IVM Groups Research Articles

Development of children born from IVM versus IVF: 2-year follow-up of a randomized controlled trial.

Is there any difference in developmental outcomes in children born after capacitation IVM (CAPA IVM) compared with conventional IVF? Overall development up to 24 months of age was comparable in children born after CAPA IVM compared with IVF. IVM has been shown to be a feasible alternative to conventional IVF in women with a high antral follicle count (AFC). In addition to live birth rate, childhood development is also a relevant metric to compare between the two approaches to ART and there are currently no data on this. This study was a follow-up of babies born to women who participated in a randomized controlled trial comparing IVM with a pre-maturation step (CAPA IVM) and IVF. Developmental assessments were performed on 231 children over 24 months of follow-up. Participants in the randomized controlled trial had an indication for ART and a high AFC (≥24 follicles in both ovaries). They were randomized to undergo one cycle of either IVM (n = 273) or IVF (n = 273). Of these, 96 women and 118 women, respectively, had live births. Seventy-six women (94 children, 79.2%) and 104 women (137 children, 88.1%), respectively, completed Ages & Stages Third Edition Questionnaire assessment (ASQ-3), and underwent evaluation of Developmental Red Flags at 6, 12 and 24 months of age. Baseline characteristics of participants in the follow-up study between the IVM and IVF groups were comparable. Overall, there were no significant differences in ASQ-3 scores at 6, 12 and 24 months between children born after IVM or IVF. The proportion of children with developmental red flags was low and did not differ between the two groups. Slightly, but significantly, lower ASQ-3 problem solving and personal-social scores in twins from the IVM versus IVF group at 6 months were still within the normal range and had caught up to the IVF group in the 12- and 24-month assessments. The number of children confirmed to have abnormal mental and/or motor development after specialist assessment was four in the IVM group and two in the IVF group (relative risk 2.91, 95% CI 0.54-15.6; P = 0.23). This study is an open-label follow-up of participants in a randomized controlled trial, and not all original trial subjects took part in the follow-up. The self-selected nature of the follow-up population could have introduced bias, and the sample size may have been insufficient to detect significant between-group differences in developmental outcomes. Based on the current findings at 2 years of follow-up, there does not appear to be any significant concern about the effects of IVM on childhood development. These data add to the evidence available to physicians when considering different approaches to fertility treatment, but require validation in larger studies. This work was funded by the Vietnam National Foundation for Science and Technology Development (NAFOSTED) under grant number FWO.106-YS.2017.02. L.N.V. has received speaker and conference fees from Merck, grant, speaker and conference fees from Merck Sharpe and Dohme, and speaker, conference and scientific board fees from Ferring; T.M.H. has received speaker fees from Merck, Merck Sharp and Dohme, and Ferring; R.J.N. has receives grant funding from the National Health and Medical Research Council (NHMRC) of Australia; B.W.M. has acted as a paid consultant to Merck, ObsEva and Guerbet and is the recipient of grant money from an NHMRC Investigator Grant; J.E.J.S. reports lecture fees from Ferring Pharmaceuticals, Biomérieux and Besins Female Healthcare, grants from Fund for Research Flanders (FWO) and is co-inventor on granted patents on CAPA-IVM methodology in the USA (US10392601B2) and Europe (EP3234112B1); T.D.P., M.H.N.N., N.A.N., T.T.L., V.T.T.T., N.T.N., H.L.T.H. and X.T.H.L. have no financial relationships with any organizations that might have an interest in the submitted work in the previous 3 years, and no other relationships or activities that could appear to have influenced the submitted work. NCT04296357 (www.clinicaltrials.gov). 5 March 2020. 7 March 2020.

OOCYTE VITRIFICATION FOR FERTILITY PRESERVATION DOES NOT DELAY THE INITIATION OF NEOADJUVANT CHEMOTHERAPY FOR BREAST CANCER

To evaluate whether oocyte vitrification for fertility preservation (FP) delays the initiation of neoadjuvant chemotherapy for breast cancer? We performed a retrospective cohort study including all breast cancer patients eligible for oocyte vitrification following COH or IVM before initiation of neoadjuvant chemotherapy between January 2016 and December 2020. Inclusion criteria were: female patients with confirmed non metastatic breast cancer, 18 to 40 years of age, with indication of neoadjuvant chemotherapy, who have had oocyte retrieval for FP after COH or IVM +/- cryopreservation of ovarian tissue. Various time-points related to cancer diagnosis, FP or chemotherapy were obtained from medical record review. A total of 198 patients with confirmed breast cancer who had oocyte retrieval following COH (n=57) or IVM +/- cryopreservation of ovarian tissue (n=141) for FP prior to neoadjuvant chemotherapy were included. Although women in IVM group were significantly younger as compared to patients who underwent COH (31.7 ± 4.2 vs. 33.3 ± 4.0 years, p=0.019), ovarian reserve parameters, BMI and cancer stage did not differ between the two groups. Overall, the average time from cancer diagnosis to chemotherapy start was similar between patients having undergone COH or IVM before oocyte vitrification (37.3 ± 13.8 vs. 36.9 ±13.5 days in COH and IVM groups respectively; p=0.857). The indication of neoadjuvant chemotherapy for breast cancer should not be considered as an impediment to urgent oocyte vitrification for FP.

Correlation between mild fetal ventriculomegaly, chromosomal abnormalities, and copy number variations

Background Lateral ventriculomegaly is the most common abnormality of the fetal nervous system. This study investigated the incidence of chromosomal abnormalities and copy number variations (CNVs) in fetuses with mild ventriculomegaly (MV) based on various ultrasonic manifestations, identifying their corresponding features via ultrasound examination. Methods A retrospective analysis was performed on ultrasound and neurosonogram (NSG) manifestations and genetic profiles of 334 cases with MV and invasive prenatal diagnosis. Results Three hundred thirty-four cases with fetal MV were assessed via karyotyping. Further chromosomal microarray analysis (CMA) was performed in 182 cases with normal chromosome karyotypes; pathogenic chromosomal copy number variations (CNVs) were found in eight cases with a prevalence of 4.4% (8/182). In this study, the incidence rate of pathogenic abnormalities of chromosomes and CNVs was 5.7% (19/334). Based on whether lateral ventriculomegaly was complicated with other ultrasonic features, the 334 patients were divided into two groups: (1) 175 cases exhibited isolated ventriculomegaly (IVM; 52.4%, 175/334 group A) including two (1.1%, 2/175) with pathogenic chromosomal karyotype abnormalities—both trisomy 21; (2) 159 cases exhibited non-isolated ventriculomegaly (N-IVM; 47.6%, 159/334) with pathogenic chromosomal abnormalities and CNVs detected in17 cases (10.7%, 17/159). The N-IVM group was further divided into two groups: 105 cases exhibited MV with undetermined ultrasonic abnormalities (31.4%, 105/334, group B) with pathogenic chromosomal abnormalities and CNVs detected in eight cases (7.6%, 8/105); 54 cases exhibited MV with structural malformations (16.2%, 54/334, group C) of which nine cases (16.7%, 9/54) presented both pathogenic chromosomal abnormalities and CNVs, and five cases (55.6%, 5/9) were diagnosed with various cortical malformations. The pathogenicity rates of the IVM and N-IVM groups were statistically different (χ 2=14.159, p = 0.000). There were significant differences (χ2=7.992, p = 0.005) among groups A, B, and C. Conclusions Combinations of various ultrasonic abnormalities significantly affect the risk of pathogenic chromosomal abnormalities and CNVs in fetuses with MV. Cases involving cortical malformations require particular attention to the occurrence of pathogenic genetic abnormalities. When fetal MV is detected, a comprehensive ultrasound examination focusing on undetermined ultrasonic abnormalities is critical. Fetal NSG should be conducted to detect potential cerebral cortical malformation easily missed by routine ultrasound.

Comparison of IVF and IVM outcomes in the same patient treated with a modified IVM protocol along with an oocytes-maturing system containing melatonin: A pilot study

AimTo compare embryonic developmental competence and clinical outcomes of oocytes matured in vivo (IVF oocytes) and those matured in vitro (IVM oocytes) from the same IVM/IVF cycles, and to analyze the clinical efficiency of a melatonin-supplemented in vitro maturation system combined with a modified IVM/IVF protocol. Main methodsWe randomly recruited 22 patients undergoing IVM/IVF treatment protocol in our medical centre. The fertilization, cleavage and blastocyst formation rates, as well as clinical pregnancy, implantation and live birth/ongoing pregnancy rates were analysed and compared between IVF and IVM oocytes. We evaluated mitochondrial function indicators by fluorescence staining and confocal microscopy, including mitochondrial membrane potential, reactive oxygen species and calcium (Ca2+) levels in 15 IVF and 15 IVM oocytes. Key findingsThere were no significant differences in fertilization or blastocyst formation rates between the IVF and IVM groups, whereas the cleavage rate was significantly higher in the IVF versus IVM group (100% vs 93.4 ± 10.9%, p = 0.03). There were no significant differences in the clinical pregnancy, implantation or live birth/ongoing pregnancy rates between the two groups. The cumulative clinical pregnancy and ongoing pregnancy/live birth rate per pick-up oocyte in the IVM/IVF treatment cycles were 68.2% (15/22) and 54.5% (12/22), respectively. The reactive oxygen species and Ca2+ levels were significantly increased, and mitochondrial membrane potential was significantly decreased, in IVM compared with IVF oocytes. SignificanceThe modified IVM/IVF protocol can be effectively applied to the treatment of some indicated patients and achieve ideal clinical outcomes, even though the developmental potential of IVM oocytes may not be as high as IVF oocytes.

In-vitro maturation of oocytes versus conventional IVF in women with infertility and a high antral follicle count: a randomized non-inferiority controlled trial.

Is one cycle of IVM non-inferior to one cycle of conventional in IVF with respect to live birth rates in women with high antral follicle counts (AFCs)? We could not demonstrate non-inferiority of IVM compared with IVF. IVF with ovarian hyperstimulation has limitations in some subgroups of women at high risk of ovarian stimulation, such as those with polycystic ovary syndrome. IVM is an alternative ART for these women. IVM may be a feasible alternative to IVF in women with a high AFC, but there is a lack of data from randomized clinical trials comparing IVM with IVF in women at high risk of ovarian hyperstimulation syndrome. This single-center, randomized, controlled non-inferiority trial was conducted at an academic infertility center in Vietnam from January 2018 to April 2019. In total, 546 women with an indication for ART and a high AFC (≥24 follicles in both ovaries) were randomized to the IVM (n = 273) group or the IVF (n = 273) group; each underwent one cycle of IVM with a prematuration step versus one cycle of IVF using a standard gonadotropin-releasing hormone antagonist protocol with gonadotropin-releasing hormone agonist triggering. The primary endpoint was live birth rate after the first embryo transfer. The non-inferiority margin for IVM versus IVF was -10%. Live birth after the first embryo transfer occurred in 96 women (35.2%) in the IVM group and 118 women (43.2%) in the IVF group (absolute risk difference -8.1%; 95% confidence interval (CI) -16.6%, 0.5%). Cumulative ongoing pregnancy rates at 12 months after randomization were 44.0% in the IVM group and 62.6% in the IVF group (absolute risk difference -18.7%; 95% CI -27.3%, -10.1%). Ovarian hyperstimulation syndrome did not occur in the IVM group, versus two cases in the IVF group. There were no statistically significant differences between the IVM and IVF groups with respect to the occurrence of pregnancy complications, obstetric and perinatal complications, preterm delivery, birth weight and neonatal complications. The main limitation of the study was its open-label design. In addition, the findings are only applicable to IVM conducted using the prematuration step protocol used in this study. Finally, the single ethnicity population limits the external generalizability of the findings. Our randomized clinical trial compares live birth rates after IVM and IVF. Although IVM is a viable and safe alternative to IVF that may be suitable for some women seeking a mild ART approach, the current study findings approach inferiority for IVM compared with IVF when cumulative outcomes are considered. Future research should incorporate multiple cycles of IVM in the study design to estimate cumulative fertility outcomes and better inform clinical decision-making. This work was partly supported by Ferring grant number 000323 and funded by the Vietnam National Foundation for Science and Technology Development (NAFOSTED) and by the Fund for Research Flanders (FWO). LNV has received speaker and conference fees from Merck, grant, speaker and conference fees from Merck Sharpe and Dohme, and speaker, conference and scientific board fees from Ferring; TMH has received speaker fees from Merck, Merck Sharp and Dohme, and Ferring; RJN has received conference and scientific board fees from Ferring, is a minor shareholder in an IVF company, and receives grant funding from the National Health and Medical Research Council (NHMRC) of Australia; BWM has acted as a paid consultant to Merck, ObsEva and Guerbet, and is the recipient of grant money from an NHMRC Investigator Grant; RBG reports grants and fellowships from the NHMRC of Australia; JS reports lecture fees from Ferring Pharmaceuticals, Biomérieux, Besins Female Healthcare and Merck, grants from Fund for Research Flanders (FWO), and is co-inventor on granted patents on CAPA-IVM methodology in the US (US10392601B2) and Europe (EP3234112B1); TDP, VQD, VNAH, NHG, AHL, THP and RW have no financial relationships with any organizations that might have an interest in the submitted work in the previous three years, and no other relationships or activities that could appear to have influenced the submitted work. NCT03405701 (www.clinicaltrials.gov). 16 January 2018. 25 January 2018.

Goat incubator: Can bovine oocytes be matured in the uterine horn of a goat?

We used a goat as a live incubator, along with associated nonsurgical embryo transfer techniques, to perform ex situ (in vivo) maturation of bovine oocytes. Immature bovine cumulus-oocyte complexes (COCs) aspirated from 3-8 mm follicles from slaughterhouse ovaries were randomly split into two groups for in vitro (IVM; n = 38) and ex situ maturation (ESM; n = 40). IVM was performed for a period of 24 h at 38.5 ºC and with 5% CO2 in the air of maximum humidity. For ESM, a presynchronized nulliparous goat (12 months old) received 40 immature COCs in the uterine horn apiece, via the transcervical route. After 24 h the structures were retrieved through uterine flushing. Analyses of nuclear maturation and lipid quantification were performed on oocytes from both groups. Fluorescent intensity was compared using the Student’s t-test. Forty-seven percent of the structures were recovered after uterine flushing (19/40). The nuclear maturation rate was 94.5% (18/19) and 81.6% (31/38) for the ESM and IVM groups, respectively. In vitro-matured COCs contained more lipid droplets, expressed as a higher amount (p < 0.05) of emitted fluorescent light than ex situ-matured COCs (858 ± 73 vs. 550 ± 64 arbitrary fluorescence units, respectively). This is the first report to associate nonsurgical embryo transfer techniques and a goat as a live incubator for the maturation of bovine oocytes. We conclude that bovine oocytes can progress meiotically in the uterus horn of a goat and that transcervical transfer of bovine oocytes to a goat’s uterus could present an alternative to nuclear maturation.

Effect of macromolecule supplement on nuclear and cytoplasmic maturation, cryosurvival and in vitro embryo development of dromedary camel oocytes

The current evaluation of oocyte vitro maturation (IVM) media has progressed toward more defined conditions in human and livestock. In this study, the replacement of fetal calf serum (FCS) with bovine serum albumin (BSA) and polyvinyl alcohol (PVA) was evaluated during IVM in dromedary camel. Nuclear maturation rates in presence of FCS and PVA were comparable (81.6 ± 1 and 75.5 ± 5%, respectively). BSA, whether used alone or in combination with FCS, significantly reduced nuclear maturation (51.6 ± 3.9 and 54.6 ± 1.1%, respectively), compared to FCS and PVA. BSA also increased the rates of chromosome aberrations compared to FCS and PVA (25.7 ± 7.4, 8.8 ± 2.3 and 6.0 ± 2.0%, respectively). IVM macromolecule differentially affected morphological aspects of cumulus expansion and FCS promoted the highest dissociation of cumulus cells, compared to all the other groups. FCS significantly increased mean lipid intensity of oocytes compared to BSA, FCS-BSA and PVA which could explain the lower cryo-survival of oocytes matured in presence of FCS compared to BSA and PVA (56.1 ± 5.2, 91.0 ± 19.5, and 87.8 ± 6.7%, respectively). Mitochondrial activity was not affected by macromolecules, but oocytes cultured with PVA had the best redox status, compared to other IVM groups. Cleavage was not affected by IVM macromolecule, but FCS promoted significantly higher rate of morula development (51.6 ± 5.2 vs. 33.6 ± 2.9% for PVA) and blastocyst development (36.8 ± 1.4 vs. 20.5 ± 2.0% for BSA). Although adding FCS during IVM supported highest hatching rate of the resulting blastocysts, differential cell number showed no long lasting effect of IVM macromolecules on blastocyst quality. Obtained results suggest the possibility to switch from undefined to more defined IVM systems for efficient in vitro maturation and subsequent vitrification of dromedary camel oocytes. Keywords: camel, oocyte maturation, protein supplement, cryosurvival.

Multi-drug resistant Haemonchus contortus in a sheep flock in Antioquia, Colombia.

A sheep farm experiencing high lamb mortality and poor body conditions of ewes due to haemonchosis was tested for the presence of anthelmintic-resistant nematodes in August 2014. A fecal egg count reduction test was performed in Colombia on this farm and a DrenchRite® Larval development Assay (LDA) was shipped to the University of Georgia in the United States for resistance testing. The fecal egg count reduction test (FECRT) was conducted on 70 out of 500 animals allocated into five groups of 14 animals each: untreated control, albendazole (ABZ; 3.5mg/kg sc), ivermectin (IVM; 0.2mg/kg sc), moxidectin (MOX; 0.2mg/kg sc) and levamisole (LEV; 5mg/kg sc) plus triclabendazol/ivermectin (TBZ; 10mg/kg and IVM; 0.2mg/kg po). Individual fecal egg counts (FEC) using the modified McMaster technique and pooled larval cultures were performed 10days after anthelmintic treatment. Following 10days the initial results from the ABZ and IVM groups, 24 animals were treated twice with trichlorfon (TCF; 50mg/kg po) on days 0 and 6, and then checked for FEC on day 10. Mean differences in FEC before and after treatment were negligible (3 to 8% change) for the control, ABZ, LEV-TBZ and IVM groups. The MOX treatment resulted a mean FEC reduction of 76.7%, which was not-significant by t-test (p=0.08). The TCF treatment showed a mean FEC reduction of 42.3%, which was also not-significant (p=0.1). The DrenchRite® LDA was used to evaluate the resistance status to benzimidazole, levamisole, ivermectin and moxidectin on this farm. Coprocultures indicated that the predominant worm species was Haemonchus contortus (Colombia 94-100%) (UGA 77% H. contortus and 23% Trichostrongylus colubriformis). Resistance was evaluated for both H. contortus and T. colubriformis. The LDA results indicated high resistance to all 4 anthelmintics for both parasite species. The current study, together with similar surveys in 4 other Antioquian goat and sheep farms, confirms the presence of multi-drug resistant H. contortus to all three classes of anthelmintics in this region of Colombia.

Colonoscopy quality with Entonox®vs intravenous conscious sedation: 18608 colonoscopy retrospective study.

AIMTo compare colonoscopy quality with nitrous oxide gas (Entonox®) against intravenous conscious sedation using midazolam plus opioid.METHODSA retrospective analysis was performed on a prospectively held database of 18608 colonoscopies carried out in Lothian health board hospitals between July 2013 and January 2016. The quality of colonoscopies performed with Entonox was compared to intravenous conscious sedation (abbreviated in this article as IVM). Furthermore, the quality of colonoscopies performed with an unmedicated group was compared to IVM. The study used the following key markers of colonoscopy quality: (1) patient comfort scores; (2) caecal intubation rates (CIRs); and (3) polyp detection rates (PDRs). We used binary logistic regression to model the data.RESULTSThere was no difference in the rate of moderate-to-extreme discomfort between the Entonox and IVM groups (17.9% vs 18.8%; OR = 1.06, 95%CI: 0.95-1.18, P = 0.27). Patients in the unmedicated group were less likely to experience moderate-to-extreme discomfort than those in the IVM group (11.4% vs 18.8%; OR = 0.71, 95%CI: 0.60-0.83, P < 0.001). There was no difference in caecal intubation between the Entonox and IVM groups (94.4% vs 93.7%; OR = 1.08, 95%CI: 0.92-1.28, P = 0.34). There was no difference in caecal intubation between the unmedicated and IVM groups (94.2% vs 93.7%; OR = 0.98, 95%CI: 0.79-1.22, P = 0.87). Polyp detection in the Entonox group was not different from IVM group (35.0% vs 33.1%; OR = 1.01, 95%CI: 0.93-1.10, P = 0.79). Polyp detection in the unmedicated group was not significantly different from the IVM group (37.4% vs 33.1%; OR = 0.97, 95%CI: 0.87-1.08, P = 0.60).CONCLUSIONThe use of Entonox was not associated with lower colonoscopy quality when compared to intravenous conscious sedation using midazolam plus opioid.

239 USE OF CYCLIC ADENOSINE MONOPHOSPHATE MODULATORS IN IN VITRO PRODUCTION OF BOVINE EMBRYOS

In vitro embryo production systems (IVP) try to emulate and enhance molecular events that occur in in vivo reproductive systems in order to increase, not only the number of embryos generated, but also their quality. Despite advances, IVP processes are still inefficient compared with in vivo systems. Several studies have attributed this deficiency to a lack of oocyte competence due to spontaneous premature resumption of meiotic maturation in the oocyte following the removal from its follicular environment. Therefore, our objective was to increase oocyte competence avoiding premature resumption of meiosis by using cyclic adenosine monophosphate modulators. Cumulus-oocyte complexes (COC) were obtained from ovaries of slaughterhouses, washed, and randomly allocated in 2 culture systems. Oocytes in the control group (IVM) were cultured for a period of 24 h in basal medium TCM-199 with EGF (1 µg mL–1) supplemented with rhFSH (25 mIU mL–1). Oocytes in the biphasic in vitro maturation (b-IVM) group were cultured for 2 h in a basal medium supplemented with a phosphodiesterase inhibitor, 3-isobutyl-1-methylxanthine (IBMX, 500 µM), and an activator of adenylate cyclase (forskolin, 100 µM). Subsequently, COC were washed and cultured in basal medium supplemented with cilostamide (20 µM) and rhFSH (25 mIU mL–1) for 24 h. Maturation rates were analysed and IVF was performed with a dose of 1 × 106 sperm cells mL–1 in IVF-SOF medium. The presumptive zygotes were cultured in continuous-single-culture medium (Irvine) supplemented with 8 mg mL–1 of BSA until they reached the blastocyst stage. No significant differences in maturation, cleavage, and cryotolerance were observed between b-IVM and IVM groups (P &gt; 0.05; Table 1). This study showed that b-IVM produced a significant increase in IVP compared with the control (IVM) at Days 7 and 8 (P &lt; 0.01). Blastocyst hatching rate was significant (P &lt; 0.05) for both treatment and day of analysis. The b-IVM group yielded an increase of 10 and 7.5% at Days 7 and 8, respectively, of IVP. The biphasic maturation showed an improvement in quality regarding the control group, in the timing analysis of production, and hatching percentages, and these results show that the use of cyclic adenosine monophosphate modulators in the oocyte maturation process enhances oocyte competence, which is reflected in increased productivity and embryo quality. We propose this treatment as an alternative to the standard protocols currently used in IVP of bovine embryos. Table 1.Effect of treatment on maturation, cleavage, and cryotolerance

Ultrastructure of in vitro Matured Human Oocytes.

Background:Approximately 20% of recovered oocytes are immature and discarded in intracytoplasmic sperm injection (ICSI) procedures. These oocytes represent a potential resource for both clinical and basic science application.Objective:The aim of this study was to evaluate the ultrastructure architecture of in vitro matured human oocytes using transmission electron microscopy (TEM).Materials and Methods:A total of 204 immature oocytes from infertile patients who underwent ICSI cycles were included in this prospective study. Immature oocytes were divided into two groups: (i) GV oocytes (n = 101); and (ii) MI oocytes (n = 103). Supernumerary fresh in vivo matured oocytes (n = 10) were used as control.Results:The rates of maturations were 61.38% for GV and 73.78% for MI oocytes in IVM medium (P = 0.07). However, the rate of oocyte arrest was significant between groups (P <0 .05). Ultrastructurally; in vitro and in vivo matured oocytes appeared round, with a homogeneous cytoplasm, an intact oolemma and an intact zona pellucida. However, immature oocytes indicated numerous large mitochondria-vesicle complexes (M-VC).Conclusions:Ultrastructural changes of M-VC in IVM groups emphasize the need for further research in order to refine culture conditions and improve the implantation rate of in-vitro matured oocytes.

When is the optimal timing of ICSI to rescue in vitro matured human oocytes in stimulated cycle?

When is the optimal timing of ICSI to rescue in vitro matured human oocytes in stimulated cycle?

Effects of vitrification on mitochondrial distribution and spindle configuration during in vitro maturation of human germinal vesicle-stage oocytes

Cryopreservation of human immature oocytes may be useful tool for fertility preservation in the future. However, vitrification of germinal vesicle (GV)-stage oocytes has poor result as for later development after fertilization. This may be due to abnormality of cytoplasmic maturation of the freeze-thawed oocytes. To elucidate this mechanism, we have investigated the distribution of mitochondria and spindle and chromosome configuration of in vitro matured oocytes with or without vitrification. GV oocytes were obtained from consenting patients undergoing ICSI treatment at our clinic. GV oocytes were randomly assigned to 1 of 3 groups: (1) GV group, GV oocytes; (2) IVM group, GV oocytes matured in vitro to metaphase II (MII); and (3) Vit group, oocytes vitrified at GV stage and matured in vitro. Confocal microscopy was applied to compared the mitochondrial distribution and configuration of the meiotic spindle and chromosomes in those oocytes. The maturation (MII) rate was 71.9% in the IVM group and 73.3% in the Vit group. There were no significant differences between the IVM and Vit groups. Compared to the IVM group (17.5%), the Vit group (45.9%) showed significant increase in diffuse mitochondria distribution. There was also a significantly lower proportion of oocytes with normal spindle configuration in the Vit group (18.9%) compared with the IVM group (47.5%). There were no significant differences in chromosome configuration between the IVM and Vit groups. This study revealed that vitrification of human GV oocytes has a detrimental effect on the spindle configuration of oocytes that have been thawed and matured in vitro which may explain decreased ability of further development.

329 SUPPRESSION OF BOVINE OOCYTE IVM BY SERUM AND FSH DEPRIVATION OR BY α-AMANITIN: EFFECT ON FERTILIZATION AND EMBRYO DEVELOPMENT

We have studied the effect of suppressed IVM on the developmental competence of bovine oocytes, aiming at elucidating the importance of cytoplasmic maturation in fertilization and embryo development. Six replicates of abattoir-derived oocytes were randomly divided into three IVM groups. Control (n = 950): TCM-199 with glutamax-I (Gibco, Grand Island, NY, USA), 0.25 mM Na-pyruvate, 100 IU mL−1 penicillin and 100 μg mL−1 streptomycin, 50 ng mL−1 FSH, and 10% fetal bovine serum (FBS) (Gibco); Serum+FSH-free (n = 944): same as control but without FSH and FBS; α-amanitin (n = 977): same as control but with 10 μg mL−1 α-amanitin. Nuclear maturation of oocytes was studied 24 h after the onset of IVM, the formation of sperm aster structure 10 hours post-insemination (hpi) and the formation of pronuclei 20 hpi. Sperm aster was visualized with β-tubulin antibody (modified from Navara et al. 1999 Dev. Biol. 162, 29–40). Presumptive zygotes were cultured until Day 7 in modified SOFaaci + 4 mg mL−1 fatty acid-free BSA in 5% O2. Cumulus cell expansion was seen only in the control group. The results of nuclear maturation, fertilization, and embryo development are summarized in Table 1. Serum and FSH deprivation did not have a statistically significant effect on the parameters studied (vs. control). α-amanitin exposure during IVM reduced nuclear maturation, fertilization, and Day 3 embryo cleavage vs. control, and resulted in total blockage of Day 7 blastocyst development. The treatment groups had significantly smaller mean diameters of male pronuclei (control: 14 ± 0.6 μ­m; serum+FSH-free: 12 ± 0.5 μ­m, P &lt; 0.05; α-amanitin: 10 ± 0.6 μ­m, P &lt; 0.001) and sperm asters (control: 86 ± 4 μ­m; serum+FSH-free: 82 ± 4 μ­m, P &lt; 0.01; α-amanitin: 49 ± 7 μm, P &lt; 0.001) (nonparametric Kruskall Wallis and Mann-Whitney U tests) vs. control group. Despite reduction in pronucleus and sperm aster diameter, serum and FSH deprivation during IVM did not affect in vitro developmental competence of bovine oocytes, suggesting a need for re-evaluation of the components of IVM. α-Amanitin exposure in IVM disturbed nuclear maturation, fertilization, and embryo development, indicating the essence of early transcription. Table 1. Average percentages ± (n) for nuclear maturation, fertilization (min two pronuclei), embryo cleavage, and blastocyst development

IVM media, oocyte diameter and donor genotype at RYR1 locus in relation to the incidence of porcine diploid oocytes after maturation in vitro

In the present study three factors were investigated that may affect the process of the first polar body extrusion in pig oocytes matured in vitro: IVM medium, oocyte diameter and donor genotype at the ryanodine receptor (RYR1) locus. In the first experiment, COCs were collected by the aspiration of slaughterhouse ovaries. Oocytes were matured in vitro at 39 °C, in humidified 5% CO 2 atmosphere for 44 h using the following media: (1) TCM199 + hCG + eCG + follicular fluid (FF), (2) TCM199 + hCG + 17β-estradiol and (3) NCSU23 + hCG + eCG + FF. According to cytogenetic analysis, 98.1% of cells reached the second metaphase stage (MII). No significant differences were observed among IVM groups in terms of diploidy level. In the second experiment, oocytes collected by the aspiration or slicing of individual ovaries were matured in vitro in groups reflecting their origin. One ovary was considered a donor. IVM was carried out under conditions described in experiment I, with the use of TCM199 + hCG + 17β-estradiol. A total of 68 ovaries/donors were included in this study. Granulosa cells collected from each ovary were used as DNA source in molecular (RFLP) analysis. Genotype frequencies at the RYR1 locus were as follows: CC, 0.46; CT, 0.48 and TT, 0.06. After maturation the diameter of each denuded oocyte was determined with the use of a computer aided system. Five size categories were distinguished: <90, 90–100, 100.1–110, 110.1–120 and >120 μm. The average diameter of haploid oocytes at MII stage was 111.7 μm, whereas that of diploid cells was 110.4 μm. According to statistical analysis, diploidy was not related to the oocyte diameter. That trait, however, was influenced by the donor genotype at the RYR1 locus. The TT genotype was associated with a higher rate of diploidy.

297 A CASE REPORT: THE OUTCOME OF IVP MAY BE RELATED TO THE BATCH OF TCM-199 IN IVM

Chemicals used in bovine IVP may be inhibitory to embryo development (see, e.g., Van Soom A et al. 1994 Theriogenology 41, 325). The present study continued earlier experiments to optimize serum-free IVM protocol for bovine oocytes (Räty M. 2004 Reprod. Fert. Dev. 16, 281). The embryo development rate was much lower in the new experiments than in our previous studies. Thorough testing indicated that the batch of TCM-199 medium used in maturation was the reason for the lower embryo development results. In total, 14,589 abattoir-derived bovine oocytes in 23 batches were matured for 24 h in TCM-199 with glutamax-I (GIBCO, Paisley, UK), 0.25 mM Na-pyruvate, 100 IU mL−1 penicillin, 100 μg mL−1 streptomycin, 10 μg mL−1 LH, 2 μg mL−1 FSH, and 1 μg mL−1 β-estradiol supplemented as follows: (1) 10% FBS (GIBCO, New Zealand); (2) 4 mg mL−1 fatty acid-free albumin (FAFBSA) (Sigma-Aldrich, Helsinki, Finland); (3) 4 mg mL−1 polyvinylpyrrolidone (PVP) (Sigma) + growth factors (GF; 100 ng mL−1 IGF-I + 100 ng mL−1 EGF); or (4) as (3) but without FSH, LH, and β-estradiol (PVPGFwoH). Fertilized oocytes were denuded and cultured in modified SOFaaci + 4 mg mL−1 FAFBSA in 5% O2. (Holm P. et al. 1999 Theriogenology 52, 683–700). Two TCM-199 batches were used in separate IVM runs, batch 1 (Lot#3075638; 10 runs) and batch 2 (Lot#3081334; 13 runs). The statistical analyses were based on generalized linear mixed models. The estimated probabilities for embryo cleavage and development are shown in Table 1. The use of TCM-199 batch 1 resulted in significantly lower embryo development rates than the use of TCM-199 batch 2 in every IVM group studied. PVP IVM groups were the most sensitive for the TCM-199 batch and resulted in reduced embryo cleavage as well as strikingly low Day 7 embryo development. Our results indicate that TCM-199 batch 1 disturbed embryo development. In defined IVM groups (PVP groups), the maturation inhibiting role of TCM-199 batch 1 was seen already at the cleavage stage. It is possible that FBS and FAFBSA may have protected the oocytes to some extent against inhibitory effects of TCM-199. There is evidence for BSA acting as a chelating agent (see, e.g., Flood L.P. and Shirley B. 1991 Mol. Reprod. Dev. 30, 226–231). Table 1. Estimated probability for embryo cleavage at 38–42 hpi and Day 7 embryo development after IVM in two different batches of TCM-199

Efficacy of long-term monthly administration of ivermectin on the progress of naturally acquired heartworm infections in dogs

This study was designed to evaluate the efficacy of prolonged monthly ivermectin treatment against Dirofilaria immitis in client-owned dogs with naturally acquired infections and to clinically monitor the animal's response to the slow killing of heartworms, with death of the worms distributed over a period of up to 2 years. A total of 17 male and female dogs of different breeds and ages were used. Prior to treatment, all of the dogs tested positive for heartworm antigen (Ag) and all but two had microfilariae (mf). The dogs were randomly allocated to one group of seven dogs which received a commercial formulation of ivermectin (minimum, 6 mcg IVM/kg) plus pyrantel (minimum, 5 mg PP/kg) (Heartgard Plus™ Chewables, Merial, Ltd.), another group of seven dogs which received a commercial formulation of IVM (min, 6 mcg/kg) (Heartgard ® Chewables, Merial Ltd.), and a group of three dogs which served as an untreated controls. All dogs were evaluated prior to initiation of treatment and thereafter at 3- to 5-month-intervals for mf, Ag, and radiographic and echocardiographic findings. All of the 17 dogs, with the exception of two dogs in the IVM group, had circulating mf of D. immitis prior to the 1st monthly dose, and a few also had mf of Dirofilaria repens. After 4 monthly doses, only one dog in the IVM/PP group and two dogs in the IVM group had a patent heartworm infection, and no heartworm mf were seen in the 14 treated dogs thereafter. After 10 monthly doses, the number of Ag-positive dogs in both of the treated groups decreased gradually. Efficacy, based on the reduction in number of Ag-positive dogs, was similar for the IVM/PP and IVM groups, with overall efficacy scores for the 14 dogs of 21, 21, 43, and 71% after 10, 14, 19, and 24 monthly doses, respectively. Two of the seven dogs treated with IVM/PP, one of the seven treated with IVM, and two of the three untreated controls showed echocardiographic evidence of a parasitic burden prior to treatment, and all of these scores had decreased by the end of the study. Only one dog (IVM/PP group) had a cardiovascular pattern of heartworm disease by echocardiography prior to treatment, but this dog's score increased to two and the scores of two additional dogs increased from zero to two (IVM group) or three (IVM/PP group) by the end of the study. Only 1 (IVM/PP group) of the 17 dogs showed a pulmonary pattern of heartworm disease by radiography prior to treatment, but this dog's score increased to three by the end of the study. The radiographic scores of two additional dogs in the treated groups increased from zero to three (IVM/PP) or two (IVM) by the end of the study. Thus, monthly administration of IVM to dogs with clinical, radiographic or echocardiographic evidence of heartworm disease is ill-advised and such treatment of even the asymptomatic dog should be done only with much caution and frequent monitoring by the veterinarian.

Effect of nuclear stages during IVM on the survival of vitrified-warmed bovine oocytes

The effect of nuclear stages during IVM on the survival of vitrified-warmed bovine oocytes was investigated. Oocytes with compact cumulus cells were cultured for 0, 6, 12 and 24 h in TCM199 supplemented with 5% fetal bovine serum (FBS) in 3% CO 2 in air. The oocytes were first exposed to 20% ethylene glycol solution and were subjected to vitrification in a solution containing 40% ethylene glycol, 18% Ficoll-70 and 0.3 M sucrose. After warming in 20 °C water, oocytes which had been vitrified at less than 24-h of IVM were again cultured to complete the 24-h of IVM period. Oocytes were then incubated with frozen-thawed spermatozoa in Brackett and Oliphant (BO) medium containing 60 μg/ml heparin and 0.25% BSA for 20 h. In vitro fertilization rates of oocytes vitrified-warmed at 0, 6, 12 and 24-h IVM were 75.2, 68.0, 82.0 and 72.4%, respectively, comparable to the rates for unvitrified control oocytes (80.6%). A higher incidence of polyspermic fertilization was observed in oocytes vitrified at 24-h IVM (44.9 vs 22.6% in the control group, P < 0.05). Vitrification of oocytes at 12-h IVM seemed to be better than that of other IVM groups, since the normal fertilization rate of all treated oocytes was the highest (36.0%) among the vitrification groups. Developmental competence of the oocytes following vitrification and in vitro fertilization (12-h IVM group) was examined by cell-free culture of presumptive zygotes up to 9 d in modified synthetic oviduct fluid (mSOF) in 5% CO 2, 5% O 2 and 90% N 2. The cleavage rate of zygotes from vitrified oocytes 48 h after insemination was 29.8%. which was lower than that of the control group (57.0%, P < 0.05). Development to blastocysts from the vitrified oocytes (4.8%) was much lower than that of the control group (27.0%, P < 0.05). These results indicate that cryopreservation of bovine oocytes by vitrification may be affected by their maturation stage in vitro, and that developmental competence to blastocysts of cleaved oocytes following vitrification may be impaired compared with unvitrified control oocytes.