Intracytoplasmic Sperm Injection Oocytes Research Articles

Benefit from luteal phase progestin primed ovarian stimulation with clomiphene citrate supplementation in young women with diminished ovarian reserve: a retrospective study.

To compare the pregnancy outcomes of luteal phase and follicular phase progestin-primed ovarian stimulation protocol with clomiphene citrate supplementation (LPPOS+CC and FPPOS+CC) in young women with diminished ovarian reserve (DOR). A total of 483 women aged ≤35 years with DOR, who underwent in vitro fertilization (IVF)/intracytoplasmic sperm injection (ICSI)/embryo transfer (ET) with controlled ovarian stimulation using LPPOS+CC (n=257) or FPPOS+CC (n=226) protocols during June 2018 and December 2021 at the First Affiliated Hospital of Wenzhou Medical University, were included in this retrospective study. The baseline characteristics, superovulation results, laboratory related indicators between the two groups, and the pregnancy outcomes of women who achieved at least one high-quality cleavage-stage embryo or good-morphology blastocyst were compared between the two groups. No statistically significant differences were identified between the groups with respect to age, duration of infertility, proportion of secondary infertility, previous failed cycles, body mass index, anti-Müllerian hormone, antral follicle count, basal luteinizing hormone level, basal progesterone level, number of oocytes retrieved, oocyte maturation rate, high-quality cleavage-stage embryo cycle rate, the percentage of women with profound pituitary suppression, live birth rate and preterm birth rate (all P>0.05). The LH levels on the day of trigger [4.0 (2.7, 5.3) vs. 5.1 (3.2, 7.2) IU/L], the percentage of women with LH levels of >10 IU/L on the trigger day (3.13% vs. 10.67%), and the two pronucleus (2PN) rate of ICSI oocytes (72.16% vs. 79.56%) were significantly lower in the LPPOS+CC group than those in the FPPOS+CC group (P<0.05 or P<0.01). The duration of stimulation [11 (9, 12) vs. 9 (8, 11) d], the consumption of total gonadotropin [2213 (1650, 2700) vs. 2000 (1575, 2325) IU], the progesterone levels on the day of trigger [1.3 (0.8, 2.9) vs. 0.9 (0.6, 1.2) ng/mL], the clinical pregnancy rate [61.88% vs. 46.84%], and implantation rate [42.20% vs. 31.07%] in the LPPOS+CC group were significantly higher than those in the FPPOS+CC group (all P<0.01). Compared to FPPOS+CC, the LPPOS+CC protocol appears to have better pregnancy outcomes for young women with DOR undergoing IVF/ICSI-ET.

Dual-Activated H2O2-Responsive AIE Probes for Oocyte Quality Assessment.

Nonobstructive azoospermia (NOA) is an important cause of infertility, and intracytoplasmic sperm injection (ICSI) is the mainstay of treatment for these patients. In cases where a sufficient number of sperm (usually 1-2) is not available, the selection of oocytes for ICSI is a difficult problem that must be solved. Here, we constructed a dual-activated oxidative stress-responsive AIE probe, b-PyTPA. The strong donor-acceptor configuration of b-PyTPA leads to twisted intramolecular charge transfer (TICT) effect that quenches the fluorescence of the probe, however, H2O2 would specifically remove the boronatebenzyl unit and release a much weaker acceptor, which inhibits TICT and restores the fluorescence. In addition, the presence of a pyridine salt makes b-PyTPA more hydrophilic, whereas removal of the pyridine salt increases the hydrophobicity of PyTPA, which triggers aggregation and further enhances fluorescence. Thus, the higher the intracellular level of oxidative stress, the stronger the fluorescence. In vitro, this dual-activated fluorescent probe is capable of accurately detecting senescent cells (high oxidative stress). More importantly, b-PyTPA was able to characterize senescent oocytes, as assessed by the level of oxidative stress. It is also possible to identify high quality oocytes from those obtained for subsequent ICSI. In conclusion, this dual-activated oxidative stress-assessment probe enables the quality assessment of oocytes and has potential application in ICSI.

Lower developmental potential of rat zygotes produced by ooplasmic injection of testicular spermatozoa versus cauda epididymal spermatozoa.

Intracytoplasmic sperm injection (ICSI) is clinically used to treat obstructive/nonobstructive azoospermia. This study compared the efficacy of ICSI with cauda epididymal and testicular sperm in Wistar (WI) and Brown-Norway (BN) rats. The transfer of ICSI oocytes with cryopreserved epididymal and testicular WI sperm resulted in offspring production of 26.2% and 3.7%-4.7%, respectively (P < 0.05). Treatments for artificial oocyte activation (AOA) and acrosome removal improved pronuclear formation in BN-ICSI oocytes; however, only AOA treatment was effective in producing offspring (3.7%-6.5%). In the case of ICSI with testicular sperm (TESE-ICSI), one offspring (0.6%) was derived from the BN-TESE-ICSI oocytes. The application of AOA or a hypo-osmotic sperm suspension did not improve the production of TESE-ICSI offspring. Thus, outbred WI rat offspring can be produced by using ICSI and less efficiently by using TESE-ICSI. Challenges in producing offspring by using ICSI/TESE-ICSI in inbred BN strain require further investigation.

Clinical and laboratory factors associated with the presence of dysmorphic oocytes in intracytoplasmic sperm injection cycles.

This study investigated the clinical and laboratory factors associated with the presence of dysmorphic oocytes in intracytoplasmic sperm injection (ICSI) cycles. The study involved 200 ICSI cycles, performed from 2020 to 2021, that yielded at least one mature oocyte. Clinical characteristics and ovarian stimulation methods were compared between 68 cycles with at least one dysmorphic oocyte (the dysmorphic group) and 132 cycles with normal-form oocytes only (the non-dysmorphic group). Dysmorphic oocytes were characterized by dark cytoplasm, cytoplasmic granularity, cytoplasmic vacuoles, refractile bodies in the cytoplasm, smooth endoplasmic reticulum in the cytoplasm, an oval shape, an abnormal zona pellucida, a large perivitelline space, debris in the perivitelline space, or an abnormal polar body. The ages of the women, indications for in vitro fertilization, serum anti-Müllerian hormone levels, and rates of current ovarian endometrioma were similar between the dysmorphic and non-dysmorphic groups. In both groups, the three ovarian stimulation regimens, two types of pituitary suppression, and total gonadotropin dose were employed similarly. However, the dual-trigger method was used more frequently in the dysmorphic group (67.6% vs. 50%, p=0.024). The dysmorphic group contained significantly more immature oocytes and exhibited significantly lower oocyte maturity (50% vs. 66.7%, p=0.001) than the non-dysmorphic cycles. Within the dysmorphic group, significantly lower oocyte maturity was found in the cycles using a dual-trigger, but not in those with a human chorionic gonadotropin trigger. ICSI cycles with dysmorphic oocytes are closely associated with reduced oocyte maturity. This association was observed exclusively in dual-trigger cycles.

P-259 Intracytoplasmic sperm injection conducted within a micro 3D printed device that requires no holding pipette or vacuum reduces porcine oocyte invagination during microinjection

Abstract Study question Can the use of a custom-designed and 3D printed microICSI™ device reduce the degree of oocyte invagination during intracytoplasmic sperm injection (ICSI)? Summary answer Invagination was significantly less in porcine oocytes when ICSI was performed in microICSI™ device compared to conventional ICSI. What is known already ICSI is a difficult procedure for embryologists to master, yet until recently, technically little had changed. In particular, the holding pipette (HP) with vacuum to hold the oocyte has not been surpassed. However, a source of shear-stress during ICSI of oocytes is the invagination of the zona pellucida and cytoplasm that occurs during microinjection, due to the pressure of the injection pipette against the small surface area of the HP. A recent study demonstrated microinjection within a two-piece 3D printed device. Here we evaluated a design refinement that displaced injection pressure more evenly across the oocyte to improve embryo quality. Study design, size, duration A control (conventional ICSI) and study group (microICSI™) were compared over three replicates investigating the level of invagination (3-point score, 1 is lowest and 3 is greatest) during ICSI. Incidence of cytoplasmic lysis was also recorded 24 h following ICSI. Participants/materials, setting, methods Porcine oocytes collected by follicle aspiration of abattoir-sourced ovaries were matured for 39-44 h in NCSU-17 medium supplemented with eCG, hCG, porcine follicular fluid, EGF and insulin. Mature oocytes, stripped of cumulus, were randomly allocated to either conventional ICSI or microICSI™. ICSI was performed, with treatment order randomised, and the injected oocytes were cultured in NCSU-17 culture medium at 39 C. Main results and the role of chance The microICSI™ device was modelled as a one-piece structure with contoured surfaces within a microwell array to support the oocyte during injection without the requirement for a holding pipette. Two-photon polymerisation 3D micro printing was used to print and assess multiple prototype geometries to optimise the support of the oocyte during ICSI. Over the three replicates (n = 10-20/treatment/replicate), the degree of invagination was consistently lower in the microICSI™ group compared with conventional ICSI (Mean ± SEM): Replicate 1: 1.40 ± 0.16 vs. 2.00 ± 0.19; Replicate 2: 1.20 ± 0.11 vs. 2.00 ± 0.13; Replicate 3: 1.42 ± 0.12 vs. 1.78 ± 0.15, with a paired t-test showing a significant difference between the two groups (p = 0.035). The incidence of lysis was not significantly different between the two treatments (ranging from 15-20% across both groups and varied between replicates). Limitations, reasons for caution The use of a porcine ICSI model may not sufficiently replicate what occurs with human ICSI as human oocytes experience greater levels of invagination (typically scored out of 4 rather than 3). Wider implications of the findings Reducing the invagination due to the injection process may reflect a less stressful procedure, with improvement to fertilization rates and embryo quality. Trial registration number not applicable

Successful Production of Offspring Derived from Phospholipase C Zeta-Deficient Sperm by Additional Artificial Activation.

During mammalian fertilization, repetitive rises of intracellular calcium called calcium oscillations are required for full activation of oocytes. Therefore, oocytes such as round spermatid injected or somatic cell nuclear transferred require additional artificial activation which mimics the calcium oscillations. It is well recognized that sperm specific phospholipase C (PLCζ) is a strong candidate as the sperm factor which can induce calcium oscillations and, at least in mammals, the genetic mutation of PLCζ in human causes male infertility due to the lack of calcium oscillations in the oocytes. Recent studies showed that the sperm lacking PLCζ (Plcz1-/-) still could induce rise(s) of intracellular calcium in the oocytes after IVF but not intracytoplasmic sperm injection (ICSI). In the ICSI oocytes, no pronuclear formation or development to the two-cell stage was observed. However, it is still unclear whether additional activation treatment can rescue the low developmental ability of Plcz1-/--sperm-derived oocytes after ICSI. In this study, we examined whether oocytes injected with a Plcz1-/- sperm can develop to term by additional artificial activation. In oocytes injected a Plcz1-/- sperm and Plcz1-/- and eCS (another candidate of the sperm factor) double knockout sperm (Plcz1-/-eCS-/-), the rates of pronuclear formation were very low (2.0 ± 2.3% and 6.1 ± 3.7%, respectively) compared to control (92.1 ± 2.6%). However, these rates were dramatically improved by additional procedures of PLCζ-mRNA injection or SrCl2 treatment (Plcz1-/- sperm + PLCζ mRNA, Plcz1-/- sperm + SrCl2 and Plcz1-/-eCS-/- sperm + PLCζ mRNA; 64.2 ± 10.8%, 89.2 ± 2.4% and 72.6 ± 5.4%, respectively). Most of the oocytes were developed to the two-cell stage. After embryo transfer, healthy pups were obtained in all these groups (Plcz1-/- sperm + PLCζ mRNA:10.0 ± 2.8%, Plcz1-/- sperm + SrCl2:4.0 ± 4.3% and Plcz1-/-eCS-/- sperm + PLCζ mRNA: 10.0 ± 5.7%). The rate in Plcz1-/- sperm + SrCl2 group was significantly lower than that in control (26.0 ± 2.4%). Taken together, our present results show that additional activation treatment such as SrCl2 and PLCζ mRNA can fully support to develop to term even in oocyte injected Plcz1-/- sperm. In addition, PLCζ-induced oocyte activation is more suitable for successful development to term compared to that such as phenomenon induced by SrCl2. These findings will contribute to improvement for male-dependent human infertility and reproductive technologies in other mammalian species.

Development of buffalo embryo derived from ICSI: effects of various somatic-cell co-culture

Context Somatic-cell co-culture of intracytoplasmic sperm injection (ICSI) buffalo embryos has not been reported earlier. Aim This study aimed to determine the effects of buffalo oviductal epithelial-cell, granulosa-cell, and cumulus-cell co-culture on in vitro culture of early embryo development as ICSI and post-activation. Methods Selected oocyte–cumulus complexes were cultured for 19–20 h in 50-μL drop of tissue culture medium (TCM199 + 10% buffalo follicular fluid, hCG 50 IU/mL, 0.02% arbitrary units (AU)/mL follicle-stimulating hormone and 1 μg/mL estradiol-17βE2). Oocytes reaching Metaphase II were subjected to ICSI with immobilised spermatozoa. All ICSI oocytes were activated with calcium ionophore for 5 min, followed by cycloheximide for 5 h. The embryos at 6–8-cell stages were co-cultured. Key results The morula, blastocyst, and hatched blastocyst rates when co-cultured with oviductal epithelial cells were 68.18%, 48.18%, and 30.00% respectively. The morula, blastocyst, and hatched blastocyst rates when co-cultured with cumulus cells were 51.49%, 34.33%, and 16.42% respectively. The morula, blastocyst, and hatched blastocyst rates when co-cultured with granulosa cells were 52.14%, 32.48%, and 13.68% respectively. Conclusions In vitro maturation buffalo oocytes can be fertilised in vitro with ICSI and co-cultured with different types of cells. Oviductal epithelial cell co-culture was shown to be superior in supporting in vitro embryo development in this study. Implications The oviductal epithelial cells are easy to prepare and may be used for co-culture to increase the efficiency of in vitro production of buffalo embryos.

P-252 Oolemma surface area impacts human pre-implantation embryo development and usage: The Rotterdam Periconception Cohort

Abstract Study question Does oolemma area change before the 2-cell stage? Is oolemma area associated with fertilization rate, embryo usage rate and preimplantation embryo development? Summary answer Oolemma area is associated with preimplantation embryo development, the odds of fertilizing and developing into a used or discarded embryo. What is known already Current morphological grading systems are based on embryo morphology and omit oocyte morphology. Fertilization and embryo potential are largely determined by the maturation and quality of the oocyte. Oocyte quality correlates to morphology, and the very early stages of embryo development are critical to changes in oocyte size. Larger oocyte size associates with higher quality blastocyst. This suggests that oocyte size can be a morphological marker of preimplantation embryo quality and assisted reproductive treatment (ART) outcomes. Study design, size, duration This study is embedded in the Rotterdam Periconceptional Cohort, an ongoing prospective tertiary hospital-based birth cohort study. From May 2017 to July 2020, a subcohort of 378 women that underwent ART was included comprising of 124 pregnancies after in vitro fertilization (IVF) and 254 after intracytoplasmic sperm injection (ICSI). Participants/materials, setting, methods Health, lifestyle and treatment factors were collected from detailed self-reported questionnaires and medical records. Oolemma area was measured at the time of fertilization (t0), pronuclear appearance (tPNa) and fading (tPNf). Oolemma area trajectories (slope surface t0-tPNf) were calculated with linear regression. Fertilization rate, embryo usage rate, and embryo morphokinetics were analyzed in association with oolemma area and trajectories. Analyses were performed with linear mixed models, mixed effects logistic regression, and adjusted for relevant confounders. Main results and the role of chance Oolemma area decreased from t0 to tPNf for oocytes that developed into both used or discarded embryos. Oolemma area at t0 was strongly associated with the rate of area shrinking, as larger oolemma areas had faster shrinking rates (Bshrinking -12.55 µm²/hour, 95%CI -14.55; -10.55, p &amp;lt; 0.001). Oocytes that resulted in a usable embryo were larger and reached tPNf faster than oocytes that resulted in a discarded embryo (tPNf 9855±595 µm2 vs 9690±721 µm2, p &amp;lt; 0.001, 24.4±3.6 h vs 26.5±5.8 h, p &amp;lt; 0.001). Oolemma area at tPNf did not differ between ICSI and IVF oocytes, however ICSI oocytes reached tPNf faster than IVF oocytes (23.6±3.2h and 25.9±3.8h, p &amp;lt; 0.001, respectively). In ICSI only, larger oolemma area showed lower odds of a successful fertilization (t0 OR 0.57, 95%CI 0.48; 0.67, p &amp;lt; 0.001), but higher odds of being cryopreserved or transferred (tPNf OR 1.66, 95%CI 1.33; 2.08, p &amp;lt; 0.001). For IVF and ICSI, larger oolemma area at tPNF was associated with faster preimplantation development up to the morula stage (t7 B -1.41, 95%CI -2.18; -0.63, p &amp;lt; 0.001). Oolemma area shrinking rate was not associated with embryo morphokinetics. Limitations, reasons for caution Small number of embryos measured at the blastocyst stage, and oolemma area measurements were not possible at t0 and tPNa for IVF oocytes. Wider implications of the findings Oolemma area is a suggested morphological marker for oocyte competence and an early marker for the success of fertilization and embryo development. The relevance of oolemma area as predictor of ART success requires further research. Cryopreservation and PCOS, which influence oocyte size, could be studied in association with oolemma area. Trial registration number not applicable

Atomic force spectroscopy-based essay to evaluate oocyte postovulatory aging.

Postovulatory aging is a process occurring in the mature (MII) oocyte leading the unfertilized ones to apoptosis. The optimal time window of fertility for different mammalian species after oocytes maturation depends on its timeliness: the higher the time elapsed from the accomplishment of the MII stage, the lower are the chances of fertilization and of development of a viable embryo. In the in vitro fertilization, the selection of competent oocytes for intracytoplasmic sperm injection (ICSI) is mostly made by the visual inspection of the MII oocyte morphology, which does not allow to determine the oocyte postovulatory age. On the other hand, more specific tests usually involve some kind of staining, thus compromising the viability of the oocyte for reproductive purposes. Hence, the need of a noninvasive analysis of oocyte aging to improve the success rate of in vitro fertilization procedures. Here, we exploit atomic force microscopy to examine the evolution of the mechanical properties of mouse oocytes during in vitro postovulatory aging. Three hours before the occurrence of any visual morphological feature related to degradation, we observe a sudden change of the mechanical parameters: the elastic modulus doubles its initial value, while the viscosity decreases significantly. These mechanical variations are temporally correlated with the release of the cortical granules, investigated by fluorescence microscopy. Interestingly, the oocyte mechanics correlates as well with the yield of embryo formation, evaluated up to the blastocyst formation stage. These results demonstrate that minimally invasive mechanical measurements are very sensitive to the aging of the oocyte and can be used as a label‐free method to detect the age of the postovulatory oocytes.

P–442 Cancer does not adversely affect oocyte maturation in vitro with the exception of breast cancer

Abstract Study question Do different types of cancer affect the success of oocyte maturation in vitro compared to infertile women included in the in vitro fertilization (IVF) program? Summary answer Cancer does not adversely affect oocyte maturation in vitro, with the exception of breast cancer, compared to infertile women in the in vitro fertilization program. What is known already Vitrification and storage of oocytes in liquid nitrogen is one of the real options for maintaining reproductive function in cancer patients. Despite careful hormonal stimulation of the ovaries, however, the proportion of oocytes is immature and lost to the patient. In vitro maturation of oocytes can play an important role in resolving immature oocytes and increasing the chances of conception in cancer patients. Moreover, it can mean a safe way to store oocytes when ovarian hormonal stimulation could worsen the disease. Therefore, the aim of this study was to determine whether different types of cancer affect oocyte in vitro maturation. Study design, size, duration After ovarian stimulation in 18 cancer patients, the number and maturity of oocytes were compared to 21 infertile patients in the IVF program over a three-year period. In both groups, 119 germinal vesicle-GV oocytes were matured in vitro to compare the maturation rate. After IVF in a subset of 17 infertile patients, the fertilization of in vitro and in vivo matured oocytes was compared in the same cycles. The procedure was considered in cancer patients. Participants/materials, setting, methods In this prospective study, forty-five GV oocytes in cancer patients and 74 GV oocytes in infertile patients underwent in vitro maturation procedure. Each oocyte was matured in vitro in the MediCult IVM System by conditioning in LAG medium and maturation for up to 28 hours in IVM medium with added hormones FSH and hCG, in coculture with cumulus cells from mature oocytes in the same patients. Oocytes were fertilized by intracytoplasmic sperm injection (ICSI). Main results and the role of chance After controlled ovarian hormonal stimulation, 198 oocytes were retrieved in cancer patients and 259 oocytes in infertile women and there were no significant differences in the number of retrieved oocytes, proportion of degenerated oocytes and proportion of GV oocytes. In cancer patients, the proportion of oocytes that matured in vitro was lower than in infertile patients (66.0 vs. 80.0%), but the difference was not significant. Among cancer patients, the oocyte maturation rate tended to be lower in patients with breast cancer than in patients with other cancers (54.5% vs. 81.2%; difference not significant). However, in patients with breast cancer, significantly fewer oocytes matured in vitro than in infertile patients (54.5% vs. 80.0%; P &amp;lt; 0.05, Chi-Square test) even though they tended to be younger (29.3 ± 7.4 vs. 33.4 ± 5.0 years; non-significant difference). After in vitro maturation, there was a 13% increase in mature oocyte yield in cancer patients and a 20.1% increase in infertile women with no significant difference observed. After ICSI in a subset of infertile women, there was approximately the same fertilization rate between oocytes matured in vitro and in vivo (55.1% vs. 57.0%) in the same cycles. Limitations, reasons for caution For ICSI in oocytes matured in vitro, we had to use semen collected the day before, while oocytes matured in vivo were fertilized with fresh semen in the same cycle. Therefore, we could not compare the development of embryos in both groups. Wider implications of the findings: In vitro maturation of oocytes in connection with their vitrification or vitrification of embryos after their fertilization appears to be a valuable way to maintain the fertility of young cancer patients, but a worse outcome is expected in breast cancer patients. Trial registration number National Medical Ethical Committee Approval, No. 0120–222/2016–2; KME 115/04/16.

P–193 First cleavage division perpendicular to the pronuclear axis adversely affects the clinical outcome in human embryos

Abstract Study question Does the direction of formation for the first cleavage plane relative to the pronuclear axis affect clinical outcome? Summary answer A first cleavage division perpendicular to the pronuclear axis adversely affects the rate of embryo utilization for transfer or cryopreservation and the pregnancy outcome. What is known already It remains unclear how the first cleavage plane is determined in human embryos. By using time-lapse monitoring, our previous study (presented in ESHRE 2019) suggested that both the axis and locations of male and female pronuclei are involved in determining the first embryonic cleavage plane. Furthermore, by using immunofluorescence analysis, it was also revealed that most analyzed zygotes showed two pericentrin signals aligned around the interface between the male and female pronuclei. Our findings suggest that the pronuclear axis strongly influences the positions of the centrosomes, which become mitotic spindle poles and define the first cleavage plane. Study design, size, duration: From January 2015 to December 2017, time-lapse imaging (EmbryoScope®) of 3397 intracytoplasmic sperm injection (ICSI) oocytes was conducted. Of those, the relationship between the pronuclear axis and the first cleavage plane was analyzed in 607 normally fertilized embryos that cleaved to two cells and were obtained in 2015. Furthermore, of 3397 ICSI oocytes, 749 transferred embryos were classified based on the first cleavage patterns relative to the pronuclear axis, and the pregnancy rate was examined. Participants/materials, setting, methods A straight line connecting the centers of the pronuclei was defined as the 2PN axis. Based on the direction of the first cleavage relative to the 2PN axis, embryos were classified into three groups: parallel, perpendicular and intermediate. Fresh embryos were transferred on Day 2/3 (fresh-ET). Frozen and thawed embryos were transferred on Day 2/3 or Day 5 (F/T-ET). Clinical pregnancy was defined as confirmed gestational sac in the uterine cavity. Main results and the role of chance Of 607 analyzed embryos, 506 produced suitable images and were assigned to one of three groups: parallel (84.4%, n = 427), perpendicular (9.7%, n = 49) and intermediate (5.9%, n = 30). Embryos that formed a cleavage furrow parallel to the 2PN axis were significantly more frequent than others (perpendicular, intermediate) (P &amp;lt; 0.001). The embryo utilization rate for transfer or cryopreservation was significantly lower in the perpendicular group than in the parallel group (30.7% vs. 69.3%, P &amp;lt; 0.01). Furthermore, of 749 transferred embryos, 504 assigned to the parallel and perpendicular groups were selected (n = 470 and n = 34, respectively), and the pregnancy outcome was analyzed. The mean maternal age was not significantly different between groups. The pregnancy rate of embryos was 24.2% (n = 45/186) from fresh-ET and 39.4% (n = 112/284) from F/T-ET in the parallel group, and 0% (n = 0/14) from fresh-ET and 15.0% (n = 3/20) from F/T-ET in the perpendicular group. Regardless of the types of embryo transfer (fresh or F/T), the pregnancy rate was significantly lower in the perpendicular group than in the parallel group (P &amp;lt; 0.01). In addition, one of three patients who became pregnant from the transfer of an embryo in the perpendicular group had a miscarriage. Limitations, reasons for caution Since only ICSI embryos were analyzed in this study, the influence of fertilization methods on subsequent development could not be investigated. Further studies including preimplantation genetic testing for aneuploidy may help determine the reasons why pregnancy rates differ between groups. Wider implications of the findings: We suggest that the 2PN axis is essential for determining the first cleavage plane because it seems to be involved in positioning the mitotic spindle poles. The direction of the first cleavage plane relative to the 2PN axis can be an important indicator for predicting embryo development and pregnancy outcome Trial registration number none

Production of viable bovine embryos by intracytoplasmic sperm injection of oocytes harvested from slaughtered old cows

Abstract: (1) Background: Intracytoplasmic sperm injection (ICSI) is currently used to increase fertilization success by avoiding several oocyte or sperm deficiencies that would normally prevent conception after in vivo fertilization or classical in vitro fertilization. This paper aimed at improving the in vitro fertilization protocol of bovine oocytes, harvested from old cows after slaughtering, using intracytoplasmic sperm injection; (2) Methods: Oocytes were harvested by puncture of follicles from ovaries obtained from slaughtered old cows, followed by aspiration. Out of the 127 cumulus-oocyte complexes that were harvested, 84 (66.14%) were declared suitable for cultivation, after morphological evaluation. Following oocyte maturation for 22 hours, 77 cumulus-oocyte complexes were morphologically intact and could undergo the steps required for intracytoplasmic injection of spermatozoa. Frozen-thawed bull semen was used for ICSI and the 77 fertilized oocytes were kept for 24 hours in an atmosphere enriched with 5% CO2.; (3) Results: Fertilized oocytes transformed into 46 zygotes (fertilization rate of 59.74%), while after 168 h of cultivation 38 transferable compact morulae or early blastocysts were obtained; (4) Conclusions: Intracytoplasmic sperm injection can represent a viable alternative to classical IVF, when oocytes or sperm with lower fertility are used.

Is intracytoplasmic sperm (ICSI) better than traditional in vitro fertilization (IVF): confirmation of higher blastocyst rates per oocyte using a split insemination design.

To explore the effects of traditional vs. intracytoplasmic sperm injection (ICSI) insemination method on the outcome of high-quality blastocyst development in a split sibling oocyte cohort. In this retrospective cohort study, we analyzed 62 ICSI/IVF split cycles. Sibling oocytes were randomly assigned to ICSI or IVF insemination. Two hundred thirty-four ICSI-only cycles and 152 IVF-only cycles were also analyzed for comparison. Blastocysts were graded by Gardner's embryo grading and were considered a high-quality blastocyst if 3BB or better (Gardner 1999). In the ICSI/IVF split group, (1) ICSI oocytes had a higher fertilization rate per oocyte allocated (73% vs 62%, p < 0.001), (2) more high-quality day 2 embryos (69% vs 55%, p < 0.005), (3) ICSI oocytes had a lower blastulation rate per 2PN (46% vs 54%, p < 0.05), but a higher blastulation rate when calculated per oocyte allocated (40% vs 32%, p < 0.05). The ICSI-only group had a lower fertilization rate (65% vs 70%, p < 0.001) but more high-quality day 2 embryos in comparison to the IVF-only group (68% vs 64%, p < .05). The total high-quality blastulation rate was higher for the IVF-only group per 2PN (49% vs 43%, p < 0.05) and per oocyte retrieved (34% vs 28%, p < 0.05). This distinctive IVF/ICSI sibling oocyte split design demonstrated a higher-quality blastulation rate in the IVF group compared to the ICSI group when calculated per 2PN, but not per oocyte allocated to each insemination procedure.

59 Genome activation in intracytoplasmic sperm injection-derived horse embryos

During pre-implantation development, embryos go through a critical period of embryonic genome activation (EGA). The timing of EGA is species specific, but little is known in horse embryos. Here, we aimed to characterise EGA in equine embryos produced by intracytoplasmic sperm injection. Embryos were produced by intracytoplasmic sperm injection of oocytes from 3 mares. Two embryos from each mare at each of 8 developmental stages (MII, zygote, 2-cell, 4-cell, 8-cell, 16-cell, morula, and blastocyst) were individually analysed by RNA-seq. Differential expression was evaluated using binomial Wald tests with an absolute logFC (fold change) threshold of 1 in the DESEqn 2R package. We found that EGA occurred in a two-step fashion. Minor EGA took place during the 2-cell to 4-cell transition, and featured up-regulation of 751 genes and discrete down-regulation of 60 genes in 4-cell embryos compared with 2-cell embryos. Differentially upregulated genes were enriched in gene ontology terms related to transcriptional activator activity, homeobox domains, and nucleosome assembly. Major EGA occurred during the 4-cell to 8-cell transition and included the largest number of differentially expressed genes (n=2,023) between consecutive stages. This period also featured the first massive transcript downregulation (n=816). Upregulated genes were enriched in gene ontology terms related to ribosomal assembly, translation, and RNA modification. Additionally, we observed that the number of intronic sequences was significantly higher from the 4-cell stage onward, indicating active transcription in comparison to oocytes, zygotes, and 2-cell embryos. To evaluate the timing of paternal genome activation, we used whole-genome sequencing data from the parents (average genome coverage of 19×) to quantify allele-specific expression. The average number of informative SNPs in exons, i.e. SNPs with alternative homozygous genotypes from the sire (AA mare - BB sire), was 26 128 per mare, corresponding to 7696 genes. Parental-specific transcript abundance was determined for each embryo, with an average of 1,911±865 informative SNPs detected per sample. Paternal alleles were considered expressed when they reached 10% of the maternal count. Across development, paternal transcripts became appreciable at the 4-cell stage, with 14.15±7.60% of the informative SNPs exhibiting paternal expression, and increased thereafter until reaching a maximum of 96.34% at the blastocyst stage. Overall, this work demonstrates that EGA in horse embryos starts at the 4-cell stage and achieves its main activation at the 8-cell stage. Further analysis will be performed to detail paternal vs. maternal gene expression at the different embryonic stages.

111 Fertilizing ability of frozen and freeze-dried semen following intracytoplasmic sperm injection of invitro-matured sheep oocytes

Freeze-drying is a novel technique that permits the storage of semen at room temperature for long time periods, retaining their fertilizing capacity. The main objective of this work was to compare the fertilization ability of frozen-thawed (FT) and freeze-dried (FD) ram semen following intracytoplasmic sperm injection (ICSI) of invitro-matured (IVM) sheep oocytes. Oocytes were recovered by slicing the ovaries of slaughtered sheep. Selected cumulus-oocyte complexes (COCs) were IVM for 24h in tissue culture medium 199 (TCM-199) supplemented with 10% heat-treated oestrous sheep serum (ESS), 0.36mM pyruvate, FSH (1IUmL−1), and luteinising hormone (LH; 1IUmL−1) under mineral oil in a humidified atmosphere of 5% CO2, at 38.5°C. Semen was collected from fertile adult rams using an artificial vagina and processed for (1) freezing and thawing (Khalifa and Lymberopoulos, 2013 Cell Tissue Bank 14, 687-698; https://doi.org/10.1007/s10561-012-9357-6) or (2) freeze-drying and rehydration according to Arav et al. (2018 J. Assist. Reprod. Genet. 35, 1149-115; https://doi.org/10.1007/s10815-018-1145-1) protocols. For FD protocol, sperm samples were diluted in a sugar solution of trehalose and sorbitol (LyoB) and dehydrated for 24h. Later, the samples were rehydrated in a warming solution and diluted in TCM-199 before ICSI. After maturation, metaphase II (MII) oocytes with a polar body were injected with FT or FD sperm. Briefly, oocytes were transferred into groups of six in an ICSI dish containing 6-µL drops of holding medium (TCM-199 + 5% fetal bovine serum) and 3-µL drops of PVP for the sperm samples. Injection was carried out with an inverted microscope (Olympus IX73) connected to a micromanipulation system (Narishige) using ICSI pipettes with 7-µm internal diameter. Within 1h, ICSI oocytes were activated with 5 µM ionomycin for 4min and invitro cultured in modified synthetic oviductal fluid medium (Bogliolo et al. 2011 Reprod. Fertil. Dev. 23, 809-817; https://doi.org/10.1071/RD11023). After 17-21h, injected oocytes were fixed and stained in a solution of ethanol Hoechst 33342 and classified as FPN (one female pronucleus and one condensed sperm head), MPN (one male pronucleus and one MII), 2PN (two pronuclei, male and female), 3PN (three or more pronuclei), and NPN (no pronuclei). Data were analysed using analysis of variance (two-way ANOVA) followed by Tukey post hoc test with SAS software, version 9.4. The ICSI-FD group had a higher number of NPN and a lower number of 2PN than did the ICSI-FT group (P&amp;lt;0.05). We think that more technical advances in the FD process as well as the rehydration procedure are necessary to improve the application of FD ovine semen for invitro fertilization by ICSI in sheep, but in any case these results have showed that FD could be a useful tool for the future of invitro embryo production. Table 1.Pronuclear formation at 17-21h post-injection1 Treatment n FPN MPN 2PN 3PN NPN FT 71 9.66±4.12 4.26±1.48 48.13±2.79a 5.97±4.16 31.98±6.75a FD 65 6.16±2.26 1.39±1.39 20.15±4.14b 10.57±6.59 61.73±6.89b a,bValues in the same column with different superscript letters differ significantly (P&amp;lt;0.05). 1Data are presented as mean±s.e.m. FPN=female pronucleus, MPN=one male pronucleus and one metaphase II oocyte, 2PN=two pronuclei, male and female, 3PN=three or more pronuclei, NPN=no pronuclei. Funding was provided by Spanish MINECO Grant AGL2017-85837-R, Spanish MECD pre-doctoral grant FPU15/00773, and Spanish MECD mobility grant EST18/00472 to Irene Menéndez Blanco.

Detrimental effects of oxidative stress in bovine oocytes during intracytoplasmic sperm injection (ICSI)

Intracytoplasmic sperm injection (ICSI) is an essential technology in animal and human reproduction. However, the developmental competence and pregnancy rate of embryos derived from ICSI are still lower than that from the conventional in vitro fertilization technique. In this report, we focused on reactive oxygen species (ROS) as a potential detrimental factor for ICSI. Experiment 1 was conducted to evaluate the effect of oxidative stress by two different oxygen concentrations (20%: control vs. 5%) in ICSI on the developmental competence (blastocyst rate: day 7, DNA fragmentation rate: day 4) and, ROS concentration and mitochondrial membrane potential of oocytes in ICSI. In the 5% O2 group, the blastocyst rate (29.5%) was higher and DNA fragmentation rate (4.8 ± 1.0%) was lower than those in the control group significantly (12.7% and 18.2 ± 2.4%, respectively, P < 0.05). Also, ROS concentration in the 5% O2 group (12.8 ± 0.7) was significantly lower than that in the control group (47.8 ± 6.9, P < 0.05). In experiment 2, we examined the supplementation of media with reduced glutathione (GSH) during ICSI procedure in an attempt to reduce the oxidative stress. The addition of GSH to the culture medium improved the blastocyst rate (17.6% vs. 30.4%, P < 0.05), and decreased the ROS levels in the oocytes (70.0 ± 7.4 vs. 23.9 ± 4.0, P < 0.05). In conclusion, our present study revealed that oocytes are under oxidative stress in ICSI procedure. Reduction of the oxygen concentration to 5% in the culture environment, or the addition of GSH in to the medium during ICSI procedure can promote the normal embryo development following the ICSI.

Impact of multiple morphological anomalies of the flagella on the outcomes of intracytoplasmic sperm injection for males with infertility

Objective: To evalute the clinical outcomes of intracytoplasmic sperm injection(ICSI) for infertility male with severe asthenospermia induced by multiple morphological anomalies of the flagella (MMAF). Methods: The clinical data of 15 patient with MMAF were retrospectively analyzed, who underwent ICSI treatment using hyponotic swelling test the "live" sperm in the Department of Reproductive Medicine of Yantai Yuhuangding Hospital from January 2011 to December 2016. Another 30 obstructive azoospermia (OA)patients are matched strictly who also accepted ICSI in the same treatment time. The two groups were compared in the couples'age, the body mass index(BMI), the duration of infertility, the retrieved oocytes, the number of ICSI oocytes, and the rates of fertilization, cleavage, transferrable embryos, good embryos, embryos implanted, clinical pregncncy, early abortion, singleton and twins. Results: After 27 cycles of ICSI, all of the MMAF patients achieved clinical pregnancy, including 11 cases of live birth, 2 cases of spontaneous abortion, and 2 cases of pregnancy maintenance. There were no significant difference between MMAF and OA groups in the couples'age and BMI, or the numbers of retrieved oocytes and ICSI oocytes(P>0.05), but the differences in the infertility duration had statistical meaning(P<0.001). No statistical differences were observed among groups in ICSI fertility rate(92.0% vs 91.6%), clesvage rate(95.4% vs 96.5%), high-quality embryonic rate(56.5% vs 57.5%), good blastocyst rate(23/61 vs 35/94), embryo implantation rate(20/48 vs 35/75), early abortion rate(4/19 vs 8/36), clinical pregncncy rate(15/27 vs 28/50), singleton rate (10/13 vs 20/25)and twinning rate(3/13 vs 5/25)(P>0.05). Conclusions: MMAF may not affect ICSI treatment outcomes, but genetic defects can be transmitted through ICSI. The affected couples should be informed of the necessity of prenatal genetic diagnosis before embryo implantation and the inevitable vertical transmission of genetic problems to the offspring.

Production of inbred offspring by intracytoplasmic sperm injection of oocytes from juvenile female mice.

The aim of the present study was to determine whether the use of oocytes from juvenile female mice would improve the efficiency of intracytoplasmic sperm injection (ICSI). In the present study, 15 adult and 14 juvenile C57BL6/J female mice were superovulated, with 17.8 oocytes per mouse harvested from adults, significantly lower than the 40.2 harvested from juveniles (P<0.01). Sixty and 233 oocytes were harvested from C57BL/6J adult and juvenile mice respectively, activated in 10mM SrCl2+5μgmL-1 cytochalasin B for 5-6h and cultured in potassium simplex optimisation medium (KSOM) for 3.5 days, with no differences in morula and blastocyst rates between groups (91.7% vs 96.6%; P>0.05). Twelve hours after injection of human chorionic gonadotrophin, oocytes were harvested from C57BL/6J juvenile mice into KSOM, randomly divided into groups and activated with the same method mentioned above at 0, 2, 4 or 6h and then cultured in KSOM for 3.5 days. There was no significant difference in morula and blastocyst rates among the different groups (P>0.05). Oocytes from juvenile mice activated in 10mM SrCl2 for 2h were subjected to ICSI and the rates of pronuclear formation and Day 1 cleavage were significantly improved compared with the control group (P<0.01). ICSI combined with activation of oocytes from inbred mouse strains (C57BL/6J, C57BL/6N and 129Svev) successfully produced pups. The fertility of some these mice resulting from ICSI was tested, and the animals proved fertile. In conclusion, superovulated juvenile mice can yield more useable oocytes than adult mice, but additional activation is essential for full development of ICSI oocytes harvested from juvenile inbred mice.

Semen residual viral load and reproductive outcomes in HIV-infected men undergoing ICSI after extended semen preparation

The aim of this study was to evaluate the residual presence of the human immunodeficiency virus (HIV) following a triple gradient extended semen wash from ejaculates of serodiscordant couples, and analyse their reproductive outcomes after intracytoplasmic sperm injection (ICSI). For this purpose, a retrospective analysis of our database was performed in serodiscordant couples, with HIV-infected men and non-infected women, using fresh or frozen sperm with ICSI in oocytes from either the patients or donors from January 2006 to September 2013. Overall, the rate of positive HIV test after semen washing was 1.86%. The positive beta human chorionic gonadotrophin, clinical and ongoing pregnancy rates in patients with their own oocytes were 47.1%, 37.5% and 30.8%, respectively, and 58.6%, 50.8% and 39.1%, respectively, in oocyte donation cycles. To summarize, the described method of sperm washing based on triple gradient sperm selection coupled with extensive centrifugations is a highly reliable technique for HIV removal, as it provides lower than reported post-wash positive tests while maintaining high pregnancy rates in assisted reproduction cycles. Despite extensive personnel training and effectiveness of the washing protocol, post-wash HIV test on semen is recommended to identify residual positive samples.

A Case Study on Vacuolated Oocytes Intracytoplasmic Sperm Injection and its Outcome

ABSTRACTThis is a case study showing effects of oocyte morphological abnormalities in the form of homogenous vacuoles on intracytoplasmic sperm injection (ICSI) outcomes and its implications. Characteristic vacuoles of different size were identified in all oocytes obtained after egg collection in this patient. Eighteen oocytes were retrieved, 15 were metaphase-II and three were metaphase-I. All the oocytes were injected with sperm; out of 18 oocytes, 12 oocytes had embryo quality of grade A and three were grade B. Fifteen embryos exhibited maturation on 24 to 36 hours of incubation and showed two to four-celled stages, and after 48 to 60 hours of incubation, the embryos showed six to eight-celled stage. Grade A quality of three embryos was transferred nearly in an ongoing singleton pregnancy was confirmedHow to cite this articleDeene V, Mudaraddi TY, Gaur SS. A Case Study on Vacuolated Oocytes Intracytoplasmic Sperm Injection and its Outcome. Int J Infertil Fetal Med 2016;7(1): 23-26.