Conventional Intracytoplasmic Sperm Injection Research Articles

P-356 Clinical implications of first-trimester ultrasound dating in singleton pregnancies obtained through in vitro fertilization

Abstract Study question Is there a correspondence between ultrasound estimation of gestational age (GAUS) and its calculation based on embryo transfer date (GAIVF), in in-vitro fertilization (IVF) pregnancies? Summary answer GAUS and GAIVF are not overlapping, being the first mildly greater than the latter, maybe due to anticipated ovulation and fertilization or accelerated embryo development. What is known already In IVF pregnancies the precise date of conception is known. However, it is possible that some factors (such as a time delay between conception and implantation, an early in-vitro embryo development, and the maternal environment itself with its altered hormonal milieu) may influence the early stages of embryonic development. Data regarding first-trimester fetal development in IVF pregnancies are in fact conflicting and both underestimation and overestimation of the true GA were reported when traditional charts were used for ultrasound pregnancy dating. Study design, size, duration A retrospective study was performed, including all singleton IVF pregnancies who underwent the first-trimester ultrasound scan for the screening of aneuploidies between January 2014 and June 2019. For each pregnancy GA was determined using two alternative methods: one based on the date of embryo transfer (GAIVF), and one based on ultrasound measurement of crown-rump length (CRL) (GAUS). GA were compared to search for any discrepancy. The impact of pregnancy dating on obstetric outcome was evaluated. Participants/materials, setting, methods All women with singleton pregnancies conceived by conventional IVF or intracytoplasmic sperm injection (ICSI), who performed first-trimester ultrasound scan for the screening of aneuploidies at the Department of Obstetrics and Gynecology ‘L. Mangiagalli', Fondazione IRCCS Ca’ Granda Ospedale Maggiore Policlinico, were included. Exclusion criteria were as follows: multiple pregnancy (i.e., the evidence of more than one gestational sac at the first ultrasound scan), abnormal karyotype and/or congenital malformations detected either in pre- or postnatal period. Main results and the role of chance Overall, 249 women were selected: 111 pregnancies (45%) by conventional IVF, and 138 (55%) by ICSI. Among the included women, 127 (51%) pregnancies resulted from frozen-thawed embryo transfer. At first trimester ultrasound scan, the discrepancy in days between GAUS and GAIVF was ≥ 4 days in 32 cases (13%, 95%CI: 9-18%). It ranged within 2 and 3 days in 100 cases (40%, 95%CI: 34-47%). In 117 cases (47%, 95%CI: 41-53%) GAUS and GAIVF overlapped or showed a discrepancy of ± 1 day. Comparing GAUS and GAIVF at first trimester ultrasound scan, the median values of GA were 88 [86 - 91] days and 87 [85 - 90] days, respectively (p < 0.001). The median difference was 1 [0 - 2] days, with GAUS systematically higher than GAIVF. This discrepancy persisted when subgroups were analyzed comparing different IVF procedures (conventional IVF versus ICSI, cleavage versus blastocyst transfer, frozen versus fresh transfer). The overall duration of pregnancy differed, with median values of 274 [269 - 281] days in GAUS group and 273 [268 - 280] days in GAIVF group, (p > 0.001). No impact of the dating method on obstetric outcomes was observed, being no differences in the rate of preterm birth or abnormal fetal growth. Limitations, reasons for caution This is a retrospective study and thus exposed to the inaccuracies of this study design. Moreover, being a single-centre study, the sample size is relatively small. Finally, ultrasound evaluations were performed by multiple operators: although they are all certified, there is some inter-individual variability that may impact on the results. Wider implications of the findings Despite a known date of conception, it is difficult to assume that the oocyte retrieval date precisely resembles the date of natural ovulation and conception. IVF seems to anticipate fertilization. On these bases, it would be advisable to date IVF pregnancies using GAUS to realign them to natural pregnancies. Trial registration number Not Applicable

P-611 In vitro fertilization outcomes of the mature and immature oocyte in mild stimulation cycle

Abstract Study question Does in vitro maturation (IVM) of immature oocytes retrieved from small antral follicles during mild stimulation protocol have an impact on future IVF outcomes? Summary answer Although fertilization, cleavage, and blastocyst rates were lower in IVM oocytes, mature and immature groups are comparable for the implantation and cumulative clinical pregnancy rates What is known already Mild-stimulation protocols are patient-friendly, cost-effective, and have acceptable outcomes, especially among poor responders. IVM obtained attention among fertility specialists for riskless ovarian hyperstimulation syndrome along with acceptable clinical pregnancy and live birth. This study is aimed to investigate IVF outcomes of IVM during mild stimulation protocols. Study design, size, duration This prospective cohort study has enrolled 949 IVF/ICSI cycles carried out with clomiphene-based mild stimulation protocol during 2018-2020 at RMC national IVF center in Mongolia. A total of 2346 oocytes were selected and divided into mature (metaphase II-MII, n = 1272) and immature (metaphase I–MI, and prophase I-GV, n = 1074) groups. Participants/materials, setting, methods 50 mg of clomiphene citrate was provided from day 3 of menstruation. The oocyte retrieval was usually performed after 30-36 hours of triggering the GnRH agonist while using a 21-G needle (Kitazato Corporation). MII oocytes were either freshly fertilized conventional insemination or intracytoplasmic sperm injection approximately after 3 and 5 hours of oocyte retrieval. MI and GV oocytes had reached the metaphase II stage in vitro after 8 to 40h of culture. Main results and the role of chance Fertilization rate was 76.3% (n = 971/1272) in mature group and 62.4% (n = 312/500) in immature group (Chi square=34.8, p < 0.01). Cleavage rate was 98.4% (n = 955/971) vs 95.5%(n = 298/312) (Chi square=8.33, p < 0.01), and blast formation rate was 62.3% (n = 595/955) vs 38.7% (n = 115/298) (Chi square=51.3, p < 0.01) which is significantly lower compared to the mature group. A total of 296 fresh or frozen single embryo transfer procedures were performed at blastocysts. Implantation rate was 58.4% (n = 149/255) in mature group and 58.5% (n = 24/41) in immature group, cumulative clinical pregnancy rate was 33.3% (n = 85/255) vs 41.5% (n = 17/41), live birth rate was 28.2% (n = 72/255) vs 41.5% (n = 17/41), which has no statistical difference between the groups Limitations, reasons for caution The sample size of embryo transfer in the immature group was small in the study, and randomized clinical trials are further required to clarify the difference between outcomes of mature and immature oocytes in mild stimulation Wider implications of the findings Data from the study has confirmed favorable clinical outcomes of in vitro maturation of the immature oocytes during mild stimulation. IVM seems to remain as an alternative option and is recommended for all IVF laboratories rather than neglecting it. Trial registration number Not applicable

Should Intra Cytoplasmic Sperm Injection (ICSI) be the primary insemination method in women undergoing IVF cycles with donor sperm?

To compare efficacy of Intra Cytoplasmic Sperm Injection (ICSI) with conventional in vitro fertilization (IVF) on treatment outcome in women undergoing in vitro fertilization with donor sperm. We examined retrospectively the outcome data from 203 patients undergoing fresh cycles of conventional IVF (cIVF) or ICSI and an additional 77 frozen-thawed embryo transfer (FET) cycles during 2003-2014, all using donor sperm. Fertilization, cleavage, pregnancy and live birth rates and number of high-quality embryos were compared between cIVF and ICSI. Altogether 185 women underwent 479 transfer cycles of fresh embryos (237 cIVF vs. 224 ICSI and 18 "rescue ICSI" cycles). In addition, 77 FET cycles were compared (24 cIVF vs. 53 ICSI cycles). No differences were found between cIVF and ICSI in fertilization, cleavage, pregnancy and live birth rates (92.6% vs 92.2%, 73.4% vs 72.4%, 25.3% vs 27.2% and 13.1% vs 14.7%, respectively). Pregnancy and life birth rates remained similar even when FET cycles were included (25.8% vs 26.2% and 13.1% vs 13.7%, respectively). The use of ICSI was associated with lower rates of high-quality embryos (52.7% vs. 63.3%, P < 0.0001). A multivariate logistic regression analysis found that patients' age, number of transferred embryos and smoking were independently associated with the chance to conceive. Patient age correlated inversely with fertilization rate (r =-0.13, P < 0.006).Non-smokers were more likely to become pregnant (OR = 2.23, P < 0.012). Our results show that ICSI does not bypass the age-related decrease in oocyte quality in patients using donor sperm for IVF. Use of ICSI was associated with lower rates of high-quality embryos. The findings imply that ICSI should not be the primary method of insemination in patients undergoing IVF with donor sperm.

Verbascoside addition during IVM improves equine blastocyst redox status after conventional and mercury-free piezo-assisted ICSI

Oxidative stress is a major problem in in vitro handling of gametes and embryos. In this study, the effects of Verbascoside (VB), a bioactive polyphenol with known antioxidant activity, added during In Vitro Maturation (IVM) of equine oocytes on blastocyst quality were analyzed after conventional or mercury-free piezo-assisted intracytoplasmic sperm injection (ICSI). Cumulus-oocyte complexes recovered from the ovaries of slaughtered mares underwent overnight holding and IVM in the presence of 0 (control) or 100nM VB. Fresh sperm of fertile stallions was subjected to swim-up procedure and used to microinject matured oocytes by conventional (Dell'Aquila et al. Biology of Reproduction 2003;68:2065-72) or piezo-assisted ICSI (Martino et al. Reproduction Fertility and Development 2019;31:1862-73). Embryos were in vitro cultured in DMEM/F-12 with 10% FCS for 9 days under 5%O 2 , 5%CO 2 , 90%N 2 atmosphere. Blastocyst quality was assessed for bioenergetic/oxidative status by confocal laser scanning microscopy after labeling mitochondria (mt) with MitoTracker Orange CMTM Ros and reactive oxygen species (ROS) with 2’,7’-dichlorodihydrofluorescein diacetate (Marzano et al. Molecular Reproduction and Development 2019;86:1388-1404). Then, blastocysts were stained with Hoechst 33258 for total cell numberevaluation and confirmed when showing more than 64 nuclei with organization of an outer trophoblast layer. Proportions of blastocyst rates after ICSI were compared between VB-exposed group and controls by the Chi-square-test, whereas blastocyst total cell number, ROS levels and mt/ROS colocalization were compared by the Student's T-test. Values with P<0.05 were considered significantly different. VB addition during IVM did not influence oocyte maturation rate (31/55, 56% vs 38/76, 50% for VB-exposed and CTRL, respectively). After both conventional and piezo-assisted ICSI, VB had no effects on blastocyst formation rate (4/21, 19% vs 3/24, 12.5%; P>0.05 and 3/10, 30% vs 2/14, 14%; P>0.05) and total cell number (187.3±104.7 vs 143.3±63.2, P>0.05; and 247.3±109.8 vs 465±17.0, P>0.05). Instead, after both microinjection procedures, VB significantly reduced ROS levels (29.7±5.1 vs 36.1±4.2, P<0.001; and 22.8±8.9 vs 30.1±4.4, P<0.01) and increased mt/ROS colocalization (0.63±0.04 vs 0.61±0.03, P<0.001; 0.79±0.06 vs 0.74±0.12, P<0.001). These findings indicate long-term beneficial antioxidant effects of in vitro exposure to nanomolar VB concentrations during oocyte IVM on equine blastocyst quality. Acknowledgments: funded by the Italian Ministry of Health: Project GR-2011-02351396.

Developmental Competence of equine MI oocytes following in vitro maturation culture

With an increasing demand for the in vitro production (IVP) of equine embryos over the past 10 years, the value of each oocyte recovered has also increased. Intracytoplasmic sperm injection (ICSI) is performed to fertilize the oocytes reaching MII stage of nuclear maturation, which is determined by microscopic evidence of an extruded polar body (PB). Cytoplasmic maturation, during which replication of organelles, proteins and RNA's necessary for postfertilization development occurs, is more difficult to evaluate. However, equine oocytes contain a large amount of cytoplasmic lipids and the migration and polarization of lipid granules can be microscopically visualized in mature oocytes. Furthermore, the distribution of lipids may reflect a level of cytoplasmic capacitation and developmental competence, although the cleavage rate of equine oocytes fertilized following maturation culture does not appear to be significantly different based on lipid polarization (Ambruosi et al. Therio 2009; 71:1093-1104). The present study evaluated the migration and polarization of cytoplasmic lipids (granularity) following in vitro maturation to determine if blastocyst development can occur in the absence of PB extrusion. Cumulus-oocyte-complexes (COC; n=411) obtained from ovarian follicles by transvaginal aspiration were stored in a holding medium overnight at 20°C before undergoing incubation at 38.5°C in 50ul drops of equilibrated maturation media as described by Foss. et al (EVJ Supp 2013;45:39-43). After 28.5 hours, oocytes were denuded followingexposure to 80IU hyaluronidase and evaluated for the presence of an extruded PB as well as lipid reorganization. The cytoplasm of MI oocytes that did not exhibit PB extrusion was graded according to the percentage with a dark granular appearance. All oocytes were fertilized by conventional ICSI with thawed spermatozoa from ICSI-fertile stallions. Fertilized oocytes were cultured as described by Foss et al (EVJ Suppl 2013;45:39-43). When examined under an inverted 1000X light microscope, 66% (N=273/411) of the denuded oocytes exhibited an intact membrane with an extruded polar body and were considered mature (MII), while 26% (N=106/411) of the oocytes without a visible polar body were classified as immature (MI). Of the MI oocytes injected, 23% (N=24/106) were deemed to have attained some level of cytoplasmic capacitation (Cc=40-70% granularity). A cleavage rate of 25.5% was recorded for all injected MI oocytes regardless of Cc status. Of the 24 MI oocytes with some Cc, nine (37%) cleaved and continued to develop, and seven of those developed to the blastocyst stage. Of the 82 MI oocytes that did not demonstrate Cc, 18 (22%) cleaved but no blastocysts (0%) were obtained. This study found a blastocyst developmental potential of 6.6% per total MI's injected and 29% per MI oocytes with at least a 40-70% Cc. Therefore, injecting oocytes that do not show nuclear maturation, yet have reached a level of cytoplasmic capacitation, may still result in blastocyst development.

Cumulative live birth rates after IVF/ICSI cycles with sperm prepared by density gradient centrifugation vs. swim-up: a retrospective study using a propensity score-matching analysis

BackgroundDensity gradient centrifugation (DGC) and swim-up (SU) are the two most widely used sperm preparation methods for in vitro fertilization (IVF) and intracytoplasmic sperm injection (ICSI). However, existing comparisons of IVF/ICSI outcomes following these sperm preparation methods are insufficient and controversial.MethodsThis retrospective study included all first autologous IVF and ICSI cycles performed between March 1, 2016, and December 31, 2020 in a single university-based center. A total of 3608 cycles were matched between DGC and SU using propensity score (PS) matching for potential confounding factors at a ratio of 1:1. The primary outcome was the cumulative live birth rate (cLBR) per aspiration.ResultsPS matching provided 719 cycles after DGC and 719 cycles after SU. After adjusting for confounders, the recovery rate, progressive motility rate after sperm preparation, fertilization rate, good-quality embryo rate, and blastocyst formation rate were similar between the DGC and SU groups. The cLBR (odds ratio [OR] = 1.143, 95% confidence interval [CI]: 0.893–1.461) and LBR per transfer (OR = 1.082, 95% CI: 0.896–1.307) were also not significantly different between the groups. Furthermore, no significant differences were found in all of the laboratory and clinical outcomes following conventional IVF or ICSI cycles between the two groups. However, a significantly higher fertilization rate (β = 0.074, 95% CI: 0.008–0.140) was observed when using poor-quality sperm in the DGC group than in the SU group.ConclusionsSperm preparation using DGC and SU separately resulted in similar IVF/ICSI outcomes. Further studies are warranted to compare the effects of these methods on IVF/ICSI outcomes when using sperm from subgroups of different quality.

Is there any effect on imprinted genes H19, PEG3, and SNRPN during AOA?

Assisted oocyte activation (AOA) has been proposed as an effective technique to overcome the problem of impaired fertilization after intracytoplasmic sperm injection (ICSI) but the safety of AOA remains a concern. We aimed to investigate if AOA induces imprinting effects on embryos. We used 13 cleavage embryos, nine blastocysts, and eight placentas from 15 patients. The subjects were divided into six groups by tissue type and with or without AOA. The methylation levels of imprinted genes (H19, paternally expressed gene [PEG3] and small nuclear ribonucleoprotein polypeptide N [SNRPN]) were tested by pyrosequencing. We observed different methylation levels among cleavage embryos. The variability was much more remarkable between cleavage embryos than blastocysts and placenta tissues. The methylation levels were especially higher in SNRPN and lower in the H19 gene in AOA embryos than those without AOA. No significant difference was found either among blastocysts or among placenta tissues regardless of AOA. The methylation levels of the three genes in blastocysts were very similar to those in the placenta. Compared to conventional ICSI, AOA changed imprinting methylation rates at H19 and SNRPN in cleavage embryos but not in the blastocyst stage and placenta. We recommend that blastocyst transfer should be considered for patients undergoing AOA during in vitro fertilization.

The Effect of Uterine Adenomyosis on IVF Outcomes: a Systematic Review and Meta-analysis.

To investigate the impact of uterine adenomyosis on in vitro fertilization (IVF)/intracytoplasmic sperm injection (ICSI) clinical outcomes and also to evaluate the impact of gonadotropin-releasing hormone agonist (GnRHa) and surgical treatments. Infertile women with adenomyosis undergoing conventional IVF or ICSI were compared with women without adenomyosis. Fertility outcomes were evaluated in two patients with adenomyosis untreated and treated surgically or medically therapy. The review protocol was registered in PROSPERO (CRD42020214586). We consider the live birth rate (LBR) as the primary outcome. The analysis showed that women with adenomyosis had lower LBR (OR 0.59, 95% CI 0.37-0.92, p = 0.02), clinical pregnancy rate (OR 0.66, 95% CI 0.48-0.90), and ongoing pregnancy rate (OR 0.43, 95% CI 0.21-0.88) compared to those without adenomyosis, and miscarriage rate was higher in women with adenomyosis (OR 2.11, 95% CI 1.33-3.33). Surgical treatment increases natural conception in women with adenomyosis although the paucity of data (only two studies), and conversely, treatment with GnRHa does not increase the IVF outcomes. Women with adenomyosis have decreased IVF clinical outcomes. Pretreatment with the use of long-term GnRHa could not be beneficial, even though only three studies were included in the meta-analysis. The major part of the studies has not divided focal and diffuse adenomyosis, and this represents a relevant source of bias: studies conducted with standardized diagnostic criteria for adenomyosis are still needed to determine if the different clinical presentations of such condition could compromise IVF outcomes.

DNA Methylation Differences Between Zona Pellucida-Bound and Manually Selected Spermatozoa Are Associated With Autism Susceptibility.

Children conceived through intracytoplasmic sperm injection (ICSI) have been reported to have a higher risk of many abnormalities and disorders, including autism and intellectual disability, which may be due to bypassing of the natural sperm selection process during ICSI. Zona pellucida (ZP)-bound spermatozoa (ZPBS) have normal morphology and nuclear DNA. Using these spermatozoa for ICSI results in better outcomes compared with conventional ICSI. However, differences besides morphology that exist between sperm selected by ZP and by an embryologist and whether these differences affect the risk of autism in offspring after ICSI are unclear. To explore these questions, we compared genome-wide DNA methylation profiles between ZPBS and manually selected spermatozoa (MSS)using single-cell bisulfite sequencing. Global DNA methylation levels were significantly lower in ZPBS than in MSS. Using gene ontology (GO) analysis, genes overlapping differentially methylated regions (DMRs) were enriched in biological processes involving neurogenesis. Furthermore, we found that 47.8% of autism candidate genes were associated with DMRs, compared with 37.1% of matched background genes (P<0.001). This was mainly because of the high proportion of autism candidate genes with bivalent chromatin structure. In conclusion, bivalent chromatin structure results in large differences in the methylation of autism genes between MSS and ZPBS. ICSI using MSS, which increases the risk of methylation mutations compared with ZPBS, may lead to a higher risk of autism in offspring.

An Open Prospective Study on Whether Intracytoplasmic Morphologically Selected Sperm Injection (IMSI) Offers a Better Outcome Than Conventional Intracytoplasmic Sperm Injection (ICSI).

ObjectiveTo differentiate the in vitro fertilization (IVF) outcomes between the two procedures, intracytoplasmic morphologically selected sperm injection (IMSI) and intracytoplasmic sperm injection (ICSI) in terms of relation to chemical pregnancy percentage, clinical pregnancy, live birth, miscarriage, and fertilization rates, respectively.Patients and methodsThis Open Prospective clinical trial was conducted during the period between Jan 2016 and Dec 2017 at one IVF unit. A total of 446 ICSI cycles and 79 IMSI cycles were conducted. Females were divided into four subgroups according to age.ResultsThe study involved 525 couples (446 first trial ICSI cycles) and (79 first trial IMSI cycles). ICSI was statistically better than the IMSI in relation to the chemical pregnancy, clinical pregnancy (CPR), live birth (LBR), and fertilization rates, respectively (p < 0.05). However, there were no statistically significant differences between the ICSI and IMSI in relation to the miscarriage rate. There were statistically significant differences favoring ICSI in all subgroups except 35-37, in relation to chemical pregnancy; and in the 38-40 and >40 subgroups in relation to CPR. There were no statistically significant differences in these subgroups regarding the live birth, miscarriage, or fertilization rates.ConclusionsThis study showed that IMSI is not superior to conventional ICSI at the first attempt. Based on the findings in this study, we would not advise couples to choose IMSI at their first treatment attempt.

P–225 The effect of rapid and delayed insemination on reproductive outcome in conventional insemination and intracytoplasmic sperm injection invitro-fertilization cycles

Abstract Study question Does rapid or delayed insemination after egg retrieval affect fertilization, blastocyst development and live birth rates in CI and ICSI cycles? Summary answer When performing CI or ICSI &amp;lt;1.5h and &amp;gt;6.5h after retrieval, detrimental effects are moderate on fertilization but do not impact blastocyst usage and birth rates. What is known already Several studies have shown that CIor ICSI performed between 3 to 5 h after oocyte retrieval has improved laboratory outcomes. However, some studies indicate that insemination of oocytes, by either CI or ICSI, within 2 hours or more than 8 hours after oocyte retrieval has a detrimental effect on the reproductive outcome. With some ART centres experiencing an increase in workload, respecting these exact time intervals is frequently challenging. Study design, size, duration A single-center retrospective cohort analysis was performed on 6559 patients (9575 retrievals and insemination cycles) between January 1st2017 to July 31st2019. The main outcome measures were live-birth rates. Secondary outcomes included analysis of fertilization per all oocytes retrieved, blastocyst utilization, clinical pregnancy, and miscarriage rates. All analyses used time of insemination categorized in both CI and ICSI cycles. Fertilization rates across categories was analyzed by ANOVA and pregnancy outcomes compared using Chi-square tests. Participants/materials, setting, methods As part of laboratory protocol, oocyte retrieval was performed 36 h post-trigger. Cycles involving injection with testicular/epidydimal sperm, donor or frozen oocytes were excluded. The time interval between oocyte retrieval and insemination was analyzed in eight categories: 0 (0- &amp;lt;0.5h), 1 (0.5-&amp;lt;1.5h), 2 (1.5-&amp;lt;2.5h), 3 (2.5-&amp;lt;3.5h), 4 (3.5-&amp;lt;4.5), 5 (4.5-&amp;lt;5.5), 6 (5.5-&amp;lt;6.5) and 7 (6.5-&amp;lt;8h). The number of retrievals in each group (0–7) was 586, 1594, 1644, 1796, 1836, 1351, 641 and 127 respectively. Main results and the role of chance This study had a mean patient age of 36.0 years and mean of 12.2 oocytes per retrieval in each category. There were 4,955 CI and 4,620 ICSI retrievals. The smallest groups were time category 7 and 0 for CI and ICSI respectively. The results showed that the mean fertilization rate per egg retrieved for CI ranged from 54.1 to 64.9% with a significant difference between time category 0 and 5 (p &amp;lt; 0.001) and category 1 and 5 (p &amp;lt; 0.0.001). Mean fertilization rate for ICSI per egg retrieved ranged from 52.8 to 67.3% with no significant difference between time categories compared to category 5. Blastocyst utilization rate for CI and ICSI were not significantly different for all time categories. In the CI and ICSI groups there were 6,540 and 6,178 total fresh and frozen transfers. The miscarriage and clinical pregnancy rate in CI and ICSI were not significantly different across time categories. The overall mean live birth rate for CI was 32.4% (range: 23.1 to 35.5%). Live-birth rates differed significantly (p = 0.04) in CI with time categories 0 and 7 the lowest. In the ICSI group, the overall mean live birth rate was 30.8% (range: 29.1 to 35.7%),with no significant differences between time categories. Limitations, reasons for caution As this is a retrospective study, the influence of uncontrolled variables cannot be excluded. The group spread was uneven with the early and late time categories having the lowest number of representative retrievals and this could have affected the results obtained. Wider implications of the findings: Our results indicate that both CI and ICSI are optimal when performed between 1.5–6.5 hours after oocyte retrieval. Further prospective studies on reproductive outcomes related to time of insemination are warranted. This data indicates a minimal detrimental effect when it is untenable to follow strict insemination time intervals. Trial registration number 2015P000122

Sperm Selection Technology in ART.

Intracytoplasmic sperm injection (ICSI) is a commonly used in vitro fertilization technique. Selection of sperm for injection is currently done using subjective assessment of morphology, which may not accurately identify the best-quality sperm. Hyaluronic acid receptors on sperm plasma membranes are a marker of maturity, and sperms which are capable of binding immobilized hyaluronic acid in vitro are of higher quality. This can be used as an advanced sperm selection technique to choose sperm for ICSI, termed physiological ICSI (PICSI). Several studies reported improved fertility treatment outcomes when using PICSI compared with conventional ICSI; however, the majority of studies are underpowered. Recently, a large, multicenter, randomized controlled trial, known as the Hyaluronic Acid Binding Sperm Selection (HABSelect) trial, found a significant reduction in miscarriage rates with PICSI, but no significant effect on live birth rate. There are still many avenues through which PICSI may provide an advantage, subject to confirmation by future research, such as improved long-term health of offspring. Other advanced sperm selection techniques include intracytoplasmic morphologically selected sperm injection, magnetic-activated cell sorting, and Zeta potential sperm selection; however, the most recent Cochrane review concluded that there is currently insufficient evidence to ascertain whether these techniques improve clinical outcomes, such as live birth rates.

Obstetrical and Neonatal Outcomes of 3,028 Singletons Born After Advanced ART Techniques: Ejaculated Sperm Intracytoplasmic Sperm Injection, Artificial Oocyte Activation, in Vitro Maturation and Testicular Sperm Extraction

Background: To evaluate obstetrical and neonatal outcomes of singletons conceived after advanced assisted reproductive technology (ART) techniques: conventional IVF pregnancies (C-IVF), ejaculated sperm intracytoplasmic sperm injection (ICSI), assisted oocyte activation (AOA), in vitro maturation (IVM), and testicular sperm extraction (TESE). Methods: The subjects were 3,028 singletons who were born after fresh or frozen embryo transfer. The subjects were separated into five groups: C-IVF ([Formula: see text]), ICSI ([Formula: see text]), AOA ([Formula: see text]), IVM ([Formula: see text]), and TESE ([Formula: see text]). We evaluated obstetrical and neonatal outcomes calculating the adjusted odds ratio (AOR) using multivariable logistic regression analyses for fresh and frozen embryos and for cleavage and blastocyst transfer. The C-IVF group was used as a background control for the ICSI group. Moreover, the TESE, AOA, and IVM groups were compared to the ICSI group to evaluate the effects of the ICSI procedure itself. Results: The incidence of perinatal complications was significantly lower in the ICSI-fresh group ([Formula: see text], 95% CI: 0.10–0.83, [Formula: see text]). Regarding sex ratio, the IVM was significantly associated with sex ratio imbalance toward female in both fresh and frozen groups ([Formula: see text], 95% CI: 0.10–0.96, [Formula: see text], 95% CI: 0.07–0.98, [Formula: see text]). On the other hand, there were no significant differences in preterm birth rate, low birth weight rate and congenital abnormalities rate between conventional IVF, ICSI, and the other groups. Conclusions: There were no negative effects on obstetrical and neonatal outcomes between conventional IVF and ICSI. Although this was a limited sample size study, advanced ART technologies such as AOA, IVM, and TESE also seem to have a low risk of adverse impact on obstetrical and neonatal outcomes but may have a slight impact on sex ratio.

Conventional ICSI vs. physiological selection of spermatozoa for ICSI (picsi) in sibling oocytes.

Physiological selection of spermatozoa for ICSI (PICSI) is a sperm selection method based on sperm binding to hyaluronic acid. Previous studies on the effect of hyaluronic acid binding assays on fertilization and embryo quality have shown inconsistent results. Previous sibling oocyte studies have not found a significant improvement in fertilization or embryo development with hyaluronic acid binding assays. To compare fertilization and embryo development between standard intracytoplasmic sperm injection (ICSI) and PICSI in sibling oocytes. This is a retrospective analysis of all in vitro fertilization (IVF) cycles between January 2017 and April 2020 in which sibling oocytes were randomly fertilized by both ICSI and PICSI. Fertilization rate and the rate of embryos eligible for transfer were compared. Forty-five IVF cycles, in which 257 oocytes were fertilized with PICSI and 294 with standard ICSI, were compared. Most of the patients included in the study had previous failures of fertilization, poor embryonic development, implantation failure, or miscarriage. All but two of the patients had at least one previous unsuccessful IVF cycle. Both fertilization rates (71% vs. 83%) and transfer eligible embryo rates (38% vs. 51%) were significantly higher in PICSI fertilized oocytes (p=0.008 and p=0.01 respectively). Our study is the largest sibling oocyte study comparing ICSI and PICSI, and the first to find a significant improvement in fertilization and embryo quality with PICSI using sibling oocytes. The fact our cohort included almost exclusively couples with previous unsuccessful IVF cycles might suggest that PICSI should be used in selected cases. PICSI improves fertilization rates and transfer eligible embryo rates in sibling oocytes in a selected study group.

Conventional ICSI improves the euploid embryo rate in male reciprocal translocation carriers.

To evaluate whether the morphologically normal spermatozoa selected for intracytoplasmic sperm injection (ICSI) under microscope had a higher rate of normal/balanced chromosome contents than that in the whole unselected sperm from reciprocal translocation carriers. Five hundred unselected spermatozoa from each of 40 male translocation carriers were performed with fluorescence in situ hybridization (FISH), to determine the rates of gametes with different meiotic contents of translocated chromosomes. Meanwhile, 3030 biopsied blastocysts from 239 male and 293 female reciprocal translocation carriers were detected with the microarray technique to analyze the rates of embryos with different translocated chromosome contents. The D3 embryo rate, blastocyst formation rate, and euploid rate of blastocysts were remarkably higher in male carriers than those in female (p = 0.001, p = 0.004, and p = 0.035, respectively). In addition, the percentages of alternate products, which contained normal/balanced chromosome contents, in embryos from male carriers were markedly higher than those in sperm FISH (p = 2.48 × 10-5 and p = 2.88 × 10-10), while the percentages of adjacent-2 and 3:1 products were lower than those in sperm FISH (p = 0.003 and p = 5.28 × 10-44). Moreover, consistent results were obtained when comparing the rates of products in embryos between male and female carriers. Specifically, the incidence of alternate products in male carriers was higher than those in female carriers (p = 0.022). However, no similar differences were seen between sperm and embryos of female carriers. ICSI facilitates the selection of spermatozoa with normal/balanced chromosome contents and improves the D3 embryo rate, blastocyst formation rate, and the euploid embryo rate in male carriers.

Sperm morphology: What implications on the assisted reproductive outcomes?

To evaluate the impact of sperm morphology (SM) on laboratory and pregnancy outcomes in conventional intracytoplasmic sperm injection (c-ICSI) cycles, using the egg donation model to minimize female confounding variables. We retrospectively collected data of oocyte donation cycles from October 2016 to February 2020. Median seminal parameters, total (1-2-3PN) fertilization rate (FR), 2PN FR, cleavage rate (CR), implantation rate (IR), pregnancy rate (PR), miscarriage rate (MR), and live birth rate (LBR) were collected. The study population was divided into three groups: Group 1 with SM<4%, Group 2 with SM between 4% and 6%, and Group 3 with SM>6%. Of 741 fresh ICSI cycles and 4507 warmed oocytes were included. Male age was 46.0 (31.0-72.0) years, and recipients' age was 44.0 (29.0-54.0) years. Normal SM was 5.0% (1.0%-15.0%). Male age was negatively correlated with normal SM (P=.002; Rho -0.113). Oocyte survival rate was 83.3% (16.7%-100.0%). Total FR was 75.0% (11.1%-100.0%), 2PN FR was 66.7% (11.1%-100.0%) %, and CR was 100% (0.0%-100%). Comparing samples with SM>6% and those with SM<4%, 2PN FR was significantly higher in the first group (P=.04). No significant associations were found among groups in terms of CR. IR was 27.7%, resulting significantly higher when normal SM was>6% (P<.01). Clinical PR was 36.0%, MR was 23.9%, and LBR was 25.9%. PR and LBR were significantly higher in samples with normal SM>6%, compared to other groups (P=.02 and P<.01, respectively). Although c-ICSI technique allows the embryologist to select the best quality spermatozoa, male factor plays a key role in achieving successful assisted reproductive outcomes. Normal SM has been shown to have implications not only for laboratory outcomes, in terms of fertilization, but also for clinical findings, as regards implantation, pregnancy, and live birth.

Perinatal and neonatal outcomes of pregnancies after early rescue intracytoplasmic sperm injection in women with primary infertility compared with conventional intracytoplasmic sperm injection: a retrospective 6-year study

BackgroundEarly rescue intracytoplasmic sperm injection (ICSI) has been used in clinic as appropriate currently. While the outcomes of children born after this method were not well assessed. The purpose of this study was to evaluate the effect of early rescue ICSI on women with primary infertility.MethodsFresh embryo transfer cycles after rescue (n = 214) and conventional (n = 546) ICSI were retrospectively evaluated from women with primary infertility who underwent their first assisted reproductive technology cycles at our center in 2012–2017. The conventional ICSI group was subdivided into ICSI-1 (semen suitable for in vitro fertilization, IVF) and ICSI-2 (poor semen quality) to minimize bias from differences in semen quality. Pregnancy, delivery and neonatal outcomes were compared between groups.ResultsThere was a higher rate of polyspermy and a lower rate of top-quality embryos (TQE) on day 3 for oocytes subject to rescue ICSI compared with conventional ICSI. This reduced the total number of TQE and the number of TQE transferred in the rescue ICSI group. There was no significant difference between groups in clinical pregnancy, ongoing pregnancy, early miscarriage and live birth. For pregnant women, gestational age, route of delivery, risk of preterm birth and gestational diabetes mellitus were also comparable. Neonatal outcomes including sex ratio, birth weight, neonatal intensive care unit admission and birth defects were also similar after rescue and conventional ICSI. Moreover, no differences were observed with the different ICSI subgroups.ConclusionsFor women with primary infertility who have a high risk of IVF fertilization failure (FF), rescue ICSI provides a safe and efficient alternative to minimize FF after initial IVF, but results in fewer TQE on day 3.

Comparison of aneuploidy rates between conventional in vitro fertilization and intracytoplasmic sperm injection in in vitro fertilization–intracytoplasmic sperm injection split insemination cycles

ObjectiveTo evaluate the influence of insemination methods on outcomes of preimplantation genetic testing for aneuploidy (PGT-A) by assessing PGT-A results in embryos that derived from conventional in vitro fertilization (IVF) versus intracytoplasmic sperm injection (ICSI) in sibling oocytes.DesignRetrospective cohort study.SettingSingle academic IVF center.Patient(s)A total of 118 couples who underwent 131 split insemination cycles from July 2016–July 2019.Intervention(s)In all cycles, sibling oocytes were allocated randomly to conventional IVF or ICSI prior to stripping. Preimplantation genetic testing for aneuploidy was performed via trophectoderm biopsy and next-generation sequencing with 24-chromosome screening.Main Outcome Measure(s)Rates of euploid, aneuploid, and mosaic embryos per biopsy.Result(s)A total of 2,129 oocytes were randomized to conventional IVF (n = 1,026) and ICSI (n = 1,103). No difference was observed in the aneuploidy rates (50.3% vs. 45.2%) and percentages of mosaic embryos (1.7% vs. 2.4%) per biopsy between conventional IVF and ICSI sibling oocytes. Percentages of different aneuploidy types and aneuploidies that involved sex chromosome abnormalities (6.2% vs. 7.2%) were similar between the two groups. In the end, the overall chance to have an euploid embryo per allocated oocyte in the two groups was similar (13.2% vs. 15.5%).Conclusion(s)Blastocysts created with conventional IVF and ICSI using sibling oocytes had similar rates of aneuploidy and mosaicism as examined using 24-chromosome screening. It is unlikely that conventional IVF caused significant contamination during PGT-A. We recommend conventional IVF as the preferred insemination method in PGT-A cycles, and ICSI should be indicated only in cases of male-factor infertility.

Effects of the sperm DNA fragmentation index on the clinical and neonatal outcomes of intracytoplasmic sperm injection cycles

BackgroundMost studies have mainly focused on the effects of the sperm DNA fragmentation index (DFI) on fertilization, embryonic developmental potential and aneuploidy, pregnancy and abortion rates after in vitro fertilization (IVF)/intracytoplasmic sperm injection (ICSI) and have remained controversial. However, few studies have reported the effects of sperm DFI on neonatal outcomes, including stillbirths, neonatal deaths, sex, gestational age, prematurity, birthweight, low birth weight (LBW) and birth defects in newborns. Our objective was to evaluate the effects of sperm DFI on the clinical and neonatal outcomes of ICSI cycles.MethodsThis retrospective study analysed a total of 2067 oocyte retrieval, 1139 transfer and 713 delivery cycles from conventional ICSI cycles, including 301, 469, and 214 live-born infants in groups segregated according to sperm DFI as the < 15%, 15–30% and > 30% groups, respectively. The clinical and neonatal outcomes were compared among the three groups.ResultsSperm DFI did not significantly affect the rates of fertilization, clinical pregnancy, miscarriage or ongoing pregnancy. Sperm DFI did not increase the risk of stillbirths or neonatal deaths. The rates of stillbirths and neonatal deaths were not significantly different among the three groups. The sex, gestational age, prematurity, birthweight and LBW of newborns in the three groups were not significantly affected by sperm DFI. Moreover, sperm DFI did not increase the number of birth defects in children.ConclusionsSperm DFI did not affect the clinical or neonatal outcomes of ICSI cycles.

Evaluation of the effect of piezo-intracytoplasmic sperm injection on the laboratory, clinical, and neonatal outcomes.

PurposeLimited research has been published on the effect of piezo‐assisted intracytoplasmic sperm injection (P‐ICSI). We evaluated the effect of P‐ICSI on the laboratory, clinical, and neonatal outcomes.MethodsThis retrospective study was based on the data collected between April 2011 and October 2016. Total 1348 mature oocytes from 145 patients were analyzed. Laboratory, clinical, and neonatal outcomes of those given conventional intracytoplasmic sperm injection (C‐ICSI) and those administered P‐ICSI were examined.ResultsP‐ICSI showed significantly more favorable results, with a survival rate of 97.0% (C‐ICSI: 94.1%, P < .010) and a fertilization rate of 83.5% (C‐ICSI: 70.6%, P < .001). There were no differences in the blastocyst development rate, implantation rate, miscarriage rate, live birth rate, gestational age, birth weight, proportion of male neonates, cesarean section rate, and congenital abnormalities between the two patient groups.ConclusionsOur comparison of P‐ICSI with C‐ICSI showed that P‐ICSI significantly improved the survival and fertilization.