovine cumulus-oocyte complexes (COC) to heat shock during in vitro maturation (IVM) reduces oocyte developmental competence. Melatonin has been reported to improve cleavage of heat-shocked oocytes after fertilization. The current study examined whether protective activity of melatonin is associated with nuclear or cytoplasmic maturation events, or both. The COC were aspirated from Holstein ovaries collected at an abattoir and randomly divided into 3 experimental groups. The control COC were matured (22h) in standard oocyte maturation medium (OMM) at 38.5°C and 5% CO2; the treatment COC were matured (22h) in OMM at 41.5°C and 6% CO2 (HS) without or with 10−7 M melatonin (HS+M). Data are presented as least squares means±s.e.m. To examine nuclear maturation, COC were denuded of cumulus cells at the end of maturation, fixed in 2% (vol/vol) paraformaldehyde, and labelled with 10μg mL−1 4′,6-diamidino-2-phenylindole (DAPI) (200 COC, 3 replicates). The meiotic status of denuded oocytes was evaluated under inverted fluorescence microscope using Nis Elements software (Nikon, Tokyo, Japan). The proportion of oocytes that resumed meiosis and reached the metaphase II (MII) stage was lower in the HS group than in the control (13.7v. 55%, respectively; chi-squared, P&amp;lt;0.05); melatonin slightly alleviated this effect, as indicated by a higher proportion of MII-stage oocytes in HS+M than in the HS group (32v. 13.7%, respectively; chi-squared, P&amp;lt;0.05). Cytoplasmic maturation was evaluated according to the distribution pattern of cortical granules (CG). Matured COC were denuded and the oocytes were stained with 100μg mL−1 fluorescein isothiocyanate-conjugated Arachis hypogaea agglutinin (FITC-PNA) and counterstained with 10μg mL−1 DAPI. Oocytes were examined under an inverted fluorescence microscope and classified as 1 of 3 types according to their cortical distribution pattern (3 replicates; 200 COC). The proportion of oocytes classified as type 3, which is associated with cytoplasmic maturation, did not differ between the experimental groups, either when analysed for total oocytes or for nuclear matured oocytes (i.e. MII stage). To evaluate the level of reactive oxygen species (ROS), matured-denuded oocytes were labelled using 2′,7′-dichlorodihydrofluorescein diacetate (H2DCFDA) and the intracellular fluorescence of DCF was immediately measured by fluorescence microscopy (3 replicates, 425 COC). Heat shock increased ROS level relative to the control (68.8±5.32v. 33.9±4.08 pixels intensity, respectively; P&amp;lt;0.05) and melatonin tended to alleviate the effect of heat stress to some extent (56.0±4.78; P&amp;lt;0.06). In summary, heat shock impairs the process of nuclear maturation, expressed by reduced proportion of MII-stage oocytes at the end of maturation. Melatonin partly overcomes this effect, perhaps through its antioxidant activity.

114 Effect of in vivo heat stress on DNA methylation and DNA hydroxymethylation of bovine oocytes

Cattle under the effect of heat stress have reduced fertility, with negative effects on the oocyte observed at the morphological, biochemical, transcriptional and developmental levels. There are no studies evaluating the effect of heat stress on the epigenetic profile of bovine oocytes, which plays a fundamental role in the regulation of gamete development. The objective of this study was to evaluate the effect of in vivo heat stress during the spring to summer transition on DNA methylation and DNA hydroxymethylation of bovine oocytes at the germinal vesicle (GV) and metaphase II (MII) stages. Ten Bos taurus crossbred nonlactating beef cows located at Saint Gabriel, Louisiana, USA (30°16′11.1″ N, 91°06′12.1″ W), were used for oocyte collection once monthly from April to August. Dominant follicle removal was performed 5-7 days before oocyte collection. Cumulus-oocyte complexes were collected through ovum pick-up from follicles &amp;gt;2mm. Germinal vesicle (GV)-stage oocytes (50% of total obtained per cow) were subjected to a standard bovine in vitro maturation protocol to obtain metaphase II (MII) stage oocytes. The DNA methylation and DNA hydroxymethylation of GV and MII oocytes was assessed by fluorescence immunohistochemistry utilising primary antibodies against 5′-methylcytosine and 5′-hydromethylcytosine. Secondary antibodies utilised were Alexa Fluor 488 goat anti-mouse IgG and Alexa Fluor 546 donkey anti-rabbit IgG. Oocytes were visualised utilising a fluorescence deconvolution microscope and immunofluorescence data were expressed as corrected relative fluorescence per nucleus. The polar body was not included for fluorescence quantification when evaluating MII stage oocytes. Results (least squares means±standard error) were evaluated as cold months (April and May) and hot months (June, July, and August). Results were analysed by the type III test of fixed effects and Tukey media separation utilising Proc Glimmix of SAS 9.4 (P&amp;lt;0.05; SAS Institute Inc., Cary, NC, USA). Maturation rates and percent of grade 1, grade 2, and grade 3 oocytes were square root arcsine transformed for statistical analysis. The number of total oocytes obtained per cow was higher in cold compared to hot months (21.88±2.34 and 14.23±2.17, respectively). Percent of grade-1 oocytes was higher in cold compared to hot months (38.25±3.69 and 27.59±3.09, respectively). There was no difference in percent of grade-2 oocytes between cold and hot months (21.80±2.44 and 22.60±2.20, respectively). There was a lower percent of grade-3 oocytes in cold compared to hot months (39.82±4.54 and 55.87±3.98, respectively). Maturation rate (in vitro maturation) was not different between cold and hot months (81.92±4.04 and 91.11±3.36, respectively). There was no difference between cold and hot months in DNA methylation (417,218.90±71,793.86 and 313,819.88±55,528.01, respectively) and DNA hydroxymethylation (444,931.10±67,920.78 and 352,254.68±56,425.96, respectively) of GV-stage oocytes. There was no difference between cold and hot months in DNA methylation (87,122.36±14,449.47 and 89,807.26±11,303.72 AU, respectively) and DNA hydroxymethylation (102,933.83±15,517.70 and 137,622.45±11,826.86 AU, respectively) of MII-stage oocytes.