Pronuclear Embryos Research Articles

Extended Injection Intervals of Gonadotropins by Intradermal Administration in IVF Treatment

Gonadotropins can be administered every 5 days under intradermal injection in in vitro fertilization (IVF) treatment. To explore the effectiveness of intradermal injection of recombinant human FSH (rhFSH) for women undergoing IVF. Women who received their first IVF treatment enrolled in this prospective intervention in 2018. All women received a bolus of 900 IU rhFSH intradermally at day 2 of the treatment cycle followed by additional dosage of rhFSH at day 7 and/or day 10. The main outcome measures included the total dose of rhFSH and number of injections required, sequential serum FSH level detected, and number of mature oocytes retrieved. Seventy women completed the study. On average, 2.31 ± 0.73 injections and 1662 ± 397 IU of rhFSH were administered. While the baseline FSH level was 5.6 ± 2.2 IU/L, the serum concentrations of FSH after rhFSH administration were 35.3 ± 7.0 on the first day (24 hours) and 10.7 ± 3.7 IU/L on the fifth day (120 hours). A total of 10.5 ± 6.6 mature oocytes were retrieved, resulting in 7.3 ± 5.1 pronuclear embryos; 1.8 ± 0.6 embryos were transferred to the uterus. Our findings resulted in 72% fertilization, 91% cleavage, 31% implantation, and 36% live birth rates. Although fewer larger follicles were found, noninferiority results were noted in the mature oocytes retrieved, good embryos available, and clinical pregnancy rate compared with those received conventional daily subcutaneous rhFSH administration. Intradermal administration of rhFSH, with a smaller dose of rhFSH and fewer injections, may achieve the goal of a cost-effective and more patient-friendly regimen.

Wenshen Shengjing Decoction Improves Early Embryo Development by Maintaining Low H3K27me3 Levels in Sperm and Pronuclear Embryos of Spermatogenesis Impaired Mice.

Many ingredients in Wenshen Shengjing Decoction (WSSJD) can cause epigenetic changes in the development of different types of cells. It is not yet known whether they can cause epigenetic changes in sperms or early embryos. Here, we investigated the role of WSSJD in epigenetic modifications of sperms or early embryos and early embryo development. A mouse model with spermatogenesis disorders was established with cyclophosphamide (CPA). WSSJD was administrated for 30 days. The male model mice after the treatment were mated with the female mice treated with superovulation. The embryo development rate of each stage was calculated. Immunofluorescence staining was used to detect the expression of H3K27me3 in sperm, pronuclear embryos, and 2-cell embryos. Western blotting was used to detect the expression of histone demethylase KDM6A and methyltransferase EZH2 in 2-cell embryos with developmental arrest. The expressions of zygotic genome activation genes (ZSCAN4, E1F1AX, HSPA1A, ERV4-2, and MYC) in 2-cell embryos with developmental arrest were analyzed with qRT-PCR. Comparing with the control group, CPA destroyed the development of seminiferous epithelium, significantly increased the expression level of H3K27me3 in sperm, reduced the expression ratio of H3K27me3 in female and male pronuclei, delayed the development of 2-cell embryos, and increased the developmental arrest rate and degeneration rate of 2-cell embryos. Moreover, the expressions of EZH2 and H3K27me3 were significantly increased in the 2-cell embryos with developmental arrest, and the expression of zygotic genome activation genes (ZSCAN4, E1F1AX, HSPA1A, ERV4-2, and MYC) was significantly decreased. Compared with the CPA group, WSSJD promoted the development of seminiferous epithelium, maintained a low level of H3K27me3 modification in sperm and male pronucleus, significantly increased the development rate of 2-cell embryos and 3-4 cell embryos, and reduced the developmental arrest rate and degeneration rate of 2-cell embryos. WSSJD may promote early embryonic development by maintaining a low level of H3K27me3 modification in sperm and male pronucleus and regulating the zygotic genome activation in mice with spermatogenesis disorders induced by CPA.

Is there still a role for a cleavage-stage embryo transfer?

ObjectiveTo determine whether pregnancy outcomes are poor or futile when an intended day 5 transfer is converted to a cleavage-stage transfer because of poor embryo development or a lower number of embryos.DesignRetrospective cohort study.SettingAcademic medical center.Patient(s)Women with a limited number of embryos, defined as ≤6 two pronuclear embryos, after in vitro fertilization.Intervention(s)Patients who had a cleavage-stage transfer were age matched with patients who had a day 5 transfer.Main Outcome Measure(s)Live birth rate.Result(s)A total of 146 women were included in the study with 73 women in each group. Cleavage-stage transfer was associated with significantly lower implantation and clinical pregnancy rates compared with those of day 5 transfer. Although the live birth rate of the cleavage-stage transfer group was lower than that of the day 5 transfer group (25% vs. 40%, respectively), the cleavage-stage transfer still resulted in a live birth rate of 25%. A subanalysis comparing women who did and did not achieve live birth after cleavage-stage transfer demonstrated a live birth rate of 27% when at least one grade A embryo was transferred vs. 17% when a lesser quality embryo (grade B or C) was transferred.Conclusion(s)As expected, the live birth rate after cleavage-stage transfer was lower than that after day 5 transfer. However, the live birth rate of cleavage-stage transfer still fell into acceptable practice, >5%, for patients who were otherwise at very high risk of having no day 5 embryo transfer. Extended culture may not be necessary for all patients.

The Effects of Two Different Freezing Method on Survival and Development of Pronuclear Stage Human Embryos

Human embryo cryopreservation is widely used in ART Programs, as it allows embryos to be stored without deterioration until needed...

The impaired development of sheep ICSI derived embryos is not related to centriole dysfunction

While intracytoplasmic sperm injection (ICSI) is an asset in human Assisted Reproduction Technologies (ART), its outcomes, in terms of blastocyst, is still unacceptably low in ruminants. The picture typically found in ICSI derived bovine and ovine embryos is an asymmetry between a high activation rate, marked by a pronuclear development, and a low first cleavage rate. Abnormal centriole function has been indicated as a possible factor which undermines embryonic development following ICSI, especially when Freeze Dried spermatozoa (FD) are used. In order to verify the hypothesis that centriole dysfunction might be responsible for low ICSI outcomes in sheep, we have investigated micro-tubular dynamics, markedly aster nucleation, in fertilized sheep zygotes by ICSI with frozen/thawed (FT) and FD spermatozoa; In Vitro Fertilized (IVF) sheep oocytes were used as control. The spermatozoa aster nucleation was assessed at different time points following ICSI and IVF by immune-detection of α-tubulin. Pronuclear stage, syngamy and embryo development were assessed. No difference was noticed in the timing of aster nucleation and microtubule elongation in ICSI-FT derived embryos with control IVF ones, while a delay was recorded in ICSI-FD ones. The proportion of 2-pronuclear stage zygotes was similar in ICSI-FT and ICSI-FD (47% and 53%, respectively), both much lower comparing the IVF ones (73%). Likewise, syngamy was observed in a minority of both ICSI groups (28.5% vs 12.5% in ICSI-FT/FD respectively) comparing to IVF controls (50%), with a high number of zygotes blocked at the 2-pronuclear stage (71.5% vs 87.5% respectively). While no significant differences were noticed in the cleavage rate between ICSI-FD, ICSI-FT and IVF groups (31%, 34% and 44%) respectively, development to blastocyst stage was markedly compromised in both ICSI groups, especially with FD spermatozoa (10% in ICIS-FD and 19% in ICSI-FT vs 33% in IVF (P < 0.005, ICSI-FD vs IVF and P < 0.05, IVF vs ICSI-FT, respectively). Hence, here we have demonstrated that the reduced cleavage, and the ensuing impaired development to blastocysts stage of ICSI derived sheep embryos is not related to centriole dysfunction, as suggested by other authors. The major recorded problem is the lack of syngamy in ICSI derived zygotes, an issue that should be addressed in further studies to improve ICSI procedure in sheep embryos.

Carboxylated ε-poly-L-lysine, a cryoprotective agent, is an effective partner of ethylene glycol for the vitrification of embryos at various preimplantation stages

It has been known that different protocols are used for embryo preservation at different stages due to different sensitivity to the physical and physiological stress caused by vitrification. In this study, we developed a common vitrification protocol using carboxlated ε-poly-l-lysine (COOH-PLL), a new cryoprotective agent for the vitrification of mouse embryos at different stages. The IVF-derived Crl:CD1(ICR) x B6D2F1/Crl pronuclear, 2-cell, 4-cell, and 8-cell, morula and blastocyst stage embryos were vitrified with 15% (v/v) ethylene glycol (EG) and 10% (w/v) COOH-PLL (E15P15) or 15% (v/v) EG and 15% (v/v) dimethyl sulfoxide (E15D15) using the minimal volume cooling method. The survival of vitrified embryos from pronuclear to blastocyst stages was equivalent between E15P15 and E15D15 groups. However, the rate of development to blastocysts was significantly lower in E15P15 than E15D15. The rates of survival and development to blastocysts were dramatically improved by a slight modification of EG and COOH-PLL concentrations (E20P10). After transferring 17 (E20P10) and 15 (E15D15) vitrified/warmed blastocysts, 8 and 7 pups were obtained (47.1% and 46.7%, respectively). Taken together, these results indicate that our vitrification protocol is appropriate for the vitrification of mouse embryos at different stages.

Ochratoxin A affects oocyte maturation and subsequent embryo developmental dynamics in the juvenile sheep model

The genotoxic and nephrotoxic mycotoxin Ochratoxin A (OTA) has also been reported to have adverse effects on oocyte maturation and embryo development. Previous studies on the effects of OTA on female fertility have used micromolar concentrations, but no information is available to date on effects in a more relevant nanomolar range. This study used a juvenile sheep model to evaluate the effects of oocyte exposure to low levels of OTA on maturation, fertilization, and embryo development. Further, it was investigated whether different mechanisms of action of OTA could be responsible for varying toxic effects at different levels of exposure. Cumulus-oocyte-complexes (COCs) were exposed to 10 μmol/L–0.1 nmol/L OTA during in vitro maturation and evaluated for cumulus viability, oocyte maturation, and bioenergetic/oxidative status. COCs were subjected to in vitro fertilization, embryo culture, and embryo quality assessment via morphology, viability, bioenergetic/oxidative status, and time-lapse monitoring. At micromolar concentrations, OTA induced cytotoxic effects, by reducing cumulus expansion and oocyte maturation. OTA altered temporospatial dynamics of zygote pronuclear formation and embryo morphokinetics. Blastocysts, even morphologically normal, were found to undergo collapse events, which were probably related to boosted blastocyst mitochondrial activity. At nanomolar concentrations, OTA did not affect COC morpho-functional parameters, but impaired oocyte ability to prevent polyspermy and increased blastocyst apoptosis. In conclusion, in the female germ cell, cytotoxic nonspecific effects characterize OTA-induced toxicity at high exposure levels, whereas fine tuning-mode effects, not associated with altered cell viability and integrity, characterize OTA toxic action at low levels.

Antinuclear antibodies in follicular fluid may reduce efficacy of in vitro fertilization and embryo transfer by invading endometrium and granular cells.

The mechanism(s) by which antinuclear antibodies (ANA) induce implantation failure are notclear, and little information regarding the function of autoantibodies in reproductive tissues is available. A total of 380 patients who underwent in vitro fertilization and embryo transfer (IVF-ET) were divided into control, serum positive, and follicular fluid (FF) positive groups based on the results of indirect immunofluorescence assay for ANA in the serum and FF. Immunofluorescence assay was performed to evaluate the existence of ANA in granular cells and endometrial tissues. Presence in FF of soluble apoptotic markers, including Bcl-2, Caspase-3, cleaved PARP, Cytochrome C, GAPDH, and p53, was assessed using magnetic bead based assays. The patients in the FF positive group had the lowest numbers of retrieved oocytes, fertilizations, and high-quality embryos. The fertilization rate, and the proportion of two pronuclear (2PN) embryos in patients in the FF positive group were significantly lower than those in the other two groups. The FF positive group also had the lowest clinical pregnancy rate, and the highest early miscarriage rate. Granulosa cells and endometrial tissues in patients in the FF positive group were ANA positive. High levels of BCL-2, Caspase-3, Cytochrome C, GAPDH, and p53 were found in the FF of patients in the FF positive group. Antinuclear antibodies in FF and endometrial tissues may cause imbalanced apoptosis, resulting in poor IVF-ET treatment outcomes. Local autoimmunity and cell apoptosis in reproductive tissues could be considered new therapeutic targets for improving IVF-ET treatment efficacy.

Development of an automated two pronuclei detection system on time-lapse embryo images using deep learning techniques.

PurposeTo establish an automated pronuclei determination system by analysis using deep learning technology which is able to effectively learn with limited amount of supervised data.MethodsAn algorithm was developed by explicitly incorporating human observation where the outline around pronuclei is being observed in determining the number of pronuclei. Supervised data were selected from the time‐lapse images of 300 pronuclear stage embryos per class (total 900 embryos) clearly classified by embryologists as 0PN, 1PN, and 2PN. One‐hundred embryos per class (a total of 300 embryos) were used for verification data. The verification data were evaluated for the performance of detection in the number of pronuclei by regarding the results consistent with the judgment of the embryologists as correct answers.ResultsThe sensitivity rates of 0PN, 1PN, and 2PN were 99%, 82%, and 99%, respectively, and the overlapping 2PN being difficult to determine by microscopic observation alone could also be appropriately assessed.ConclusionsThis study enabled the establishment of the automated pronuclei determination system with the precision almost equivalent to highly skilled embryologists.

Improvement of genome editing by electroporation using embryos artificially removed cumulus cells in the oviducts

Many genome-edited animals have been produced using the CRISPR/Cas system. Genome-edited strains were produced by introducing nucleases into pronuclear stage embryos. Recently, a new electroporation technique (TAKE: Technique for Animal Knockout system by Electroporation) was developed for the production of genome-edited animals by introducing nucleases into intact embryos using electroporation instead of the microinjection method. Furthermore, this method, which can introduce nucleases into intact embryos, enables genome editing of mouse embryos in the oviducts. However, the present protocol required improvements for low litter size and restriction of operation time. In this study, the influence on the development and genome editing of mouse embryos in the oviducts by electroporation and operation time was examined. The genome-editing rate was higher in the embryos electroporated at 16:00–17:00 (PM) (54%) on the following day of natural mating compared to that of embryos at 10:00–11:00 (AM) (27%). The embryos at AM formed a complex with cumulus cells, and cumulus cells were freed from embryos by treatment with hyalronidase before electroporation. The results showed that the genome-editing rate was significantly increased in the embryos treated with hyalronidase at AM, because the cumulus cells surrounding the embryos interfered with the introduction of nucleases into embryos. This study demonstrated that it was possible to adjust the operation time for the introduction of nucleases into embryos in the oviducts by treatment with hyalronidase before electroporation. However, litter size and development of embryos after electroporation was quite low in all experiments (5–7) compared with the control without operation (11).

Aberrant DNA and histone methylation during zygotic genome activation in goat cloned embryos

In somatic cell nuclear transfer (SCNT) embryos, developmental defects first appear at the time of zygotic genome activation (ZGA), a process that is under the control of DNA and histone methylation. However, dynamics of 5-mC and 5-hmC during ZGA differ between porcine and bovine SCNT embryos, and histone methylation during ZGA in goat SCNT embryos remains poorly understood. Therefore, in the present study, we investigated the dynamic changes of 5-mC, 5-hmC, H3K4me2/3, and H3K9me3, as well as the expression of key genes related to these epigenetic modifications, during ZGA in goat cloned embryos. Compared with the IVF embryos, the 5-mC signal intensity was significantly increased at the 2- and 4-cell stage SCNT embryos, and the H3K4me3 and H3K9me3 signal intensity was significantly increased at 2- to 8-cell stage SCNT embryos, while the 5-hmC and H3K4me2 signal intensity was significantly lower at the 4- and 8-cell stage SCNT embryos. Of note, the H3K9me3 level was also significantly higher, whereas H3K4me3 signal intensity showed no statistical difference in the pronuclear stage SCNT embryos. Moreover, the expression of TET2, DNMT3B, KDM4A, SUV39H1, G9A, and SETDB1 was significantly increased, while the expression of UHRF1, PCNA, KDM4B, KDM4D, KDM5A, KDM5B, and KDM5C was significantly decreased at the 8-cell stage SCNT embryos. Our data revealed aberrant DNA and histone methylation during ZGA in goat cloned embryos. We further inferred that the abnormally higher level of 5-mC, H3K4me3, and H3K9me3 might serve as epigenetic barriers of the reprogramming and modifying these aberrant modifications might be a promising strategy to improve cloning efficiency in goat.

Simple induction of pseudopregnancy by artificial stimulation using a sonic vibration in rats

Embryo transfer has been used as one of the essential reproductive technologies for production of new strains and maintenance of genetic resources in animals. Mating with vasectomised male rats is a requirement for inducing pseudopregnancy in female rats selected for embryo transfer. Although this procedure has been used routinely, large breeding space and high expenditure are required to maintain a sufficient number of females and vasectomised males. This study was performed to induce pseudopregnancy in females by artificial stimulation using sonic vibration instead of vasectomised males. The females continued to be in the dioestrus stage for at least 14 days after artificial stimulation was performed. Of fresh 2-cell embryos that transferred into the oviducts of females after artificial stimulation, 56% was implanted and 50% was developed to offspring. Approximately 46% of the frozen 2-cell embryos were implanted and 24% developed into offspring. Furthermore, 66% of the fresh pronuclear embryos were implanted and 60% developed into offspring. This study successfully induced pseudopregnancy in rat females by artificial stimulation using a sonic vibration. This method, ‘Easy-ET’, was useful for efficient production and maintenance of rat strains.

Reparative effects of lycium barbarum polysaccharide on mouse ovarian injuries induced by repeated superovulation

To explore the repair effect of lycium barbarum polysaccharide (LBP) on ovarian injuries induced by repeated superovulation in mice, a model of ovarian injury was established, and ovarian repair was assessed after intragastric administration of LBP. The oocyte quality and blastocyst rates of pronuclear embryos in vitro were observed. The levels of 8-hydroxydeoxyguanosine (8-OHdG) and lipid peroxide (LPO) in ovarian tissue were measured, and ovarian damage was assessed in paraffin sections. The groups with significant injury were selected according to the above observation, mice in the significant injury group were intragastrically administered with LBP (low dose, 25 mg/kg; medium dose, 35 mg/kg; and high dose, 45 mg/kg) for 30 days. The above measurements and anti-Müllerian hormone (AMH) expression were detected in the mouse ovaries and the breeding verification was carried out. Our results showed that repeated superovulation could cause mouse oocyte quality to drop, significant differences started from 4 superovulation events (P < 0.05). The levels of 8-OHdG and LPO in the ovary increased gradually as the number of superovulation events increased, and significant differences were observed after 4–6 superovulations (P < 0.05). The ratios of primordial follicles, primary, tertiary and mature follicles decreased and the ratio of atresia follicles increased as the number of superovulation events increased, especially in 4–6 superovulation groups. Thus, the groups of superovulation 4–6 events were considered as significant injury groups. LBP-medium dose groups significantly improved the number and quantity of oocytes and embryo blastocyst rate (P < 0.05), significantly decreased 8-OHdG and LPO levels in mice ovary (P < 0.05), also improved the ratios of all stages follicles and reduced the rate of atresia follicles, increased the numbers of litter size, live birth, weaning survival, and repaired the expression of AMH in ovary significantly (P < 0.05). In conclusion, the degree of ovarian injury was affected by the number of superovulation. LBP repaired ovarian injuries most likely through scavenging oxidative products 8-OHdG and LPO and increasing AMH protein expression.

Effect of single and combined treatments with MPF or MAPK inhibitors on parthenogenetic haploid activation of bovine oocytes

In bovine, correct oocyte artificial activation is a key step in ICSI and other reproductive biotechnologies, and still needs to be improved. The current study was designed to compare the activating efficiency of ionomycin (Io) followed by: a 4 h time window and ethanol (4h-Et), roscovitine (Rosc), dehydroleucodine (DhL), cycloheximide (CHX) or PD0325901 (PD), each as a single treatment, and then combine them in novel protocols. Parthenogenetic haploid activation was evaluated in terms of pronuclear (PN) formation, second polar body (2PB) extrusion, ploidy of day 2 embryos and in vitro development. Combined treatments with Io-4h-Et-Rosc and Io-Rosc/CHX increased PN formation (92.2% and 96%, respectively) compared with Io-Rosc, Io-CHX or Io-4h-Et, which were equally efficient at inducing PN formation (82–84%) and 2PB extrusion (62.1–70.5%). Oocyte activation with Io-DhL and Io-Rosc/DhL resulted in higher 2PB extrusion rates (90% and 95.9%, respectively) but lower PN formation (49.4–58.8%) and cleavage rates (36–57.9%), as occurred with Io-CHX/DhL (76.4% and 70.4%, respectively). For the first time, results show that Io followed by the MAPK inhibitor PD induces PN formation and 2PB extrusion, but PD combined with Rosc or CHX resulted in low rates of haploid day 2 embryos. In conclusion, DhL strongly induces 2PB extrusion but leads to poor PN formation and embryo development. PD induces bovine oocyte activation but results in low rates of haploid embryos. In contrast, the improved PN formation rates after treatment with combined Io-4h-Et-Rosc and Io-Rosc/CHX suggest they should be further evaluated in ART, aiming to increase success rates in bovine.

The role of glyphosate in infertility: the mechanistic link

In light of the recent Roundup lawsuit, glyphosate has been widely accepted as a significant environmental toxin that may affect humans in various ways including cancer and infertility. Exposure to even low doses of glyphosate-based herbicides during pregnancy has been found to impair fertility, cause intrauterine growth restriciton and induce fetal malformations. In this study we sought to determine the underlying mechanism by which glyphosate negatively impacts oocyte quality, fertilization rates as well as embryo development. Experimental case-control study of mouse oocytes and pronuclear embryos, exposed in vitro to increasing glyphosate concentrations and followed through day 5 of development. We utilized multiple assays including reactive oxygen species (ROS) generation and zinc depletion to examine the possible underlying detrimental mechanisms. Metaphase II mouse oocytes (n=200) were retrieved from 8-10 week female mice and a subset (n=100) were fertilized using IVF. The oocytes as well as the fertilized mouse embryos were then exposed to increasing concentrations of glyphosate (0-200 μM) for 2h - 4h as per protocol. The oocytes were divided into four groups that were treated as follows: Group A: ROS detection assay, Group B: Zinquin ethyl ester assay, Group C: fixed, stained and scored based on the spindle structure (microtubule morphology -MT and chromosomal alignment - CH) as indicators of the oocyte's capacity to sustain exposure. All groups were compared to Group 4: untreated controls. Exposed embryos were incubated for up to 120 hours post fertilization and evaluated for full and hatching blast rate conversion. They were photographed and graded daily based on their appearance and development using published embryo grading protocols. A subset of the treated embryos (n = 10 for each concentration) were treated, in a similar fashion as the oocytes, in order to evaluate for ROS overproduction and zinc depletion. Confocal microscopy was used to assess the embryos. Statistical analysis was performed using t-test, ANOVA and chi-square. A p-value < 0.05 was considered statistically significant. Oocytes treated with increasing glyphosate concentrations > 50 μΜ were found to have poor scores for MT and CH and that difference was statistically significant as compared to controls (p< 0.05). Embryos followed to 96 hours post fertilization (early blastocyst) and 120 hours (full and hatching blastocyst) after glyphosate exposure (0-200 μM) were assessed and those exposed to glyphosate concentrations > 100 μM showed significantly increased arrest rates and poor morphology scores compared to controls. ROS overproduction as well as zinc depletion was evident in embryos treated with high glyphosate concentrations. These observations were statistically significant compared to untreated controls (p<0.05). This work suggests the possible underlying mechanisms by which glyphosate negatively affects reproductive health in the mouse model. Possible fertility implications in humans will require further research.

Melatonin improves the efficiency of super-ovulation and timed artificial insemination in sheep.

It has been well proved that melatonin participates in the regulation of the seasonal reproduction of ewes. However, the effects of short term treatment of melatonin on ewe’s ovulation are still to be clarified. In this study, the effects of melatonin on the number of embryos harvested from superovulation, and the pregnant rate in recipients after embryo transferred have been investigated. Hu sheep with synchronous estrus treatment were given melatonin subcutaneously injection (0, 5, and 10 mg/ewe, respectively). It was found that the estrogen level in the group of 5 mg melatonin was significantly higher than that of other two groups at the time of sperm insemination (p < 0.05). The pregnant rate and number of lambs in the group of 5 mg melatonin treatment was also significantly higher than that of the rests of the groups (P < 0.05). In another study, 31 Suffolk ewes as donors and 103 small-tailed han sheep ewes as recipients were used to produce pronuclear embryo and embryo transfer. Melatonin (5 mg) was given to the donors during estrus. The results showed that, the number of pronuclear embryos and the pregnancy rate were also significantly higher in melatonin group than that in the control group. In addition, 28 donors and 44 recipient ewes were used to produce morula/blastocyst and embryo transferring. Melatonin (5 mg) was given during estrus. The total number of embryos harvested (7.40 ± 1.25/ewe vs. 3.96 ± 0.73/ewe, P < 0.05) and the pregnant rate (72.3 ± 4.6% vs. 54.7 ± 4.0%, P < 0.05) and number of lambs were also increased in melatonin group compared to the control group. Collectively, the results have suggested that melatonin treatment 36 hours after CIDR withdrawal could promote the number and quality of embryos in vivo condition and increased the pregnant rate and number of lambs.

Comparison of Oocyte Maturation Trigger Using Follicle Stimulating Hormone Plus Human Chorionic Gonadotropin versus hCG Alone in Assisted Reproduction Technology Cycles.

BackgroundThe goal of this study was to investigate oocyte maturation, fertilization and pregnancy rates among infertile women, by concomitant follicle stimulating hormone (FSH) administration at the time of human chorionic gonadotropin (hCG) trigger, compared to hCG trigger alone.Materials and MethodsIn this prospective randomized controlled trial, 109 infertile women between the ages of 20 and 40 years, received gonadotropin-releasing hormone (GnRH) antagonist and fresh embryo transfer. Following the procedure, the subjects were randomly divided into two groups on the oocyte-triggering day. In the experimental group, final oocyte maturation was achieved by 5000 IU hCG plus 450 IU FSH. In the control group, however, oocyte triggering was performed by 5000 IU hCG, only. The primary outcome was clinical pregnancy and the secondary out- comes included oocyte recovery rate, oocyte maturity rate, fertilization proportion rate, fertilization rate, implantation rate and chemical pregnancy rate.ResultsFifty-four women were appointed to the group with the FSH bolus injection at the time of hCG trigger and 55 women were assigned to the hCG alone group. Women in the FSH group had a significantly higher metaphase II (MII) oocyte (7.17 ± 3.50 vs. 5.87 ± 3.19), 2 pronuclear embryos (2PNs) (5.44 ± 3.20 vs. 3.74 ± 2.30) and total em- bryos (4.57 ± 2.82 vs. 3.29 ± 2.13) compared to hCG alone group, respectively. Furthermore, fertilization rate (0.75 ± 0.19 vs. 0.68 ± 0.25), implantation rate (14.2 vs. 8.5%) as well as clinical (27.9 vs. 15.9%) and chemical (32.6 vs. 20.5%) pregnancy rates were higher in the FSH group, but no statistically significant difference was found (P>0.05).ConclusionCombination of FSH and hCG for oocyte triggering improves oocyte maturity and fertilization propor- tion rates without increasing the chance of implantation, chemical and clinical pregnancy rates (Registration number: IRCT2017082724512N5).

Tolerance to vitrification of rat embryos at various developmental stages

Numerous genetically engineered rat strains have been produced via genome editing. Although freezing of embryos is helpful for the production and storage of these valuable strains, the tolerance to freezing of embryos varies at each developmental stage of the embryo. This study examined the tolerance to freezing of rat embryos at various developmental stages, particularly at the pronuclear stage. Embryos that had developed to the pronuclear, 2-cell, and morula stages were frozen via vitrification using ethylene glycol- and propylene glycol-based solutions. More than 90% of the embryos at all developmental stages survived after warming. The developmental rates to offspring of thawed embryos at the pronuclear, 2-cell, and morula stages were 19%, 41%, and 52%, respectively. Pronuclear stage embryos between the early and late developmental stages were then vitrified. The developmental rates to offspring of the thawed pronuclear stage embryos collected at 24, 28, and 31 h after the induction of ovulation were 17%, 21%, and 23%, respectively. These results indicated that the tolerance to vitrification of rat embryos increased with the development of embryos. The establishment of vitrification method of rat embryos at various developmental stages is helpful for improving the production and storage of valuable rat strains used for biomedical science.

Effect of low-dose dexamethasone on patients with elevated early follicular phase progesterone level and pregnancy outcomes in IVF-ET treatment: A randomized controlled clinical trial.

To evaluate the effect of low-dose dexamethasone on patients with elevated early follicular progesterone levels in IVF-ET treatment. Randomized controlled trial. In vitro fertilization (IVF) centre. A total of 459 patients undergoing a first IVF/ICSI cycle. If progesterone concentration exceeded 1.9nmol/L on days 3-4 of ovarian stimulation, the patients in dexamethasone (DEX) group were treated with oral dexamethasone 0.75mg/d, and the patients in control group received no extra treatment. The cumulative live-birth rate (per cycle started) in 2years. The total dose of gonadotropins (1987±536 IU in DEX group vs 2135±701 IU in control group, P=0.009) and the serum concentrations of progesterone on human chorionic gonadotropin (HCG) day (3.1±1.4nmol/L in DEX group vs 4.0±1.3nmol/L in control group, P<0.001) were both significantly lower in DEX group than that in control. No significant differences between the two groups were observed in the number of oocytes, two pronuclear (2PN) embryos and clinical pregnancy rate. In addition, the cumulative live-birth rate was significantly higher in the DEX group than that in controls (70.0% vs 61.1%, P=0.029, 95% confidence interval: 1.01-2.19). Progesterone secretion can be suppressed by dexamethasone, and dexamethasone may sensitize the ovary to gonadotropin stimulation in IVF treatment. In addition, the cumulative live-birth rate was significantly higher in the DEX group than in controls, and the obstetric and neonatal outcomes support the safety of DEX treatment in IVF.

AANAT transgenic sheep generated via OPS vitrified-microinjected pronuclear embryos and reproduction efficiency of the transgenic offspring.

BackgroundThe open pulled straw (OPS) vitrification method has been successfully applied in mouse, pig, and goat embryos as well as in buffalo oocytes, but it has not yet been applied to the microinjected embryos. This study examined the effects of OPS vitrification on embryo development and the reproductive capacity of the transgenic offspring in order to establish a method for preservation of microinjected embryos.MethodsOvine pronuclear embryos were microinjected with the exogenous aralkylamine N-acetyltransferase gene (AANAT), frozen by the OPS method, and subsequently thawed for embryo transplantation. Pregnancy rate, lambing rate, survival rate, average birth weight and transgenic positive rate as well as reproduction efficiency and hormone level of the transgenic offspring were investigated to analyze the effect of OPS vitrification on microinjectd pronuclear embryos.ResultsNo significant differences were observed in the birth rate, lamb survival rate and transgenic positive rate between the frozen and non-frozen AANAT-microinjected pronuclear embryos. The average birth weight of the frozen embryos offspring was greater than that of the non-frozen embryos. Importantly, the transgenic offspring that overexpressed the AANAT gene showed improved ovulation efficiency and lambing rate by regulating their hormone levels.ConclusionsThe OPS vitrification approach may be a valuable method in microinjected- embryo transfer technology, which could reserve embryos and result in fewer unnecessary animal sacrifices. In addition, the AANAT+ transgenic offspring exhibited improved reproductive capacity on account of regulation effect of melatonin on reproductive hormone. These data may provide available references for human-assisted reproduction.