Oocytes Of Donors Research Articles

Effect of Endometrial Thickness and duration of Estrogen Supplementation on In Vitro Fertilization-Intracytoplasmic Sperm Injection Outcomes in Fresh Ovum/Embryo Donation Cycles.

Background:There is no consensus regarding optimal endometrial thickness and duration of estrogen supplementation in embryo transfer cycles, at present.Aims:To observe the effect of endometrial thickness and/or duration of estrogen supplementation on in vitro fertilization (IVF)/intracytoplasmic sperm injection (ICSI) outcomes in fresh ovum/embryo donation cycles.Settings and Design:This was a retrospective observational study. The study was conducted from January 2015 to November 2017.Subjects and Methods:Nine hundred and fifty seven fresh blastocyst transfer cycles in the recipients of oocyte/embryo donation regardless of reproductive history and diagnosis conducted at Nova IVF Fertility, Ahmedabad, Gujarat, India. Of these, 315 women had single embryo transfer (SET), while 642 had double embryo transfer (DET). Only fresh blastocysts derived from oocytes of young donors (≤30 years) and transferred in a uniform hormone replacement therapy (HRT) cycle were included. The effect of endometrial thickness and duration of estrogen on live birth rate (LBR) and other IVF/ICSI outcomes were analyzed.Statistical Analysis:Univariate logistic regression.Results:A significant improvement in LBR was noted in the recipients with each millimeter increase in endometrial thickness starting from 6 mm after transfer of either single (odds ratio [OR] = 1.3, P = 0.003) or double (OR = 1.14, P = 0.0218) blastocysts. Lower LBR was observed in recipients having SET and who received estrogen supplementation of <10 days (OR = 0.72; P = 0.02). Implantation rate and clinical pregnancy rate also improved significantly with endometrial thickness, but there was no change in clinical abortion rate and ectopic pregnancy rate.Conclusions:After minimizing the possible oocyte factor by including only donor oocytes and that of COH using a uniform HRT protocol, LBR improved with each millimeter increase in endometrial thickness starting from 6 mm. Shorter duration of estrogen supplementation (<10 days) reduced the chances of live birth in recipients after transfer of a single blastocyst.

Subcortical maternal complex (SCMC) expression during folliculogenesis is affected by oocyte donor age in sheep

The age-associated decline in female fertility is largely ascribable to the decrease in oocyte quality. The subcortical maternal complex (SCMC) is a multiprotein complex essential for early embryogenesis and female fertility and functionally conserved across mammals. The present work evaluated expression dynamics of its components during folliculogenesis in relation to maternal age in sheep. The expression of the SCMC components (KHDC3/FILIA, NLRP2, NLRP5/MATER, OOEP/FLOPED, PADI6, TLE6 and ZBED3) was analyzed by real-time PCR in pools of growing oocytes (GO) of different diameters (70-90 μm (S), 90-110 μm (M), or 110-130 μm (L)) derived from non-hormonally treated adult (Ad; age < 4 years), prepubertal (Pr; age 40 days), or aged ewes (age > 6 years). Specific expression patterns associated with donor age were observed during folliculogenesis for all genes, except ZBED3. In oocytes of adult donors, the synthesis of NLRP2, NLRP5, PADI6, and ZBED3 mRNAs was complete in S GO, while FILIA, TLE6, and OOEP were actively transcribed at this stage. Conversely, Pr GO showed active transcription of all mRNAs, except for ZBED3, during the entire window of oocyte growth. Notably, aged GO showed a completely inverse pattern, with a decrease of NLRP2, TLE6, FILIA, and PADI6 mRNA abundance during the latest stage of oocyte growth (L GO). Interestingly, MATER showed high expression variability, suggesting large inter-oocyte differences. Our study describes the SCMC expression dynamics during sheep oogenesis and reports age-specific patterns that are likely involved in the age-related decline of oocyte quality.

Performance of zebu donor cows in vitro production of embryos

In vitro production (IVP) of embryos is a biotechnology capable of increase bovine production. Thus, the performance of Brahman, Gir, and Nellore donor cows in IVP was evaluated. Data from 2012 to 2017 were collected from donors subjected to ovum pick up (OPU) for IVP at the In Vitro Acre Company, Rio Branco, Acre, Brazil. The numbers of aspirated oocytes, viable oocytes, viable embryos, and pregnancy per OPU, and the cleavage, viable embryo, and pregnancy rates were analyzed considering the breed, location, time, number of OPU/day, and number of OPU/donor. The numbers were compared by the Student t test, and the rates by the chi-square test. Brahman donors presented higher rates of cleavage, and viable embryos, followed by Nellore, and Gir (p&lt;0.05), and lower number of pregnancies (p&lt;0.05). Donors raised in the state of Acre, Brazil, had higher numbers of aspirated oocytes, viable oocytes, and viable embryos (p&lt;0.05), whereas those raised in Peru had higher cleavage and pregnancy rates (p&lt;0.05). Pregnancy rates were higher in the rainy season (p&lt;0.05). Cleavage rates were higher when more than 20 OPU/day were performed (p&lt;0.05), and intermediate when performing 11-20 OPU/day. The numbers of aspirated and viable oocytes of donors subjected to one OPU were higher than those of donors subjected to four to seven OPU (p&lt;0.05). Cleavage rates of donors subjected to two OPU were higher than those of donors subjected to more than three OPU (p&lt;0.05). The breed, location, number of OPU/day, and number of OPU performed in the same donor affected, with different intensities, the performance variables of Brahman, Gir, and Nellore donors in IVP.

Follicle diameter predicts oocyte maturity but not formation of blastocysts or ploidy of blastocysts arising from mature oocytes of donors

Follicle size during controlled ovarian hyperstimulation is the only measure helpful to the clinician in deciding when to trigger final maturation. Controversy exists over whether the largest follicle(s) or the complete cohort of follicles best predicts outcome for IVF. Past efforts have concentrated on predictors of pregnancy and live birth following IVF with fresh transfer. Now there is increasing interest in retrieving oocytes for cryopreservation or for embryo production with preimplantation genetic testing for aneuploidy (PGT-A). It remains unclear whether there is a preferred size of follicles to obtain euploid oocytes or oocytes that will become euploid blastocysts. Retrospective Analysis of Embryo Outcomes. Consented oocyte donors (N = 22) underwent retrieval of oocytes, one-by-one, from follicles with diameters measured during the retrieval. Oocytes were cultured individually, fertilized by intracytoplasmic sperm injection (ICSI) and monitored for development. Quality blastocysts, achieving Gardner grades of AA, AB, BA, BB or BC, underwent trophectoderm biopsy on days 5 or 6 and biopsies were sent to a commercial PGT-A lab in the US. Results of maturity, fertilization, development and ploidy were considered with reference to the size of the follicle from which the oocyte was retrieved. Analysis of data involved Student’s T tests, and receiver operator characteristic (ROC) curves with significance determined using Mann-Whitney U test. Oocytes were retrieved from follicles with measured diameters averaging 17.4 +/- 2.9 mm (N = 315). Of the oocytes, 80.4% had 1 polar body (MII), 9.8% had a germinal vesicle (GV) and 9.1% had neither a polar body nor a GV (MI). The sizes of the follicles from which these oocytes came were significantly different: MII, 18.3 +/- 2.2 mm; GV, 12.5 +/- 1.6 mm; and MI, 15.3 +/- 3.2 mm. ROC curves indicated that follicle diameter was a “grade A” predictor of GV oocytes (AUC = 0.96; P < 0.0001, Mann-Whitney U test) and a “grade B+” predictor of MII oocytes (AUC = 0.87; P<0.0001, Mann-Whitney U test). Among MII oocytes, follicle size did not predict fertilization by ICSI (ROC AUC = 0.54, not significant), formation of quality blastocysts (ROC AUC = 0.53, not significant), or blastocyst ploidy ROC AUC = 0.53, not significant). Significant AUCs for ROC curves indicate that follicle diameter is an excellent predictor of oocyte maturity. However the diameter of the follicle from which an MII oocyte was retrieved did not predict its quality as assessed by its fertilizability with ICSI, its ability to develop into a quality blastocyst or its ploidy. Whereas follicle diameter can predict maturity of the oocytes retrieved from them quite well, follicle diameter is a poor predictor of oocyte quality including blastocyst ploidy. Since oocyte ploidy was not directly assessed, it remains unclear whether oocyte ploidy is associated with follicle diameter. However, with most embryo aneuploidy arising from errors in meiosis, we believe that follicle size is unlikely to be a good predictor of oocyte ploidy.

Delay in maternal transcript degradation in ovine embryos derived from low competence oocytes.

Oocytes from prepubertal animals have a reduced ability to undergo embryo development and produce viable offspring. The present work used an ovine model consisting of oocytes derived from adult and prepubertal donors to assess the molecular status of oocytes and preimplantation embryos with different developmental competence. The lower potential of oocytes of young donors was confirmed in terms of in vitro developmental capabilities and kinetics. A panel of genes including maternal effect (DPPA3, GDF9, NMP2, ZAR1) and housekeeping genes (ACTB, RPL19, SDHA, YWHAZ, ATP1A1), genes involved in DNA methylation (DNMT1, DNMT3A, DNMT3B), genomic imprinting (IGF2R), pluripotency (NANOG, POU5F1) and cell cycle regulation (CCNB1, CDK1, MELK) was relatively quantified. Temporal analysis during oocyte maturation and preimplantation embryo development evidenced patterns associated with donor age. With a few gene-specific exceptions, the differential model showed a reduced transcript abundance in immature prepubertal oocytes that completely reversed trend after fertilization, when higher mRNA levels were consistently observed in early embryos, indicating a delay in maternal transcript degradation. We propose that the molecular shortage in the prepubertal oocyte may affect its developmental potential and impair the early pathways of maternal mRNA clearance in the embryo. While confirming the different potential of oocytes derived from adult and prepubertal donors, our work showed for the first time a consistent delay in maternal transcript degradation in embryos derived from low competence oocytes that interestingly recalls the delayed developmental kinetics. Such abnormal transcript persistence may hinder further development and represents a novel perspective on the complexity of developmental competence.

205 DEVELOPMENT OF OOCYTES FROM COWS TREATED WITH RETINOL IS COMPROMISED PRIOR TO IMPLANTATION

In the cow, sheep, and gilt, a retinoid support enhances the oocyte development (reviewed by Hidalgo C et al. 2003 Reproduction 125, 409–416). The shortest effective time interval for these animals injected with retinol (ROH) is four days, but the reasons for this have not been described. Furthermore, the development after transfer of embryos derived from oocytes of donors treated with ROH is not known. The objective of the present work was to elucidate the two above questions. We analyzed the ROH and retinyl palmitate (REP) contents in plasma and follicular fluid (FF). The estrus cycles of heifers (n = 12) were synchronized with progestagen and PGF2α. Blood samples were taken at progestagen removal (Day 0). Animals were injected with 1 × 106 ROH units (n = 6) or corn oil (vehicle; n = 6) on Days 0, 1, and 4. Contents of follicles between 3 and 10 mm were aspirated on Day 4 by an ultrasound-guided procedure. All samples were analyzed for ROH and REP by HPLC. To produce embryos for transfer, follicles were aspirated from donors treated four times with 1 × 106 ROH units/week or vehicle, starting four days before the first aspiration. Both groups of cumulus-oocyte complexes (COCs) were matured in vitro with or without 5 nM 9-cis-retinoic acid (RA). The presence of RA prolonged the exposure to retinoids (cows treated with ROH) or acted as a positive control (heifers with vehicle). Oocytes were fertilized and cultured in mSOF + 5% FCS. Embryo transfer (ET) to recipients was performed with fresh (one) or vitrified (two) good-morphology Day 7 embryos, and pregnancy monitored on Days 21, 35, and 60. Data analysis was by GLM (ROH and REP concentrations) or CATMOD (pregnancy monitoring), and Duncan's test (a,bP &lt; 0.05; v,x,y,zP &lt; 0.02). Average FF volume recovered were 351 ± 127 μL (controls; range 100–700) and 393 ± 127 μL (ROH; range 80–950). Concentrations of REP were unaffected by timing, follicle or blood, and ROH treatment (data not shown). Concentrations of ROH (μg/dL) for vehicle and ROH-treated cows were (LSM ± SE) 42.0 ± 1.8vx and 42.0 ± 2.4vx (plasma-Day 0, before ROH injection), 37.3 ± 1.8v and 47.64 ± 2.4x (plasma-Day 1), 42.6 ± 1.9vx and 45.5 ± 2.3vx (plasma-Day 4), and 6.1 ± 3.0y and 16.8 ± 2.6z (FF-day 4), respectively. Cumulatively, embryos from donors receiving ROH did not exhibit pregnancy on Day 21 (0/15x; confirmed on Day 35), which differed from vehicle donors on Day 21 (44%, 8/18y) and Day 35 (33%, 6/18y), and tended to differ (P = 0.07) on Day 60 (22%, 4/18). Pregnancy rates were independent of fresh or vitrified embryos within ETs. Injected ROH elevated blood concentrations of ROH, although values became normalized on Day 4. However, a coasting period of 4 days for ROH administration seems to be justified by increased intrafollicular levels of ROH. Development into blastocysts is disrupted before implantation, showing that ROH directly affects the oocyte during its intrafollicular growth. This work was supported by MCYT-AGL-2002-0117.

Kinetics of the functional loss of different muscarinic receptor isoforms in Xenopus oocytes.

Native Xenopus oocytes express two isoforms of muscarinic receptors that mediate qualitatively different physiological responses. Oocytes of the majority of donors (common) express M3-like receptors (M3Rs) at comparable densities at both the animal and vegetal hemispheres of the cell. Rare (variant) donors possess oocytes that express mainly M1-like receptors (M1Rs), localized predominantly at the animal hemisphere. We have investigated the apparent degradation of these two isoforms and its relationship to their hemispheric distribution. Cycloheximide (CHX) caused a time-dependent decrease in receptor-mediated responses and [3H]quinuclidinyl benzylate (QNB) binding in oocytes from both types of donors. The t1/2 values ranged between 3 and 7 h. Removal of CHX resulted in rapid recovery of the response. This implied rapid degradation and turnover of both types of receptors. The loss of M1Rs was more than that of M3Rs. Moreover, the decrease was more rapid and more extensive on the animal hemisphere in both types of donors. Injection of oocytes expressing either receptor isoform with specific antisense oligonucleotides complementary to either m1 or m3 muscarinic receptors (from mouse) showed receptor loss at approximately the same rate as that calculated from experiments with CHX. Furthermore, oocytes of variant donors express M1Rs exclusively on the animal hemisphere, while the residual activity found on the vegetal hemisphere of the cell was mediated by M3Rs. Inhibition of putative receptor glycosylation with tunicamycin caused a rapid decrease in receptor-mediated responses and radioligand binding on M1Rs, but had virtually no effect on M3Rs. The expression of cloned m1 muscarinic receptors, however, was not affected by tunicamycin, suggesting that glycosylation is not a general prerequisite for the functional expression of muscarinic receptors.

Native Xenopus oocytes express two types of muscarinic receptors

We have recently described two types of muscarinic responses in native Xenopus oocytes of different donors (common and variant) that display qualitative and quantitative differences (Lupu-Meiri et al., 1990). Here we characterized the muscarinic receptors mediating these two types. The anti-muscarinic toxins from Dentroaspis significantly inhibited responses in oocytes of common donors, but had little effect on responses in oocytes of variant donors, possibly indicating expression of different receptor subtypes. Using specific muscarinic antagonists, we found that oocytes of common donors exhibit a pattern compatible with the M3 subtype of muscarinic receptors, while oocytes of variant donors appear to possess receptors of the M1 subtype. To more directly determine the subtypes of muscarinic receptors in oocytes of both populations of donors, we have microinjected antisense oligonucleotides into native oocytes. Antisense oligonucleotides to unique sequences in the N-terminal and the third cytoplasmic loop of M3 muscarinic receptors caused a significant inhibition of the response of common oocytes, but had virtually no effect on responses in oocytes of variant donors. Conversely, oligonucleotides complementary to the unique sequences of the m1 muscarinic receptors inhibited the response in variant oocytes, but not in oocytes of common donors. We conclude that native Xenopus oocytes of different donors phenotypically express either M3-like (majority) or M1-like (minority) muscarinic receptor subtypes. The differences in receptor subtype expression may explain the different characteristics of responses in the two populations.

Two types of intrinsic muscarinic responses in Xenopus oocytes

Oocytes of 40% of Xenopus laevis frogs respond to acetylcholine (ACh). Oocytes of the majority of responders exhibit the common two-component depolarizing muscarinic response (mean amplitude of the rapid component, 54 nA). Oocytes of approximately 10% of the responders ("variant" donors) exhibit a muscarinic response characterized by a very large transient, rapid current (mean amplitude 1242 nA, reversal potential -33 mV). Responses in oocytes of variant donors exhibit further qualitative differences: pronounced desensitization (absent in oocytes of common donors), characteristic prolonged latency (5.4 vs 0.9 s in oocytes of common donors) and marked inhibition of the response by activators of protein kinase C. Rapid responses in oocytes of variant donors are usually increased by treatment with collagenase, which, in common oocytes, often results in a complete loss of the response that correlates with the loss of muscarinic ligand binding. The number of muscarinic receptors was similar in oocytes of both types of donors (2.2 vs 3.0 fmol/oocyte). Also, the responses of oocytes of variant donors to microinjections of CaCl2 or inositol 1,4,5-trisphosphate were similar to those found in cells of common donors. These findings imply that altered receptor number, calcium stores and/or chloride channel density are not responsible for the variant responses. However, ACh caused an sixteen-fold greater efflux of 45Ca in oocytes of variant donors (35 vs 2.2% of total label in oocytes of common donors). Hence, the characteristics of the variant response may be related to a more efficient coupling between receptor stimulation and the mobilization of cellular calcium.

Two types of intrinsic muscarinic responses in Xenopus oocytes

Fully grown Xenopus laevis oocytes display marked morphological asymmetry. The giant cell is divided into animal (pigmented) and vegetal hemispheres. We have developed methodology aimed at easy determination of hemispheric responses to the application of acetylcholine (ACh) and determination of the distribution of muscarinic receptors. Oocytes of common donors exhibit muscarinic responses that are similar when either the animal or the vegetal hemisphere of the cell is exposed to ACh. Oocytes of variant donors, however, exhibit markedly larger muscarinic responses when the animal hemisphere is exposed to ACh (ratio animal/vegetal, 5.8). The differences in hemispheric responsiveness correlate well with the hemispheric distribution of muscarinic receptors. While oocytes of common donors exhibit a modest excess of receptor number at the animal hemisphere (ratio animal/vegetal hemispheres, 1.4), oocytes of variant donors exhibit a large excess of receptors on the animal hemisphere (ratio animal/vegetal, 5.6). Upon further examination, we have found that the distribution of muscarinic receptors is non-homogeneous in either hemisphere in oocytes of both common and variant donors. The asymmetric distribution of receptors may be related to increased efficiency of signal transduction coupling in oocytes of variant donors.