Intracytoplasmic sperm injection in dysmorphic human oocytes

Clinical-pregnancy outcome after vitrification of blastocysts produced from in vitro maturation cycles

Polarity and early human embryo development

Selection of high potential embryos using time-lapse imaging: the era of morphokinetics

Three hypotheses were tested: (i) the distance between first and second polar bodies (PB) may relate to embryo morphology, (ii) that the orientation of pronuclei (PN) relative to PB may relate to embryo morphology, (iii) that the placement of a spermatozoon in a fixed plane relative to the first PB [intracytoplasmic sperm injection (ICSI)] may alter PN/PB orientation relative to in-vitro fertilization (IVF). A total of 251 two pronuclear (2PN) embryos (124 ICSI, 127 IVF) from 64 patients was studied. Angles were measured between the PN axis and the nearest PB (alpha), the furthest PB (beta), and between the two PB (gamma). On day 2, the morphological grades of embryos were recorded. gamma ranged from 0 to 150 degrees and was not significantly different for ICSI or IVF embryos of different grades; however, an unusual distribution of gamma suggested different populations of oocytes. The first hypothesis was rejected. alpha and beta ranged from 0 to 90 degrees : alpha did not relate significantly to embryo grade, but beta increased significantly with decreasing quality of ICSI embryos (P < 0.05) and the total group (P < 0.01), supporting hypothesis (ii). The difference in beta between ICSI and IVF embryos was not significant, so hypothesis (iii) was unproven. Significant differences between ICSI and IVF embryos in PN positions, irregular cleavage, and cleavage failure were noted.

Objective To investigate the suitable culture medium and culture time for human immature oocytes. Methods Three hundred and thirty-two oocyte-cumulus-complexes (OCCs) and 393 denuded oocytes (DOs) were randomly cultured in TCM199 culture system and PI culture system. The mature condition of OCCs was observed at time point of 48 h after culture in vitro. After DOs were cultured for 24 h, 30 h,48 h, expulsion of first polarbody was overviewed. Maturing rates(MR) of OCCs and DOs in vitro were calculated. Mature oocytes from OCCs and DOs were subjected to intracytoplasmic sperm injection (ICS1). Main outcomes were compared including fertilization rate (FR), eleaverage rate (CR) and blastulation rate (BR) between two culture systems. Results There was no statistical difference in MR of OCCs,CR and FR of mature oocytes from OCCs in TCM 199 and P1 culture system (all P > 0.05). The BR of mature oocytes from OCCs in TCMI99 culture system was higher than that of P1 culture system (53.7% vs 37.8%, P < 0. 05). For DOs when comparing TCM199 culture system with PI culture system,no significant difference was found in MR after cultured for 24 h,30 h and 48 h in vitro. The FR,CR and BR of mature oocytes from DOs in PI culture system were higher than those in TCM199 culture system,but only FR was significantly different (73.5% vs 62.9%, P <0.05). The MR of DOs in two culture systems was all significantly higher after culture in vitro for 30 h and 48 h than that for 24 h (P < 0.05), but MR was no difference at 48 h compared with 30 h. Conclusions OCCs are suitably cultured in TCM199 culture system whereas DOs in PI culture system. Culture time of human mature oocytes from DOs in vitro can be shortened to 30 h. Key words: Oocytes; In vitro maturation; Culture media; Sperm injections, intracytoplasmic; Oocyte-cumulus-complex; Denuded oocyte

Objective: Human oocytes collected for in vitro fertilization (IVF) vary in morphologic features and measurements. This study aimed to describe the morphology of human oocytes that failed to cleave after intracytoplasmic sperm injection (ICSI). Materials and Methods: Oocytes that failed to cleave post-ICSI were collected from IVF cycles. The oocytes were microscopically examined and the cytoplasmic diameter, zona pellucida thickness, perivitelline space (PVS) size, and first polar body (PBI) size were measured. The granularity pattern of cytoplasm; the color of cytoplasm; the presence of smooth endoplasmic reticulum (sER) aggregates, vacuoles, refractile bodies, and pronuclei; and, the status of polar bodies were all recorded. Results: A total of 142 oocytes were analyzed. The mean cytoplasmic diameter was 119.15±9.06 μm (range: 90.66-137.79 μm). The mean zona pellucida thickness was 19.29±3.69 μm (range: 12.86-33.69 μm). The mean PVS size was 3119.08 μm2 (range: 765-6448 μm2). The mean PBI size was 225.48±106.03 μm2 (range: 53.56-703.65 μm2). The cytoplasm showed fine (42.7%), dispersed (40.3%), central (14.5%), and uneven (2.54%) patterns of granulation. Other cytoplasmic abnormalities included refractile bodies (33.3%), sER aggregates (5.6%), vacuoles (5.6%), and dark cytoplasm (14.5%). A significant inverse correlation was found between the oocyte cytoplasmic diameter and PVS size. Conclusion: The morphologic abnormalities in oocytes that failed to cleave after ICSI are not significantly different from those observed in the general population of human oocytes. The oocyte cytoplasmic diameter was found to significantly inversely correlate with PVS size.

Successful in vitro maturation of human oocytes not exposed to human chorionic gonadotropin during ovulation induction, resulting in pregnancy

Three-parent in vitro fertilization: gene replacement for the prevention of inherited mitochondrial diseases

Artificial oocyte activation using calcium ionophores and enhancement of embryonic developmental potential by the granulocyte-macrophage colony-stimulating factor (GM-CSF) have already been reported. In this study, we evaluated the synergistic effect of these two methods on aged human unfertilized oocytes after intracytoplasmic sperm injection (ICSI). Then, we cultured the resulting embryos to the blastocyst stage and screened them for chromosomal abnormalities, to assess the safety of this protocol. Aged human oocytes deemed unfertilized after ICSI were activated, either by briefly applying the calcium ionophore A23187 alone (group A) or by briefly applying the ionophore and then supplementing the culture medium with recombinant human GM-CSF (rhGM-CSF) (group B). Next, the development was monitored in a time-lapse incubator system, and ploidy was analyzed by array comparative genomic hybridization (aCGH), after whole embryo biopsy and whole genome amplification. Differences between oocytes and resulting embryos in both groups were evaluated statistically. Oocytes unfertilized after ICSI can be activated with the calcium ionophore A23187 to show two pronuclei and two polar bodies. Addition of rhGM-CSF in the culture medium of A23187-activated oocytes enhances their cleaving and blastulation potential and results in more euploid blastocysts compared to the culture medium alone. This study shows that activating post-ICSI aged human unfertilized oocytes with a combination of a calcium ionophore and a cytokine can produce good-morphology euploid blastocysts.

Study question Are there differences in fertilization, blastocyst development, or euploidy rates when comparing ICSI versus conventional insemination among patients with male and non-male factor infertility? Summary answer Fertilization rate was lower for male factor ICSI. There were no differences in blastocyst development. Euploidy rate was lower for non-male factor ICSI. What is known already ICSI was developed for male factor infertility due to its requirement for a very small number of viable sperm. Its use has expanded beyond male factor due to some studies suggesting enhanced fertilization, and lower risks of failed fertilization or inadvertent contamination of embryo biopsy specimens. There are potential risks associated with ICSI, including imprinting disorders, birth defects, and significantly increased cost. It is also unclear whether ICSI contributes to genetic abnormalities by bypassing the egg’s natural sperm selection and potentially disrupting the meiosis apparatus. Study design, size, duration This retrospective cohort study of 576 patients was conducted at a single clinic from January 2021 to December 2021. Patients were grouped into (1) ICSI with male factor (n = 201), (2) ICSI with non-male factor (n = 160), and (3) conventional insemination with non-male factor (n = 215). Primary outcome was ploidy status, determined by calculating the percentage of euploid, aneuploid, and mosaic (both low and high mosaic) among the three groups. Secondary outcomes included fertilization and blastocyst development rates. Participants/materials, setting, methods Patients undergoing autologous IVF at a single clinic, with PGT-A performed at a single lab were included. Fertilization rate was defined as the percentage of 2PNs per number of mature eggs injected/inseminated. Blastocyst rate was defined as the percentage of blastocysts per 2PNs. Ploidy rate was defined as the percentage of euploid/aneuploid/mosaic blastocysts per total biopsied blastocysts for which a result was obtained. Percentage data was compared using N-1 Chi-squared test. Main results and the role of chance There were no significant differences in patient age, number of oocytes retrieved, or number of mature oocytes among the three groups. Fertilization rate for male factor ICSI was significantly lower compared to both non-male factor ICSI (74.2% vs 77.8%, p = 0.005) and conventional insemination (74.2% vs 76.8%, p = 0.018). There was no significant difference in fertilization rate between non-male factor ICSI and conventional insemination. There were no significant differences between blastocyst rates on Day 5, 6, or 7, or total blastocyst rate among the three groups. The euploidy rate for conventional insemination was significantly higher than that for non-male factor ICSI (53.4% vs 46.8%; p = 0.008). Non-male factor ICSI yielded lower euploidy rate than male factor ICSI (46.8% vs 52.4%, p = 0.034). Aneuploidy rate for non-male factor ICSI was significantly higher than both male factor ICSI (36.1% vs 31.0%, p = 0.041) and conventional insemination (36.1% vs 31.0%, p = 0.030). Mosaicism levels were similar between the three groups. There were no significant differences in number of biopsied embryos with no results: 5 for ICSI male factor group (0.59%), 6 for ICSI non-male factor (0.96%), and 10 for conventional insemination (0.89%). Limitations, reasons for caution This was a retrospective analysis of patients from a single clinic, with a relatively small sample size so results may not be generalizable to all populations. Extensive demographic information and baseline characteristics (including reason for ICSI performed among non-male factor patients) were not analyzed so there may be confounding factors. Wider implications of the findings This is the first study that we are aware of to investigate the ploidy status of embryos created among non-male factor patients undergoing ICSI. Conservative use of ICSI can significantly decrease the cost of IVF. Larger studies are needed to further elucidate the role for ICSI among these patients. Trial registration number not applicable

Proteins and mRNA produced in oogenesis support embryonic development until the zygotic transition, 3 days after fertilization. Since polar bodies can be biopsied with little if any harm to the oocyte, we tested the hypothesis that mRNA originating from expression in the meiotic oocyte is present and detectable in a single polar body prior to insemination. Human oocytes were obtained from patients undergoing controlled ovarian hyperstimulation and intracytoplasmic sperm injection. Immature oocytes were cultured overnight and inspected the following day for maturation. Metaphase II oocytes underwent polar body biopsy followed by reverse transcription without RNA isolation. Sibling oocytes were similarly prepared. Complementary DNA from all samples were pre-amplified over 15 cycles for candidate genes using selective primers. Real-time PCR was performed to detect and quantify relative single-cell gene expression. Polar body mRNA was detected in 11 of 12 candidate genes. Transcripts that were present in greater abundance in the oocyte were more likely to be detected in qPCR replicates from single polar bodies. Pre-amplification of cDNA synthesized without RNA isolation can facilitate the quantitative detection of mRNA in single human polar bodies.

Monochorionic triplet gestation after in vitro fertilization using donor oocytes: Case report and review

To identify and characterize amyloid-like substance (ALS) in human and mouse oocytes and preimplantation embryos. An experimental prospective pilot study. A total of 252 mouse oocytes and preimplantation embryos and 50 immature and in vitro matured human oocytes and parthenogenetic human embryos, from 11 consenting fertility patients, ages 18-45. Fluorescence intensity from immunofluorescent staining and data from confocal microscopy were quantified. Data were compared by one-way analysis of variance, with the least square-MEANS post-test, Pearson correlation coefficients (r), and bivariate analyses (t tests). ALS morphology was verified using transmission electron microscopy. Immunostaining for ALS appears throughout the zona pellucida, as well as in the cytoplasm and nucleus of mouse and human oocytes, polar bodies, and parthenogenetic embryos, and mouse preimplantation embryos. In mouse, 2-cell embryos exhibited the highest level of ALS (69000187.4 ± 6733098.07). Electron microscopy confirmed the presence of ALS. In humans, fresh germinal vesicle stage oocytes exhibited the highest level of ALS (4164.74088 ± 1573.46) followed by metaphase I and II stages (p = 0.008). There was a significant negative association between levels of ALS and patient body mass index, number of days of ovarian stimulation, dose of gonadotropin used, time between retrieval and fixation, and time after the hCG trigger. Significantly higher levels of ALS were found in patients with AMH between 1 and 3ng/ml compared to < 1ng/ml. We demonstrate for the first time the presence, distribution, and change in ALS throughout some stages of mouse and human oocyte maturation and embryonic development. We also determine associations between ALS in human oocytes with clinical characteristics.

In order to identify the real contribution of early fertilization check (EFC) for reproductive outcome, we compared the developmental potential of embryos derived from intracytoplasmic sperm injection (ICSI) after EFS with those from conventional insemination in sibling oocytes. Between April 2009 and April 2012, a total of 3249 oocytes in 386 patients were recruited following conventional insemination. Oocytes showing a second polar body (2ndPB) after an EFC were considered to be fertilized oocytes (IVF group), but, oocytes not showing a 2ndPB after EFC were placed into the ICSI group. The incidence of morphologically good embryos (MGE) on day 3, the blastocyst formation (BL), and the development of full blastocysts (full-BL) on day 5 were compared between the two groups. The clinical pregnancy rate was compared between the cycles with only conventional insemination or ICSI after EFC of the embryos being transferred. The fertilization rates in both the IVF and the ICSI groups were 48.1 and 73.9 %, respectively. The percentage of MGE in the ICSI group (40.8 %) was significantly lower than that in the IVF group (56.1 %, p < 0.01). The percentages of BL and full-BL in the ICSI group were significantly lower than those in the IVF group. The pregnancy rates were similar in both the groups. Checking fertilization earlier than the usual period contributed to an avoidance of lower fertilization. Moreover, the embryos derived from ICSI after EFC possessed a normal developmental potential.

Four healthy children from frozen human oocytes and frozen human sperms.