Embryo Formation Rate Research Articles

Micropropagation of Tulip via Somatic Embryogenesis

An effective method of tulip regeneration via somatic embryogenesis (SE) was developed. Explants, flower stem slices excised from cooled bulbs were incubated in darkness on MS modified media containing auxins alone (2,4-dichlorophenoxyacetic acid—2,4-D, 1-naphthalene acetic acid—NAA and 4-amino-3,5,6-trichloro-2-pyridine carboxylic acid—picloram) or combined with thidiazuron (TDZ) at 0.1 and 0.5 mg L−1. Yellowish-white callus with a granular structure was developed in the presence of all auxins on the cut surface from the tissues of the vascular bundles. From this, lines of embryogenic calli were derived. The addition of TDZ to the medium with auxins significantly stimulated somatic embryo formation. Cyclic and the most intensive proliferation of embryogenic callus as well as embryo formation was obtained in the presence of 2,4-D at 0.1 mg L−1 combined with TDZ at 0.5 mg L−1. Addition of proline enhanced either callus proliferation rate or frequency of embryo formation. The best quality embryos with cotyledons longer than 10 mm able to form bulbs were recorded when TDZ was replaced with 6-benzylaminopurine (BAP) at the concentration of 0.1 mg L−1. Histomorphology showed that the development of somatic embryos could have either external or internal origins. Embryos of external origin were initiated by cell division on the edge of embryogenic calli. Embryos of internal origin resulted from the division of parenchyma cells inside the tissue. Embryonic cells were characterized by their small volume, regular shape, dense cytoplasm and large nuclei. The globular embryos were covered by a distinct layer of periderm. Then, the embryos developed into structures having leaf-shaped cotyledons with a procambial strand and a sideward-orientated meristem of the vegetative apex (stolon). Cotyledon embryos did not show vascular connections with the parent tissue, and they did not develop embryonic roots.

AMH has no role in predicting oocyte quality in women with advanced age undergoing IVF/ICSI cycles

It has been widely acknowledged that anti-Müllerian hormone (AMH) is a golden marker of ovarian reserve. Declined ovarian reserve (DOR), based on experience from reproductive-aged women, refers to both the quantitative and qualitative reduction in oocytes. This view is challenged by a recent study clearly showing that the quality of oocytes is similar in young women undergoing IVF cycles irrespective of the level of AMH. However, it remains elusive whether AMH indicates oocyte quality in women with advanced age (WAA). The aim of this study was to investigate this issue. In the present study, we retrospectively analysed the data generated from a total of 492 IVF/ICSI cycles (from January 2017 to July 2020), and these IVF/ICSI cycles contributed 292 embryo transfer (ET) cycles (from June 2017 to September 2019, data of day 3 ET were included for analysis) in our reproductive centre. Based on the level of AMH, all patients (= > 37 years old) were divided into 2 groups: the AMH high (H) group and the AMH low (L) group. The parameters of in vitro embryo development and clinical outcomes were compared between the two groups. The results showed that women in the L group experienced severe DOR, as demonstrated by a higher rate of primary diagnosis of DOR, lower antral follicle count (AFC), higher level of basal follicle stimulating hormone (FSH) and cancelation cycles, lower level of E2 production on the day of surge, and fewer oocytes and MII oocytes retrieved. Compared with women in the H group, women in the L group showed slightly reduced top embryo formation rate but a similar normal fertilization rate and blastocyst formation rate. More importantly, we found that the rates of implantation, spontaneous miscarriage and livebirth were similar between the two groups, while the pregnancy rate was significantly reduced in the L group compared with the H group. Further analysis indicated that the higher pregnancy rate of women in the H group may be due to more top embryos transferred per cycle. Due to an extremely low implantation potential for transfer of non-top embryos from WAA (= > 37 years old) in our reproductive centre, we assumed that all the embryos that implanted may result from the transfer of top embryos. Based on this observation, we found that the ratio of embryos that successfully implanted or eventually led to a livebirth to top embryos transferred was similar between the H and the L groups. Furthermore, women with clinical pregnancy or livebirth in the H or L group did not show a higher level of serum AMH but were younger than women with non-pregnancy or non-livebirth. Taken together, this study showed that AMH had a limited role in predicting in vitro embryo developmental potential and had no role in predicting the in vivo embryo developmental potential, suggesting that in WAA, AMH should not be used as a marker of oocyte quality. This study supports the view that the accumulation of top embryos via multiple oocyte retrieval times is a good strategy for the treatment of WAA.

The Embryo Toxicity Research of Manufacturing Water of Medical Device of in Vitro Fertilization Technology

Water is an important component in liquid medical device products for human assisted reproductive technology. Water traits, conductivity, microbial limits, total organic carbon, easy oxides, heavy metal content, bacterial endotoxin and other indicators have an important impact on sperm, egg and embryo development in vitro, so for such products, the quality of water control is extremely important. The production water for producing such products is generally prepared by MilliQ purification system. In this research, we used four different types of water to fabricate the IVF liquids. It included deionized reverse osmosis water, ultra purified water and ultra purified water without endotoxin or nucleic acid, and compared with tap water. The in vitro rat embryo test system was used to study the embryotoxicity of this four different culture liquid production waters. From the result, the group of the super purified water without endotoxin and nucleic acid has the best result of the embryo formation rate, the number of total cell number and the inner cell number. This study proved the importance of removing endotoxin and nucleic acid from the water used for the preparation of the liquid products for assisted reproduction, and provided the basis for the selection of water quality for the liquid products for assisted reproduction.

Being on the side of old findings: progesterone elevation on the day of oocyte maturation induction does not affect embryological parameters throughout the blastocyst culture period.

To investigate whether there is any detrimental effect of progesterone elevation (PE) on the day of oocyte maturation induction on embryological development potentials. This retrospective single-center cohort study included a total of 1485 individual intracytoplasmic sperm injection (ICSI) cycles between January 2014 and December 2018. Serum progesterone (P) levels were measured on the day of oocyte maturation induction following the GnRH antagonist suppression protocol. Embryological parameters such as maturation, fertilization rate (FR), top-quality embryo (TQE) formation rate per 2PN on day 3, and excellent-quality blastocyst (EQB) formation rate per 2PN on day 5/6 were recorded. The inclusion criteria for women were an age ≤ 37years, a BMI ≤ 30kg/m2, and access to a total sperm concentration ≥ 2 million. Groups were stratified according to the serum P levels using the cut-off levels of < 0.8ng/ml; 0.8-1.49ng/ml; and ≥ 1.5ng/ml. Peak E2 level and total number of oocytes retrieved were significantly related to PE (p < 0.001). FR did not display a significance difference between groups (p = 0.108). The TQE and the blastulation rates were not affected by PE (p = 0.82 and p = 0.68, respectively). Chi square analysis revealed a significant relationship between PE and the EQB formation rate (p = 0.01). GEE analysis failed to present any statistical significance regarding the effect of PE on neither the TQE nor the EQB formation rates per 2PN [OR 1.07; 95% (0.98-1.16) p = 0.113 and OR 0.93; 95% (0.80-1.07) p = 0.32, respectively]. In accordance with previously published papers, our study could not find any detrimental effect of PE on embryological outcomes throughout the blastocyst culture period.

Growth hormone alleviates oxidative stress and improves the IVF outcomes of poor ovarian responders: a randomized controlled trial

BackgroundOxidative stress (OS), defined as an imbalance between excessive reactive oxygen species (ROS) and/or reactive nitrogen species (RNS) production and antioxidant insufficiency, has been suggested to be involved in the pathogenesis of poor ovarian response (POR). Growth hormone (GH) can reduce OS in some cell types. This study investigated whether GH can improve OS and the in vitro fertilization and embryo transfer (IVF-ET) outcomes of poor ovarian responders.MethodsThis study enrolled 105 patients with POR and 58 patients without POR (controls) who were diagnosed according to the Bologna criteria and underwent conventional IVF-ET. Poor ovarian responders were randomly assigned to two groups: the POR-GH group, which received pretreatment with GH 4 IU/d on day 2 of the previous menstrual cycle before IVF until the trigger day, and the POR-C group, which received no pretreatment. OS markers in follicular fluid (FF), ROS levels in granulosa cells (GCs), and the IVF outcomes of the groups were compared.ResultsEndometrial thickness on trigger day, the number of cleaved embryos, the number of higher-quality embryos, and the rates of embryo formation, higher-quality embryo formation, implantation and clinical pregnancy were significantly increased in the POR-GH group compared with the POR-C group (P < 0.05). Moreover, compared to those in the non-POR group, FF malondialdehyde (MDA), total oxidant status (TOS), oxidative stress index (OSI) and ROS levels in GCs were significantly higher, whereas superoxide dismutase (SOD) and the total antioxidant capacity (TAC) were significantly lower in the POR-C group (P < 0.05). Furthermore, compared with those in the POR-C group, the FF TAC was significantly increased in the POR-GH group, and TOS, OSI and intracellular ROS levels were significantly reduced (P < 0.05).ConclusionsPretreatment with GH alleviates OS and improves oocyte quality and IVF outcomes of poor ovarian responders.Trial registrationChinese Clinical Trial Registry. ChiCTR1900021269. Registered 8 February 2019, http://www.chictr.org.cn/edit.aspx?pid=35837&htm=4.

Comparison of Orchard-grass and Sweet Maize for Doubled Haploid Plant Production via Wide Hybridization in Bread Wheat

In this study, the potential of haploid regeneration was investigated in hybridization of six bread wheat F1 hybrids known response to another culture with orchard-grass (Dactylis glomerata L.) and sweet maize varieties (Baron, Challenger and Merit). A total of 150 wheat spikes were pollinated with orchard grass and sweet maize and 2730 pseudo-seeds were produced. Although the high rate of developed pseudo-seeds was developed from bread wheat F1 hybrids × orchard-grass, no embryos were produced. Developed pseudo-seeds (2057 number) of bread wheat × sweet maize produced 53 haploid embryos and only 8 of them were regenerated. Developed green plantlets were vernalized and applied colchicine and only four of them produced fertile seeds. The highest rate (5.9) of haploid embryo formation within wheat genotypes was determined in DH20 × Kate A-1. Although the highest haploid embryo formation was observed in Challenger with 3.5% among sweet maize genotypes, it had no effect on plant regeneration. Also, the mixture of pollen of sweet maize varieties increased haploid plant regeneration. It has been observed that some F1 hybrids such as DH20 × Kate A-1 and DH6 × Altay 2000 with low anther response gave better results in terms of haploid embryo formation and regeneration. The means of fertile spike percentages and number of seeds per fertile spike were 26.75% and 9.83, respectively in developed green plants. As a result, bread wheat × sweet maize hybridization will be a good alternative to obtain a homozygous line in a short time in bread wheat genotypes with low anther response.

Methotrexate impaired in-vivo matured mouse oocyte quality and the possible mechanisms

BackgroundMethotrexate (MTX) is an antifolate agent which is widely used in clinic for treating malignancies, rheumatoid arthritis and ectopic pregnancy. As reported, MTX has side effects on gastrointestinal system, nervous system and reproductive system, while its potential damages on oocyte quality are still unclear. It is known that oocyte quality is essential for healthy conception and the forthcoming embryo development. Thus, this work studied the effects of MTX on the oocyte quality.ResultsWe established MTX model mice by single treatment with 5 mg/Kg MTX. Both morphological and molecular biology studies were performed to assess the in-vivo matured oocytes quality and to analyze the related mechanisms. The in-vivo matured oocytes from MTX-treated mice had poor in-vitro fertilization ability, and the resulting embryo formation rates and blastocyst quality were lower than the control group. We found that the in-vivo matured MTX-treated mouse oocytes displayed abnormal transcript expressions for genes of key enzymes in the folate cycles. MTX increased the rate of abnormal chromosome alignment and affected the regulation of chromosome separation via disrupting the spindle morphology and reducing the mRNA expressions of MAD2 and Sgo1. MTX reduced the DNA methylation levels in the in-vivo matured oocytes, and further studies showed that MTX altered the expressions of DNMT1 and DNMT 3b, and may also affect the levels of the methyl donor and its metabolite.ConclusionsMTX impaired the in-vivo matured mouse oocyte quality by disturbing folate metabolism and affecting chromosome stability and methylation modification.

Short term temperature elevation during IVM affects embryo yield and alters gene expression pattern in oocytes, cumulus cells and blastocysts in cattle

Heat stress causes subfertility in cattle by inducing alterations in steroidogenic capacity, follicular function and ovulation defects, which eventually negatively affect oocyte quality and embryo survival. Here, the effects of short, moderate temperature elevation during IVM, on embryo yield, and on the expression of various genes was evaluated. In 8 replicates, cumulus oocyte complexes (COCs) were matured for 24 h at 39 °C (controls n = 605) or at 41 °C from hour 2 to hour 8 of IVM (treated, n = 912), fertilized, and presumptive zygotes were cultured for 9 days at 39 °C. Cleavage and embryo formation rates were evaluated 48 h post insemination and on days 7, 8, 9 respectively. Cumulus cells, oocytes and blastocysts from 5 replicates were snap frozen for the relative expression analysis of genes related to metabolism, thermal and oxidative stress response, apoptosis, and placentation. In treated group, cleavage and embryo formation rates were statistically significantly lower compared with the control (cleavage 86.7% vs 74.2%; blastocysts: day 7, 29.9% vs 19.7%, day 8, 34.2% vs 22.9% and day 9 35.9% vs 24.5%). Relative mRNA abundance of three genes in cumulus cells (HSP90AA1, CPT1B, G6PD) and three genes in blastocysts (DNMT3A, PLAC8, GPX1) indicated significantly different expression between groups (p < 0.05)., The expression of G6PD, SOD2, GXP1 in oocytes and PTGS2 in blastocysts tended to differ among groups (0.05<p < 0.08). Heat stress altered (p < 0.05) the correlation of expression between HSPs and other genes in oocytes (G6PD, GPX1, CCNB1), cumulus cells (LDH, CCNB1) and blastocysts (AKR1B1, PLAC8). These results imply that exposure of oocytes to elevated temperature, even for only 6 h, disrupts the developmental competence of the oocytes, suppresses blastocyst yield and significantly alters the coordinated pattern of gene expressions.

The influence of balanced complex chromosomal rearrangements on preimplantation embryonic development potential and molecular karyotype

BackgroundBalanced complex chromosome rearrangements (BCCR) are balanced chromosomal structural aberrations that involve two or more chromosomes and at least three breakpoints. It is very rare in the population. The objective is to explore the difference of influence of three types of BCCR on early embryonic development and molecular karyotype.ResultsTwelve couples were recruited including four couples of three-way rearrangements carriers (group A), three couples of double two-way translocations carriers (group B) and five couples of exceptional CCR carriers (group C). A total of 243 oocytes were retrievedin the seventeen preimplantation genetic testing (PGT) cycles, and 207 of these were available for fertilization. After intracytoplasmic sperm injection, 181oocytes normally fertilized. The rates of embryos forming on day3 in three groups were 87.88, 97.78 and77.14%, which was significantly different (P = 0.01). Compared with group B, the rate of embryo formation was statistically significantly lower in group C (P = 0.01). Furthermore, the rates of high-quality blastocysts in three group were 14.71, 48.15 and 62.96%, respectively, which was significantly different (P = 0.00). Compared with group B andC, the rate of high-quality blastocysts in group A was statistically significantly lower (P = 0.00;P = 0.00).Comprehensive chromosome analysis was performed on 83 embryos, including 75 trophectodermcellsand 8 blastomeres. Except 7 embryos failed to amplify, 9.01%embryos were diagnosed as euploidy, and 90.91% were diagnosed as abnormal. As for group A, the euploid embryo rate was 10.71%and the abnormal embryo rate was 89.29%. In group B,the euploid embryo rate was 3.85%, the abnormal embryo rate was 96.15%. The euploid embryo rate was 13.04%, the abnormal embryo rate was 86.96% in group C. There were no significant differences among the three groups (P = 0.55).ConclusionsThe lowest rate of high quality blastocysts has been for three-way rearrangements and the lowest rate of euploidy has been for double two-way translocations, although no significant difference. Different types of BCCR maybe have little effect on the embryonic molecular karyotype. The difference of influence of BCCR on early embryonic developmentandmolecular karyotypeshould be further studied.

Oocyte Vitrification: A Comparative Analysis Between Fresh and Cryopreserved Oocytes in an Oocyte Donation Program

Background: Oocyte Cryopreservation has become an important part of infertility treatment for various reasons such as fertility preservation in women going for oncological treatment; in oocyte donation cycles; in eliminating several religious, ethical, and legal concerns of embryo freezing and in women who wish to delay childbirth. The newer ”vitrification” technique for freezing has further improved the success rates for actual conception than the earlier method of slow freezing. A successful oocyte freezing program can help in establishment of oocyte banks, which would help to provide compatible oocytes immediately, thus would eliminate the several problems of fresh donor cycles. Methods: In this retrospective observational study, total 60 oocyte donation cycles were included (38 were fresh and 22 were vitrified oocytes cycle, respectively). After a thorough screening, controlled ovarian hyperstimulation for donors was performed using flexible antagonist protocol. All mature oocytes were allocated into “vitrified oocytes” and “fresh oocytes” groups. Vitrification technique using Cryotop method was used for oocyte freezing. Both clinical and laboratory outcomes of vitrified and fresh oocytes in donor cycles were compared. Results: A total of 600 oocytes (226 “vitrified oocytes” and 374 fresh oocytes), were studied. After warming 218 oocytes survived resulting in survival rate of 96.4%. Fertilization rate and embryo formation rate was 86.2% and 93.6%, respectively. Results of frozen-thawed oocyte donor cycles were compared with fresh donation cycles. For fresh oocyte group, fertilization rate and embryo formation rate was 83.4% and 92.6%, respectively. On comparing clinical outcomes, clinical pregnancy rate was 60.5% in fresh group and 63.6% in vitrified group. Conclusions: Both clinical and laboratory results obtained in the study suggest that oocyte cryopreservation can be performed with reproducible success, thus vitrification technique can be provided as a useful tool for achieving highly successful outcomes in an oocyte donor program.

Sperm enrichment from poor semen samples by double density gradient centrifugation in combination with swim-up for IVF cycles

Sperm preparation in IVF cycles using density gradient centrifugation (DGC) in combination with swim-up (SU) has been widely adopted in reproductive centres worldwide. It is a fact that the sperm recovery rate following one DGC from poor semen samples (showing liquefaction defects/containing too many unresolvable clots or rare sperm) is relatively low. Our results showed that double DGC (DDGC) is effective at increasing the sperm recovery rate from poor semen samples. However, DDGC may increase the mechanical stress of sperm, thereby potentially impairing embryo development. Therefore, it is necessary to evaluate the safety of using sperm prepared by DDGC/SU for IVF cycles. In this study, we retrospectively analysed the data generated from a total of 529 IVF cycles (from June 2017 to June 2018), and these IVF cycles contributed 622 transfer cycles (from June 2017 to December 2018) in Changzhou Maternal and Child Health Care Hospital. Of them, 306 IVF cycles and the related 355 transfer cycles (normal semen samples prepared by DGC/SU) were set as the normal group, while 223 IVF cycles and the related 267 transfer cycles (poor semen prepared by DDGC/SU) were set as the observation group. The main outcome measures, including the normal fertilization rate, top D3 embryo formation rate, blastocyte formation rate, clinical pregnancy rate and live birth rate, birth weight and duration of pregnancy, were compared between the two groups. Compared to semen in the DGC/SU group, semen in the DDGC/SU group showed increased levels of the DNA fragmentation index (DFI) and reduced sperm concentration, percentage of progressive motility (PR) sperm, and percentage of normal morphology sperm. The indicators reflecting in vitro embryo development and clinical outcomes were similar in the DGC/SU group and DDGC/SU group, including the normal fertilization rate, top D3 embryo formation rate, blastocyte formation rate, pregnancy rate, implantation rate, spontaneous abortion rate, live birth rate, birth weight and duration of pregnancy. Furthermore, we found that the 1PN zygote formation rate was significantly lower in the DDGC/SU group than that in the DGC/SU group. We concluded that oocytes fertilized by sperm from poor semen samples separated by DDGC/SU achieved the same outcomes as oocytes fertilized by sperm from normal semen separated by DGC/SU, suggesting that DDGC/SU is an effective and safe method of sperm enrichment for poor semen samples in IVF. The main contribution of the present study is the verification of the effectiveness of DDGC/SU in improving sperm recovery from poor semen samples and the safety of using sperm prepared by DDGC/SU for IVF.

Comparative Analysis of Surgically Retrieved Sperms in Cases of Obstructive and Nonobstructive Azoospermia Treated for Infertility.

Background:Azoospermia is a highly evolving subject in the last few decades. In the past, use of donor sperm was the only option providing a realistic chance of conception for couples affected by azoospermia. Introduction of sperm retrieval techniques and assisted reproductive technologies, especially intracytoplasmic sperm injection (ICSI), has provided these men a chance to father their genetically own child and changed the management approach significantly.Objective:The objective was to compare the sperm retrieval rate (SRR) and ICSI outcomes of surgically retrieved sperms in cases of obstructive and nonobstructive azoospermia (NOA) as well as to evaluate the efficacy of sperm retrieval techniques.Materials and Methods:A total of sixty azoospermic patients were included in the study. The patients were divided between OA (16) and NOA groups (44). A retrospective outcome analysis was done on SRR and ICSI results among them.Results:The overall SRR in patients with NOA and OA was 47.7% and 100%, respectively (P < 0.001). On subgroup analysis, higher serum follicle-stimulating hormone has shown significantly decreased sperm retrieval. The size of testes was not found to be related to sperm retrieval. Fertilization and embryo formation rate were found to be higher in OA cases in comparison to those of NOA cases. Clinical pregnancy rate showed no significant difference.Conclusion:Various sperm retrieval techniques can provide new dimensions for successful ICSI and managing azoospermia patients. Although SRRs as well as ICSI outcomes are lower in NOA patients than patients with OA, still they are potentially fertile. A systematic approach especially in patients with NOA is an important step. Microdissection testicular sperm extraction is an attractive option for NOA patients in order to increase the chances of successful sperm retrieval.

Growth differentiation factor 9 and cumulus cell supplementation in in vitro maturation culture media enhances the viability of human blastocysts.

ObjectiveIn vitro maturation (IVM) of immature oocytes can be useful for some infertile patients. In IVM programs, the rates of embryo formation and pregnancy are low. Therefore, it is essential to recognize the main factors involved in regulating oocyte maturation in vitro. The purpose of this study was to investigate the effects of growth differentiation factor 9 (GDF9) and cumulus cell (CC) supplementation in IVM medium on the rates of embryo formation and viability of human blastocysts.MethodsA total of 80 germinal vesicle oocytes from stimulated cycles underwent an IVM program. The oocytes were divided into four groups, where group I consisted of IVM media only and served as the control, group II consisted of IVM+CCs, group III consisted of IVM+GDF9 (200 ng/mL), and group IV consisted of IVM+CCs+GDF9 (200 ng/mL). Intracytoplasmic sperm injection was performed on the IVM oocytes, and the cleavage embryos that were generated were vitrified. Following thawing, the embryos were cultured for 3 additional days, and the viability rates of the developed blastocysts were determined.ResultsThe maturation rate of the oocytes did not differ significantly across the four groups. The fertilization rate in group II was significantly higher than that in the control group (76.5% vs. 46.2%). Embryo formation was significantly more frequent in all experimental groups than in the control group, while blastocyst formation did not show significant differences in the three experimental groups compared to the control. The mean viability rates in groups II, III, and IV were 58.16%, 55.91%, and 55.95%, respectively, versus 37.78% in the control group (p<0.05).ConclusionSupplementation of IVM culture media with GDF9 and CCs enhanced the fertilization, embryo formation, and viability rates of blastocysts generated from vitrified cleavage embryos.

Explant type and hormone regime influences somatic embryogenesis and regeneration of cassava

Cassava (Manihot esculenta Crantz) is a tropical root crop that serves as a food staple and a vital income source to resource deprived farmers in the tropics. Despite its importance, cassava production and consumption is faced by a myriad of biotic/abiotic constraints. Genetic transformation which can be applied to mitigate these challenges however has as a prerequisite the availability of robust regeneration systems. This study evaluated the effect of explant type and hormone regime on somatic embryogenesis and regeneration of Kenyan cassava cultivars. The embryogenic competence of immature leaf lobe and stem explants of three cassava cultivars was determined by culturing them in MS medium supplemented with picloram (4, 6, 8 and 10 mg/l). The optimum media composition for embryo maturation, germination and plant recovery was assessed by culturing embryos in media supplemented with varying concentrations of 6-Benzylaminopurine (BAP), α–Naphthalene acetic acid (NAA) and Gibberellic acid (GA3). Somatic embryo formation frequencies in leaf explants were significantly higher (P≤0.05) than in stem explants. Embryo formation rates were found to increase with increasing concentrations of picloram. Cultivar 08/080 gave the least response to embryogenesis while embryogenesis rates for cultivar 08/274 were similar to the control cultivar TMS 60444. Maturation and plant recovery rates differed significantly with regard to the ratios of BAP, NAA and GA3 in the maturation media. The optimal medium for embryo germination and plant recovery was supplemented with 1 mg/l BAP, 0.02 mg/l NAA and 1.5 mg/l GA3. This optimized regeneration protocol can be coupled with mutation breeding or genetic transformation to improve the cassava germplasm. Key words: Cassava, explant, regeneration, somatic embryogenesis, plant growth regulator.

The Impact of In vitro Oocyte Maturity on Developmental Potential of Embryos Derived from Controlled Ovarian Stimulation Cycles

Background and Aims: One of the major subjects for improving in vitro fertilization (IVF) outcome is the quantity and quality of retrieved oocytes. In vitro maturation (IVM) provides an opportunity for using immature oocytes routinely discarded in clinics. This study aimed at evaluating the quality of embryos derived from in vivo and rescue in vitro matured oocytes. Materials and Methods: Totally, 462 immature oocytes as cases and 466 mature (MII) oocytes as controls were included for study of their developmental competence. Oocytes underwent intracytoplasmic sperm injection insemination and then denuded oocytes were microscopically assessed regarding cytoplasmic and nuclear maturity and quality. Results: The morphological assessments showed fertilization rate of 60.9 and 61.4%, the embryo formation rate of 86.7% and 90.9% and arresting rate of 27.3% and 25.6% for the case and control oocytes, respectively. Evaluating embryo quality in the cleavage stage indicated that 63% of the embryos in the case group and 68% of the embryos in the control group were of good quality. There was no significant difference between fertility rate and arresting rate of oocytes matured in both groups, although the embryo formation rate and the quality of embryos differed significantly. Conclusions: Our findings suggest that IVM is a valuable and practical option for patients who had to cancel IVF treatment cycles because of severe responses or resistance to routine hormonal therapies or those with low functional ovarian reserve.

Preconditioning of sperm with sublethal nitrosative stress: a novel approach to improve frozen-thawed sperm function.

Can sublethal stress induced by nitric oxide on fresh human spermatozoa protect the functional properties of post-thaw human spermatozoa? Semen samples were obtained from 46 donors. Twenty semen samples were used to determine toxicity level of nitric oxide by incubation of semen with different concentrations of nitric oxide (0.01 to 400 μM). Then, 26 semen samples were cryopreserved with optimized ranges of nitric oxide: control (NO-0.00), 0.01 μM nitric oxide (NO-0.01), 0.1 μM nitric oxide (NO-0.1), 1 μM nitric oxide (NO-1), 10 μM nitric oxide (NO-10), 100 μM nitric oxide (NO-100). Frozen-thawed spermatozoa were assessed for motion characteristics, viability, morphology, apoptosis-like changes, caspase 3 activity, DNA fragmentation and intracellular reactive oxygen species levels. Fertilization potential was investigated by heterologous piezo-intracytoplasmic sperm injection (piezo-ICSI) of human spermatozoa into mouse oocytes. In fresh spermatozoa, nitric oxide did not induce a negative effect, except a significant reduction in motility and viability at 200 µM and 400 µM (P < 0.05). Cryopreservation significantly reduced sperm motility and increased reactive oxygen species, apoptosis-like changes, caspase 3 activity, and DNA damage (P < 0.05). NO-0.01 significantly increased total and progressive motility versus the other groups (P < 0.05). The lowest percentage of caspase 3 activity was in the NO-0.01 and NO-0.1 compared with the other freezing groups. In the fertilization trial, the rate of two-cell embryo formation after heterologous piezo-ICSI was higher (P < 0.05) in NO-0.01 (69%) versus controls (42%). Sublethal oxidative stress induced by nitric oxide might improve human sperm function after cryopreservation.

Effect of Sperm DNA Fragmentation on Embryo Quality in Normal Responder Women in In Vitro Fertilization and Intracytoplasmic Sperm Injection.

PurposeTo investigate the associations between sperm DNA fragmentation (SDF) and embryo formation rate in normal responder women to in vitro fertilization/intracytoplasmic sperm injection (IVF/ICSI).Materials and MethodsFifty-three consecutive, fresh IVF/ICSI cycles performed from 2014 to 2017 were selected. All women were normal responders (4 to 14 mature oocytes were retrieved) and at least one normally fertilized oocyte with two pronuclei was obtained in all cycles. Semen was collected on the day of oocyte retrieval, and SDF levels were measured by sperm chromatin dispersion test (Halosperm assay). At day 3 after insemination, embryo quality was evaluated by morphologic criteria and categorized as A/B/C/D. Top quality embryo were defined as grade A embryos with seven cells or more.ResultsSDF levels showed a positive linear correlation with the male's age (r=0.307, p=0.025) and a negative linear correlation with sperm motility (r=−0.491, p<0.0001). To achieve top-quality or a grade A embryo formation rate >70%, the cut-off value SDF was <30.7% for each. Among individuals with SDF <30.7%, the median top-quality or grade A embryo formation rate was significantly higher than that among individuals with SDF ≥30.7% (38.1% vs. 20.0%, p=0.038; 50% vs. 25.0%, p=0.017).ConclusionIn normal responder women, high SDF level resulted in low day 3 embryo formation rates. Our results suggest a paternal effect on embryo quality in IVF/ICSI cycles.

Effect of the Air Filtration System Replacement on Embryo Quality in the Assisted Reproduction Laboratory.

Improving infrastructural conditions of the in vitro fertilization laboratory, such as the air quality, has profound positive effects on embryo culture. Poor environmental conditions reduce the rate of embryo formation and, therefore, of pregnancy. This review article presents important publications regarding the impact of air quality in human reproduction laboratories on embryo quality, pregnancy success, and live births. The studies demonstrate that the replacing the air filtration system improves significantly the environmental air quality, and, consequently, improves laboratory parameters, such as the fertilization rate, the number of blastocysts, the embryo implantation rate, and the number of live births. On the other hand, improving air quality decreases the number of abortions. Therefore, environmental parameters that improve embryo quality and increase healthy child birth rates must be the main targets for the assisted reproduction laboratory quality control.

Male factor & mosaicism: assessing the contribution of abnormal semen parameters to rate of mosaicism in next generation sequencing (NGS) preimplantation genetic screening (PGS) intracytoplasmic sperm injection (ICSI) cycles

Advances in preimplantation genetic screening (PGS) have led to practices changes in assisted reproductive technology (ART), enabling fertility centers to transfer single embryos while maintaining excellent ongoing pregnancy rates, reducing miscarriage rates, and dramatically reducing ART-associated multiple pregnancies [1,2]. The introduction of next generation sequencing (NGS) has allowed PGS laboratories to assess for embryo mosaicism – although the true incidence and reproductive potential of mosaic embryos is controversial. Severe male factor is known to increase risk of aneuploidy [3]. However, the influence of male factor infertility on mosaicism is unknown at this time. While aneuploidy is mostly due to maternal meiotic errors, mosaicism is likely caused by post-fertilization mitotic errors [4] and abnormal sperm may contribute to an embryo's risk of being mosaic. We sought to examine the influence of abnormal semen parameters on mosaicism in NGS PGS-tested embryos. The purpose of this study was to evaluate rate of mosaic embryo formation in NGS PGS cycles according to semen abnormality. Secondary aims were to assess sex ratio and subtypes of mosaicism in NGS PGS cycles according to semen abnormality. We performed a retrospective review of women undergoing PGS at a single academic fertility center from July 1, 2015 to September 1, 2017. Only intracytoplasmic sperm injection (ICSI) PGS cycles were included. In all cycles, PGS was performed via trophectoderm biopsy on day 5 or 6 and analyzed using NGS at a single reference lab. Patient demographics, fertility testing, cycle characteristics and PGS outcomes were collected. PGS outcomes were compared between oligospermic and non-oligospermic cohorts, between asthenospermic and non-asthenospermic cohorts, and between oligoasthenospermic and normospermic cohorts. 228 ART-ICSI cycles with PGS testing were included for analysis: 130 normospermic, 32 oligospermic, 47 asthenospermic, and 12 oligoasthenospermic cycles, resulting in 374 normospermic, 106 oligospermic, 162 asthenospermic, and 38 oligoasthenospermic biopsied embryos. Baseline characteristics were similar between cohorts with the exception of semen parameters (Tables 1-3). PGS cycles with oligospermia demonstrated a higher proportion of mosaic embryos (36/100, 34%) versus PGS cycles without oligospermia (115/617, 18.6%), p<0.001. PGS cycles with oligoasethenospermia demonstrated a higher proportion of mosaic embryos (15/38, 39.5% versus 88/384, 23.5%, p<0.001) compared to PGS cycles without oligoasthenospermia. Additional PGS outcomes were similar between cohorts (Tables 1-3). Oligoasthenospermia and oligospermia, but not asthenospermia, may increase the risk of mosaic embryos following ART-ICSI cycles with NGS PGS.

Characterization of oocyte retrieval cycles with empty zona pellucida.

PurposeTo identify the factors that characterize cycles with empty zona pellucida (EZP).MethodsThirty‐six oocyte retrieval cycles from which EZP were collected and another 36 cycles from which no EZP was collected were compared. The patients were divided into three groups: those with no EZP collected during any cycle, those with EZP collected during all cycles, and those experiencing cycles both with and without EZP.ResultsThe mean number of oocytes collected per cycle was higher in the cycles with EZP than without EZP. The fertilization rate of the collected oocytes and the rate of good embryo formation were significantly lower in the cycles with EZP. No significant difference was observed between the three groups in terms of age, number of oocytes collected, or hormone levels before and after the oocyte retrieval. The fertilization and pregnancy rates were highest in the patients with no EZP being collected during any cycle, followed by those experiencing cycles both with and without EZP, and then by those with EZP collected during all cycles.ConclusionThe observation of lower fertilization, poor embryo formation, and a low pregnancy rate in the patients with EZP suggests the poor quality of oocytes that were collected with EZP in the same cycle.