Intracytoplasmic Sperm Injection Embryos Research Articles

Performance of preimplantation genetic testing for aneuploidy for patients with unexplained recurrent pregnancy loss and repeated implantation failure

ObjectiveThe primary objective was to investigate whether the utilization of next-generation sequencing (NGS) for preimplantation genetic testing for aneuploidy (PGT-A) could enhance the reproductive outcomes in patients with unexplained recurrent pregnancy loss (uRPL) or unexplained repeated implantation failure (uRIF) undergoing intracytoplasmic sperm injection (ICSI) cycles. Materials and MethodsWe studied the reproductive outcomes of uRPL or uRIF sufferers in Chengdu women and children’s central hospital from July 2020 to Jan 2024 retrospectively. These patients were categorized into two groups based on whether they underwent PGT-A or not. As the patients in the PGT-A group all had ICSI and frozen-thawed embryo transfer (FET), only patients who underwent ICSI and FET were included in the non-PGT-A group for comparison. Demographic characteristics and reproductive outcomes were compared in uRPL or uRIF sufferers. ResultsFor uRPL group, a significant increased ongoing pregnancy rate (63.6% vs 26.1%, p=0.002) and reduced pregnancy loss rate (18.4% vs 73.3%, p<0.001) were found in the PGT-A group in comparison with those in the non-PGT-A group. For uRIF group, no significant difference was noted in the HCG-positive rate, ongoing pregnancy rate, or pregnancy loss rate between the two groups. It is noteworthy that the maternal age in the PGT-A group was significantly higher than that in the non-PGT-A group (p=0.048). ConclusionsNGS-based PGT-A effectively optimized the reproductive outcomes in uRPL sufferers. Although its benefits in uRIF appeared to be limited, there is a potential advantage for those with advanced maternal age. Considering the small sample size, further randomized controlled trials are warranted to validate these findings.

Secretomes from Conventional Insemination and Intra-Cytoplasmic Sperm Injection Derived Embryos Differentially Modulate Endometrial Cells In Vitro.

Conventional Insemination (CI) and Intra-Cytoplasmic Sperm Injection (ICSI) are routinely used insemination methods in clinical Assisted Reproductive Technologies (ART) settings. However, the existing data on the developmental competence and implantation potential of CI and ICSI derived embryos are not unequivocal. This prospective study on 23 patients undergoing ART treatment explored whether the secretomes of CI- and ICSI-derived embryo differentially alter the expression of integrins (αv and β3 integrin) and MUCIN-1 (MUC-1) in a human endometrial epithelial cell line (Ishikawa). Immunocytochemical data demonstrated that the secretome of CI-derived top quality (GI) embryos induced higher (p < 0.05) expression of ɑv β3 compared to sibling ICSI derived G1 embryos in Ishikawa cells. Though, relative levels of the transcript for MUC-1, anti-adhesion molecule did not show a significant difference between the study groups, immunocytochemical analysis demonstrated significantly (p < 0.0001) higher expression of MUC-1 in cells treated with ICSI-derived embryo secretome, compared to that treated with CI -derived embryo secretome. These results suggest that secretomes from CI and ICSI embryos differentially modulate the endometrial cells in vitro. This hints at differences in the ability of CI- and ICSI- derived embryos to alter endometrial profile.

A sonographic endometrial thickness

BackgroundThe prevalence of placenta accreta spectrum (PAS), a potentially life-threatening condition, has exhibited a significant global rise in recent decades. Effective screening methods and early identification strategies for PAS could enable early treatment and improved outcomes. Endometrial thickness (EMT) plays a crucial role in successful embryo implantation and favorable pregnancy outcomes. Extensive research has been conducted on the impact of EMT on assisted reproductive technology cycles, specifically in terms of pregnancy rates, live birth rates, and pregnancy loss rates. However, limited knowledge exists regarding the influence of EMT on PAS. ObjectiveThis study aims to evaluate association between pre-implantation EMT and the occurrence of PAS in women undergoing assisted reproductive technology cycles. Study DesignA total of 4637 women who had not undergone prior caesarean section, conceived by in vitro fertilization or intracytoplasmic sperm injection embryo transfer treatment and subsequently delivered at the Third Affiliated Hospital of Guangzhou Medical University between January 2008 to December 2020 were included in this study. To explore the relationship between EMT and PAS, we employed smooth curve fitting, threshold effect, and saturation effect analysis. Multivariate logistic regression analysis was performed to evaluate the independent association between EMT and PAS while adjusting for potential confounding factors. Propensity score matching (PSM) was performed to reduce the influence of bias and unmeasured confounders. Furthermore, we employed causal mediation effect analysis to investigate the mediating role of EMT in the relationship between gravidity and ovarian stimulation protocol and the occurrence of PAS. ResultsAmong the 4637 women included in this study, pregnancies with PAS (159, 3.4%) had significantly thinner EMT (non-PAS 10.08±2.04 vs PAS 8.88±2.21 mm，P<0.001) during the last ultrasound before embryo transfer. By smooth curve fitting, the saturated effect was found on EMT and PAS, with an inflection point at 10.9mm. Then the EMT was divided into four groups: ≤7mm, >7 to≤10.9mm, >10.9 to≤13mm, >13mm, and the absolute rates of PAS in each group was 11.91%, 3.73%, 1.35%, and 2.54%, respectively. When compared to women with an EMT ranging from between 10.9-13mm, the odds of PAS increased from adjusted odds ratio (aOR) of 2.27 (95% CI, 1.33-3.86) for EMT ranging from 7-10.9mm to aOR of 7.15 (95% CI, 3.73-13.71) for EMT <7mm after adjusting for potential confounding factors. Placenta previa remained as an independent risk factor for PAS (aOR, 11.80; 95% CI, 7.65-18.19). Moreover, EMT <7mm was still an independent risk factor for PAS (aOR, 3.91; 95% CI, 1.57-9.73) in the matched cohort after PSM. Causal mediation analysis revealed that approximately 63.9% of the total effect of gravidity and 18.6% of the total effect of ovarian stimulation protocol on PAS were mediated by the EMT. ConclusionsThe findings from our study indicate that thin EMT is an independent risk factor for PAS in women without prior caesarean section undergoing assisted reproductive technology treatment. The clinical significance of this risk factor is slightly lower than placenta previa. Furthermore, our results demonstrate that EMT plays a significant mediating role in the relationship between gravida or ovarian stimulation protocol and PAS.

Efficacy of Dan'e Fukang Soft Extract in Moderate Ovarian Hyperstimulation Syndrome for Concurrent Treatment of Blood and Fluid Guided by the "Triple Prevention" Principle.

This study aimed to evaluate the therapeutic efficacy and safety of Dan'e Fukang soft extracts in moderate ovarian hyperstimulation syndrome (OHSS) for the simultaneous treatment of blood and fluid, guided by the traditional Chinese medicine principle of "triple prevention". This study conducted a retrospective analysis of clinical data from outpatients who underwent in vitro fertilization (IVF)/intracytoplasmic sperm injection embryo transfer (ICSI-ET). A total of 2245 cases were included and divided into a treatment group (1002 cases) and a control group (1243 cases). Patients in the treatment group were administered Dan'e Fukang soft extracts orally in addition to conventional Western medicine. Comparative assessments were made between the two groups on pelvic ascites volume, maximum ovary diameter, dysmenorrhea incidence post-oocyte retrieval, and safety indicators. There were no statistically significant differences between the treatment group and the control group in terms of general characteristics or the levels of follicle-stimulating hormone (FSH), luteotropic hormone (LH), estradiol (E2), or progesterone (P) at the time of gonadotropin (Gn) initiation. The groups did not differ significantly when we compared the levels of LH, E2, or P on the day of human chorionic gonadotropin (hCG) injection and during ovarian hyperstimulation protocols (P > 0.05 for all indicators). The differences in the volume of pelvic ascites, the maximum ovarian diameter, and the incidence of dysmenorrhea after oocyte retrieval were statistically significant between the treatment group and the control group (P < 0.05 in both). There were no instances of adverse reactions in either group. Based on the traditional Chinese medicine principle of "triple prevention", the use of Dan'e Fukang soft extracts for the simultaneous treatment of blood and fluid in moderate OHSS significantly improved the absorption of pelvic ascites, promoted ovarian recovery, and reduced the incidence of dysmenorrhea after oocyte retrieval.

#166 : Investigating the Effect of Parental Age on Intracytoplasmic Sperm Injection Reproductive Outcomes in Indonesia: Prognostic Tools and Prediction Approaches to Maximize Human Embryo Implantation

Background and Aims: Reproductive issues impact one in eight Indonesian married couples. Intra Cytoplasmic Sperm Injection (ICSI) is still expensive; hence many couples don’t get evaluated or enroll fertility programs. We decided to study the correlation between age, hormonal status, and ICSI embryo quality because Indonesia’s infertility rate is rising. This is the first investigation into the factors behind ICSI outcomes in Indonesia. Method: In 2022, two fertility centers in Surabaya and Tangerang, Indonesia, performed ICSI on couples. 150 couples receiving ICSI at two research sites comprise the sample. Surgically retrieved or frozen sperm cycles and incomplete medical records were excluded. The Spearman test was used to investigate the correlation between parental age, hormonal status, and ICSI embryo quality on day 1 and day 3. (D1 and D3). Results: 150 ICSI cycles were studied. The hormonal baseline varied from normal to subnormal, while paternal and maternal age ± SDs were 37.03 ± 6.08 and 34.28 ± 5.11. The Spearman test demonstrated a significant negative connection between paternal age and embryo quality D1 (p&lt;0.005, r=-0.363) and D3 (good category: p&lt;0.005, r=-0.172; intermediate category: p&lt;0.005, r=-0.250; poor category: p&lt;0.005, r=-0.221). The Spearman test showed a significant negative connection between maternal age and embryo quality D1 (p&lt;0.005, r=-0.413) and D3 (good category: p&lt;0.005, r=-0.231; intermediate category: p&lt;0.005, r=-0.294; poor category: p&lt;0.005, r=-0.291). Conclusion: This study reveals that married couples have a substantial impact on their hormonal condition, the rate of fertilization, the quality of D1 and D3 embryos, and the number of embryos generated following ICSI. To increase the success rate of ICSI procedures, it is necessary to maximize fertility at a younger age.

TSH levels after fresh embryo transfer are associated with reproductive outcomes in euthyroid women undergoing the first IVF/ICSI cycles

To investigate whether there is a relationship between TSH levels on the 14th day post embryo transfer (D14 TSH levels) and the reproductive outcomes in euthyroid women who are free from levothyroxine (LT4) treatment and undergo the first in vitro fertilization /intracytoplasmic sperm injection embryo transfer (IVF/ICSI-ET) cycles with the homogeneous ovarian stimulation protocols. This was a prospective study including a total of 599 euthyroid women undergoing the first IVF/ICSI ET cycles. Serum samples were collected and frozen on the 14th day post embryo transfer. TSH levels were measured after the confirmation of clinical pregnancy. The patients were divided into three groups (low-normal ≤ 2.5 mIU/L; high-normal 2.5–4.2 mIU/L; and high > 4.2 mIU/L) based on D14 TSH levels. Reproductive outcomes were compared among the three groups. Binary logistic regression analyses and generalized additive mixed models with smoothing splines were used to investigate the relationship between TSH levels and reproductive outcomes. D14 TSH levels were significantly elevated compared to basal TSH levels, and the degree of TSH elevation was significantly higher in pregnant women compared to that in non-pregnant women. The clinical pregnancy and live birth rates increased significantly in the high-normal D14 TSH groups, and doubled in the high D14 TSH groups compared to the low TSH groups. When adjusted by age, basal TSH, AMH, E2, endometrial thickness, type and causes of infertility, and transferred embryos, the dose-dependent relationships between D14 TSH and clinical pregnancy and live birth were observed. Obstetric outcomes in singleton or twins live birth among the different D14 TSH groups were similar. Elevated D14 TSH levels were associated with better clinical pregnancy and live birth rates, and were not associated with worse obstetric outcomes. The mechanisms to explain the phenomenon remained to be studied.

Centrosome fragmentation and malposition is common in the first cell division of equine ICSI embryos

In vitro production of embryos via Intra-Cytoplasmic-Sperm-Injection (ICSI) has become popular in the horse industry. Although improving, the blastocyst yield in clinical practice remains sub-optimal (∼20%: Stout. JEVS 2020; 89:103011). The high incidence of developmental arrest of ICSI embryos is suggested a result of mitotic errors during the first zygotic cell divisions. A better understanding of the cause of these errors could help develop strategies to prevent mitotic errors in equine in vitro produced (IVP) embryos. In somatic cells, the centrosomes, composed of two centrioles surrounded by pericentriolar material are the major microtubule-organizing centers and play essential roles in spindle assembly and chromosome segregation. Mammalian oocytes lack centrosomes and although two centrioles are re-introduced by the spermatozoon at fertilization, centrosomes make only a minor contribution to zygotic spindle assembly (Schneider et al., J.Cell. Biol. 2021; 220(11):e202010106). Although not essential for spindle assembly, the role of the centrosomes in ensuring fidelity of chromosome segregation during zygotic cell division is unclear. The aim of this study was to evaluate the position and number of centrosomes in horse ICSI zygotes during the first mitotic division. Cumulus-Oocyte-Complexes (COCs) were obtained from Warmblood mares by Ovum-Pick-Up, maintained in H-SOF at room temperature overnight and then matured in vitro, fertilized by ICSI and cultured as described previously (Ducheyne et al. Reprod. Fertil. Dev. 2019; 31(12):1830). After 24h, injected oocytes were fixedand immunostained for alpha-tubulin, acetylated tubulin, (peri-)centrosome material (NEDD1) and chromatin (DAPI). For each zygote, confocal microscopy was performed using a Leica SPE II microscope and a 0.17µm interval z stack generated. Of 326 COCs collected, 177 reached MII and were injected and cultured further. Of the 177, 136 zygotes were fixed and imaged, 13 were at the two pronucleus, 11 at prophase/prometaphase, 14 at metaphase, 11 at anaphase or telophase, 54 at the two cell stage and 33 were either degenerated or poorly stained. During the two pronucleus and prophase/prometaphase stages, centrosomes mainly localized at the interface between the two pronuclei. In 56% (n=14/25) of zygotes in pro-metaphase and metaphase, independent spindles formed around each of the two parental pronuclei. The majority of dual spindles were closely adjacent and synchronous. Moreover, in 64.2% (n=9/14) of the metaphase zygotes, the (peri-)centrosomes were either malpositioned (e.g. not at the poles of the spindle) or fragmented. Fragmented or multiple centrosomes are known to predispose to abnormal chromosome-microtubule attachment and aberrant chromosome segregation. We therefore hypothesize that malposition and fragmentation of the centrosomes contributes to mitotic errors and developmental arrest of equine ICSI embryos. Acknowledgements to CAPES, Brazil, for financial support (Code 88882.433332/2019-01 and 88887.467895/2019-00)

Sperm vitrification supports in vitro blastocyst development after ICSI in horses

In the last decade, the introduction of advanced assisted reproductive techniques has significantly impacted the horse breeding industry. Specifically, in vitro embryo production by intracytoplasmic sperm injection (ICSI) has gained relevance and begun to be used to conserve wild equids. Kinetic sperm vitrification permits cell cryopreservation without permeating cryoprotective agents, facilitating the international commercialization and transportation of samples. This study aimed to assess embryo morpho-kinetic dynamics up to the blastocyst stage of ICSI embryos produced by injection of frozen (F) or vitrified (V) spermatozoa into horse eggs. Semen collection, as well as sperm vitrification and analysis were done as described by Consuegra et al. (Animal Reproduction Sciences. 2019; 206:69-77). Computer-assisted sperm analysis (Sperm Class Analyzer® CASA) and flow cytometry with double stain PNA-FITC (peanut agglutinin-fluorescein isothiocyanate) and PI (propidium iodide) were performed to assess acrosomal integrity and sperm viability respectively. Frozen and vitrified sperm cells from a single ejaculate of a horse and a donkey were used. Oocyte collection, in vitro maturation, ICSI, time-lapse imaging andembryo culture were performed as reported by Bragulat et. al. (Theriogenology 2023; 195:199-208). Sperm analysis before cryopreservation showed: total motility (TMOT) 98.34%; progressive sperm motility (PMOT) 78.60%; intact-membrane sperm (IMS) 86.00%, and normal form (NF) 79.37%. After thawing, frozen sperm (F) analysis showed: TMOT 84.36%; PMOT 61.28%; IMS 77.50% and NF 86.21% and for vitrified sperm (V): TMOT 80.64%; PMOT 61.28%, IMS 81.00% and NF 68.97%. A total of 89 matured oocytes were injected. No significant differences were found in cleavage rates (F: 35/43, 81.39%; V: 35/46, 76.08%) or blastocyst rates at day 8 (F: 3/43, 6.97%; V: 8/46, 17.39%). Preliminary data from time-lapse imaging showed similar embryo morpho-kinetic dynamics between groups. Average hours after ICSI for time to cleavage were (mean ± SEM): F, n = 4, 27.00 ± 1.41; V, n = 3, 27.67 ± 1.20. Second cell division: F, n = 3, 38.33 ± 6.64; V, n = 3, 32.33 ± 3.38. Morula stage: F, n = 1, 85; V, n = 1, 120. Blastocyst stage: F, n = 1, 150; V, n = 1, 180. Our results demonstrate for the first time that vitrified sperm cells can be used successfully to produce blastocysts by ICSI without compromising developmental competence in horses.

Effect of pre-maturation oocyte timing on equine ICSI embryo development rates

Within the last 5 years, client adoption of ovum-pick-up (OPU) paired with equine intracytoplasmic sperm injection (ICSI) and in vitro embryo production (IVP) in Brazil has been rapid. Equine ICSI has facilitated the use of expensive semen be it imported, rare, poor-quality or low motility for IVP. A pre-maturation step occurs between OPU on the farm and transport to the lab. Oocytes are kept in 5 ml vials of pre-maturation medium, held at 22°C until placement in maturation medium. Oocytes in pre-maturation medium are kept at this low temperature to inhibit the onset of germinal vesicle breakdown and the onset of oocyte maturation. If maturation begins during transport, oocyte maturation rates and oocyte quality would be severely reduced. Recently, (Merlo, B et al 2022 Anim Reprod Sci 107071) reported different rates of equine ICSI embryo development based on timing of oocyte pre-maturation and maturation. In our IVP system, oocyte maturation time is tightly controlled between 28 and 32 hours, so we decided to evaluatethe timing of pre-maturation since it can vary between 0 and 24 hours. This variation is based on the time required for OPU completion by multiple teams, aspirating on the same day in different locations. We tested the potential impact of oocyte pre-maturation in 3-time intervals 0-8, 9-16 and 17-24 hours, in the 2021-2022 breeding season. The average blastocyst formation rate based on total oocytes collected was 17%. Embryo development from oocytes aspirated in the 3 pre-maturation time intervals was: 0-8 hours, 522 embryos produced from 3413 oocytes aspirated (15.3%); 9-16 hours, 471 embryos produced from 2667 oocytes aspirated (17.7%) and 17-24 hours, 3931 embryos produced from 22,896 oocytes aspirated (17.2%). There were no significant differences observed (Mann-Whitney test p >0.05), demonstrating that up to 24 hours, the holding time of pre-maturation oocytes, held at 22.5°C had no significant impact on embryo development in our equine ICSI embryo production system.

Physiological intracytoplasmic sperm injection does not improve the quality of embryos: A cross-sectional investigation on sibling oocytes.

This study aimed to compare the efficacy of physiological intracytoplasmic sperm injection (PICSI) and intracytoplasmic sperm injection (ICSI) in terms of the fertilization rate and embryo quality using sibling oocyte cycles. This prospective, cross-sectional study collected data from 76 couples who underwent their first cycle at the Hue Center for Reproductive Endocrinology and Infertility, Vietnam, between May 2019 and November 2021. The inclusion criteria were cycles with at least eight oocytes and a sperm concentration of 5×106/mL. Sperm parameters, sperm DNA fragmentation (SDF), fertilization, and the quality of cleavage-stage embryos on day 2 and blastocysts on day 5 were examined. From 76 ICSI cycles, 1,196 metaphase II (MII) oocytes were retrieved, half of which were randomly allocated to either the PICSI (n=592) or ICSI (n=604) treatment group. The results showed no significant difference between the two groups in terms of fertilization (72.80% vs. 75.33%, p=0.32), day 2 cleavage rate (95.13% vs. 96.04%, p=0.51), blastulation rate (52.68% vs. 57.89%), and high-quality blastocyst rate (26.10% vs. 31.13%, p=0.13). However, in cases where SDF was low, 59 cycles consisting of 913 MII oocytes produced a considerably higher blastulation rate with PICSI than with ICSI (50.49% vs. 35.65%, p=0.00). There were no significant differences between the pregnancy outcomes of the PICSI and ICSI embryo groups following embryo transfer. Using variable sperm quality provided no benefit for PICSI versus ICSI in terms of embryo outcomes. When SDF is low, PICSI appears to be able to produce more blastocysts.

Sperm deoxyribonucleic acid fragmentation index at the time of intracytoplasmic sperm injection and standard in vitro fertilization is correlated with lower fertilization but not with blastocyst genetic diagnosis

To determine the effects of sperm deoxyribonucleic acid (DNA) fragmentation at the time of fertilization on invitro fertilization (IVF) outcomes and genetic diagnosis using next generation sequencing. Prospective double-blinded study. Private Clinic. Couples (n = 150). Invitro fertilization with preimplantation genetic testing for aneuploidy and sperm DNA fragmentation assay, as in sperm chromatin structure assay the day of retrieval. Laboratory outcomes are listed in the results section. Statistical analysis was performed using JMP, XYLSTAT, and STATA version15. The sperm DNA fragmentation index (DFI) in the neat ejaculate did not predict fertilization rate, quality, blastulation, or genetic diagnosis. No statistically significant results were obtained comparing <15% with >15%, <20% with >20%, <30% with >30% except for DFI. No statistically significant differences in oocyte source age or male age were observed. No statistically significant differences comparing <15% with >15%, <20% with >20%, <30% with >30% DFI at the time of standard IVF or intracytoplasmic sperm injection (ICSI) were observed for % euploid, aneuploid, mosaic, blastulation, biopsied, or D5/total biopsied. The DFI of >15% had more good quality D3 embryos than the <15% group, as did the >20% group compared with the <20% group. The ICSI fertilization was significantly higher in all 3 lower percentage groups compared with the higher counterpart. Standard IVF had significantly more blastocysts/fertilized suitable for biopsy and more D5/total number biopsied than ICSI embryos despite no difference in DFI. The DFI at fertilization is correlated with decreased fertilization for ICSI and IVF.

How Could One Sperm and One Oocyte Proceed To Molar Pregnancy? Prevalence of Molar Pregnancy during ICSI Procedure in Over 25,000 Fresh Embryo Transfers: A Retrospective Cross-Sectional Study.

Gestational trophoblastic disease (GTD) is a heterogeneous group of diseases characterized by excessive proliferating trophoblastic tissue. The prevalence of GTD has a varied geographical distribution. However, its frequency following intracytoplasmic sperm injection (ICSI) cycles has not yet been reported. This study aimed to estimate GTD frequency and prevalence after ICSI cycles. This retrospective cross-sectional study included all patients diagnosed with GTD subsequent to ICSI and segmental embryo transfer procedure during 2011-2019 at Royan Institute. GTD diagnosis was established for patients who met all three criteria: beta-human chorionic gonadotropin (β-hCG) levels greater than 100,000 mIU/mL, vesicular ultrasonographic pattern, and presence of pathologic features of hydatidiform mole. Although we assessed the GTD frequency in all ICSI cycles, GTD cases were only observed following fresh embryo transfer ICSI procedures. We evaluated 25,667 fresh embryo transfer ICSI procedures out of 41,540 ICSI cycles. This study identified a total of 10 GTDs confirmed by all criteria which were mentioned previously. Of these 10 GTDs, nine cases had hydatidiform mole, and one had gestational trophoblastic neoplasia. The frequency of GTD was calculated 10 cases in 41,540 (0.240 per 1000) ICSI procedures and 10 in 25,667 (0.389 per 1000) fresh embryo transfers following ICSI cycles. Also, we detected 10 GTD cases in 8,196 (1.220 per 1000) clinical pregnancies. We discuss that the possibility of GTD after ICSI procedure is not as low as expected. Thus, the previous theses are insufficient to explain all aspects of molar pregnancy, and more research is required.

Assisted reproductive technology treatment and risk of multiple sclerosis – a Danish cohort study

To compare the incidence of multiple sclerosis (MS) among women who had undergone assisted reproductive technology (ART) treatment with the women who had conceived a child without previous ART treatment. A register-based nationwide cohort study. Women with a first ovarian stimulation cycle before invitro fertilization (IVF) or intracytoplasmic sperm injection (ICSI) (i.e., ART treatment) recorded in the Danish IVF register between 1996 and 2018; and women recorded in the Danish Medical Birth Register with the birth of their first child where date of conception is between 1996 and 2018. The cohort was observed until March 10,2021. Mainly included IVF, ICSI, and fresh embryo transfer with hormone stimulation. A diagnosis of MS recorded in the Danish Multiple Sclerosis Registry. Crude and adjusted hazard ratios (aHRs) with 95% confidence intervals (CIs) were calculated. A total of 585,716 women were included in the cohort of which 63,791 (11%) were exposed to at least one initiated IVF or ICSI cycle during the study period. Cycles with oocyte donation were excluded. The median follow-up time for the entire cohort was 12.4 years (Q1-Q3= 6.6-18.1). Compared with women conceiving without previous ART, ART treated women were older (31.8 years vs. 27.5 years), more often had a university degree (45% vs. 36%), and more often had received other fertility treatments than IVF or ICSI before cohort entry (26% vs. 3%). We found no association between incident MS and exposure to ART compared with non-ART pregnancy (aHR=1.08; 95 % CI, 0.93-1.25). An analysis following intention-to-treat principle on a propensity score matched sub cohort confirmed our results. In subgroup analysis including all ART cycles among the ART treated women, we found no increased risk of MS within 2 years of ART cycle start for successful ART cycles (pregnancy) compared with failed ART cycles (no pregnancy) (aHR=1.01; 95% CI, 0.58-1.76). We found a non-significant trend toward increased risk of MS with increasing numbers of ART cycles although based on small numbers. Women treated with ART do not seem to be at increased risk of developing MS compared with the women not exposed to ART.

Elevated serum uric acid level is associated with adverse reproductive outcomes in women with polycystic ovary syndrome undergoing in vitro fertilization or intracytoplasmic sperm injection embryo transfer cycles: a retrospective cohort study

Serum uric acid levels are elevated in polycystic ovary syndrome, however, the relationship between serum uric acid level and reproductive outcomes in women with polycystic ovary syndrome remains unclear. This study aimed to investigate the association between serum uric acid level and the reproductive outcomes in women with polycystic ovary syndrome undergoing invitro fertilization or intracytoplasmic sperm injection embryo transfer cycles. This was a retrospective cohort study performed at a university-affiliated reproductive medicine center. A total of 1903 women with polycystic ovary syndrome undergoing their first invitro fertilization or intracytoplasmic sperm injection embryo transfer cycles between January 2010 and January 2021 were initially included. The trends for reproductive outcomes in polycystic ovary syndrome across quartiles of serum uric acid levels were assessed. A logistic regression analysis was performed to obtain the odds ratios for invitro fertilization outcomes based on the quartiles of serum uric acid with or without adjusting for potential confounding variables. Using generalized additive models, serum uric acid was further treated as its original continuous property to visualize its nonlinear relationship with invitro fertilization outcomes. The live birth rate was the main outcome. After exclusions, a total of 883 women with polycystic ovary syndrome with their first fresh-embryo transfer cycles were included. In quartiles of serum uric acid levels, there was a significant decreasing trend in the live birth rate from the lowest quartile (Q1: 61.8%) to the highest (Q4:45.9%) (Ptrend=.002). The percentage of low birthweight increased from Q1 (22.3%) to Q4 (31.7%) (Ptrend=.049). Compared with those in Q1, women in Q4 showed a significant lower probability of live birth and clinical pregnancy and a higher risk for low birthweight (all P<.05). Both the unadjusted and adjusted generalized additive models indicated that as the serum uric acid level increased, the probability of clinical pregnancy and the live birth rate exhibited an overall decreasing profile, and the risk for low birthweight showed an increasing profile. An elevated serum uric acid level is associated with decreased probabilities of live birth and clinical pregnancy and an increased risk for low birthweight in women with polycystic ovary syndrome. However, these associations may be confounded by other factors and more well-designed studies are needed to confirm these findings in the future.

Time-lapse imaging and developmental competence of donkey eggs after ICSI: Effect of preovulatory follicular fluid during oocyte in vitro maturation

Equus members exhibit very divergent karyotype, genetic plasticity, and significant differences in their reproductive physiology. Despite the fact that somatic cell nuclear transfer and intracytoplasmic sperm injection (ICSI) has gained relevance in the last few years in horses, few reports have been published exploring ovum pick up (OPU) and in vitro maturation (IVM) of cumulus-oocyte complexes (COCs) in donkeys. Yet, some donkey species and breeds are considered endangered, and these assisted-reproductive technologies could help to preserve the genetic of valuable individuals. In this study, we tested the hypothesis that supplementation with jenny preovulatory follicular fluid (PFF) during IVM could improve oocyte developmental competence in the donkey. For this, in vitro nuclear maturation rates, cumulus cell expansion, and embryo development after ICSI of donkey COCs matured in culture media supplemented with fetal bovine serum (FBS) or donkey PFF, with a known metabolomic profile, were assessed. Time-lapse imagining was performed after ICSI of horse and donkey oocytes. Eight OPU sessions were done in five jennies with an average recovery rate of 69.2% (n = 45 COCs). Although lower cumulus cells expansion was observed in oocytes of PFF group (P = 0.0010), no significant differences were described in nuclear maturation rates and preimplantation embryo development between groups. Donkey ICSI embryos showed similar morphokinetics to horse ICSI embryos. Our study shows that supplementing IVM media with FBS or donkey PFF supports nuclear maturation and early preimplantation embryo development after ICSI in donkeys. To our knowledge, the present study is the first report of ICSI, time-lapse imaging and in vitro blastocyst production in donkey.

ICSI in non-male factor infertility patients does not alter metabolomic signature in sibling embryos as evidenced by sensitivity enhanced nuclear magnetic resonance (NMR) spectroscopy.

Intracytoplasmic sperm injection (ICSI) was developed to overcome male factor infertility, however, there recently has been an increasing trend in ICSI usage irrespective of the etiology, demonstrating an overuse of this insemination technique. There is a limited knowledge on the behaviour of ICSI derived embryos in non-male factor infertility patients. Metabolomic assessment of preimplantation embryos in conjunction with morphological evaluation can provide better understanding of embryonic behaviour. Hence, this study was undertaken to explore if there are any metabolomic differences between IVF and ICSI derived sibling day-5 blastocysts from non-male factor infertility patients. This prospective study included nineteen couples with non-male factor infertility undergoing Assisted Reproductive Technology. The sibling oocytes retrieved from each patient were randomly assigned to two groups and inseminated either by IVF or ICSI. Spent culture media (SCM) in which embryos were cultured up to day 5 were collected and investigated using sensitivity enhanced NMR based metabolite profiling utilizing high resolution (800 MHz) NMR equipped with cryogenically cooled micro-coil (1.7 mm) probe. The metabolomic signature between IVF and ICSI derived sibling blastocysts was assessed. A significant reduction in the concentrations of pyruvate, citrate, glucose and lysine were observed in both IVF and ICSI sibling embryos compared to medium control (P< 0.05–0.001). Further, histidine and valine level was found lower in ICSI embryos compared to medium control (P<0.05) during 96 hours of in vitro culture. Notably, between IVF and ICSI SCM, no significant difference in the concentration of the metabolites was found. Our results suggest that ICSI in non-male factor does not alter the SCM metabolomic signature during 96 hours of embryonic development.

Non-invasive preimplantation genetic testing for conventional IVF blastocysts

BackgroundPrevious studies suggested that non-invasive preimplantation genetic testing (niPGT) for intracytoplasmic sperm injection (ICSI) blastocysts can be used to identify chromosomal ploidy and chromosomal abnormalities. Here, we report the feasibility and performance of niPGT for conventional in vitro fertilization (IVF) blastocysts.MethodsThis was a prospective observational study. In the preclinical stage, whole genome amplification and NGS were performed using the sperm spent culture medium (SCM). Then, trophectoderm (TE) biopsies and corresponding SCM derived from 27 conventional IVF monopronuclear embryos were collected. In the clinical stage, samples from 25 conventional IVF cycles and 37 ICSI cycles from April 2020–August 2021 were collected for performance evaluation.ResultsPreclinically, we confirmed failed sperm DNA amplification under the current amplification system. Subsequent niPGT from the 27 monopronuclear blastocysts showed 69.2% concordance with PGT results of corresponding TE biopsies. In the clinical stage, no paternal contamination was observed in any of the 161 SCM samples from conventional IVF. While maternal contamination was observed in 29.8% (48/161) SCM samples, only 2.5% (4/161) samples had a contamination ratio ≥ 50%. Compared with that of TE biopsy, the performances of NiPGT from 161 conventional IVF embryos and 122 ICSI embryos were not significantly different (P > 0.05), with ploidy concordance rates of 75% and 74.6% for IVF and ICSI methods, respectively. Finally, evaluation of the euploid probability of embryos with different types of niPGT results showed prediction probabilities of 82.8%, 77.8%, 62.5%, 50.0%, 40.9% and 18.4% for euploidy, sex-chromosome mosaics only, low-level mosaics, multiple abnormal chromosomes, high-level mosaics and aneuploidy, respectively.ConclusionsOur research results preliminarily confirm that the niPGT approach using SCM from conventional IVF has comparable performance with ICSI and might broadening the application scope of niPGT.

P-018 Serum Follicle-Stimulating Hormone (FSH) levels as a predictor of outcomes after Surgical Sperm Retrieval (SSR) for male infertility

Abstract Study question Is there an association between pre-operative FSH levels and successful outcomes following Surgical Sperm Retrieval (SSR), and subsequent fertility treatment? Summary answer Higher levels of pre-operative FSH are associated with unsuccessful SSR, and, in the cases of successful retrieval, sperm less likely to produce healthy live births. What is known already Surgical Sperm Retrieval (SSR) techniques combined with Intracytoplasmic Sperm Injection (ICSI) offer men with azoospermia a chance to father biological children. However, the variable success rates of these physically, psychologically, and financially costly procedures highlights the potential value of pre-operative predictors of success. A literature review indicated serum FSH a promising pre-operative prognostic factor. However, only very few papers investigated pregnancy outcomes as an endpoint. Study design, size, duration An extensive literature review was undertaken examining literature between 2015-2020. This led to determination of the serum cut-off level to apply to the different groups’ outcomes. The study was a retrospective clinical analysis of SSR offered by a university Hospital between 2014-2021. Sample size was 118 (mainly Testicular Sperm Aspiration (TESA) and Percutaneous Epididymal Sperm Aspiration (PESA) patients. Patients with Klinefelter’s (KF) (N = 3) were excluded from analysis due to the genetic nature of their azoospermia. Participants/materials, setting, methods Databases from the University Hospital's Theatres and Andrology Department provided data on patients who underwent SSR 2014-present. A retrospective evaluation of physical and electronic notes identified preoperative hormone profiles and other potential influencers. Pregnancy outcomes were ascertained from the ICSI and Frozen Embryo transfer (FET) databases. Using a serum FSH cut-off of 15IU/mL (chosen based on previous literature review) data was analysed for differences between the groups’ outcomes. ROC analysis generated potential alternate cut-offs. Main results and the role of chance Using the initial 15IU/mL cut-off analysis demonstrated significantly more patients had unsuccessful SSR and sperm that did not produce healthy live birth where FSH was ≥15IU/mL at a 95% confidence level. The ROC curves for both SSR and pregnancy outcomes also proved significant – suggesting various potential cut-offs from our data. One potentially valuable cut-off for identifying men unlikely to produce sperm capable of producing a healthy live birth following all treatment is 14.0IU/mL (FSH above this predicting failure in pregnancy outcomes) (sensitivity 40.8%, specificity 90.5%), i.e., above this level the model predicts 90.5% of men will not have success. The lower bound 95% CI for this curve was above 0.5, indicating the curve to be statistically significant at a 95% confidence level. Limitations, reasons for caution SSR is rare and a lack of documentation available for some patients resulted in a relatively small sample size. Our data comprises primarily TESE and PESA patients. Different surgeon technique or service standards may influence outcomes. Our findings did not consider KF patients, who may have favourable SSR outcomes. Wider implications of the findings SSR is a costly procedure financially and physically with variable success rates. Hopefully these findings can help counsel men as to their likelihood of success in having a healthy live birth following SSR and subsequent treatment, helping to reduce both a financial burden on services and emotional burden on patients. Trial registration number Not applicable

P-359 Effect of estradiol pretreatment on antagonist ICSI cycles outcome

Abstract Study question Does estrogen pretreatment in antagonist ICSI cycles improves the outcome? Summary answer Pretreatment with estrogen in antagonist cycles reduced the days of stimulation, the number of hMG ampoules and enhanced the implantation rate. What is known already It has been previously reported that, programming of antagonist cycles with E2 only, started in the luteal phase and continued beyond the menses till the first day of stimulation, yielded similar results as the long agonist protocol. E2 pre-treatment in the luteal phase has an inhibiting role on follicular growth through its negative feedback on the FSH increase during the luteal follicular transition phase. This effect stops as soon as E2 administration is terminated. Study design, size, duration This was a prospective randomized controlled trial performed in a private IVF centers from June 2020 to February 2021. One hundred and fourteen infertile couples undergoing intracytoplasmic sperm injection embryo transfer cycles (ICSI-ET) were included. Participants/materials, setting, methods Group A includes 57 patients were pretreated with a dose of 4 mg estradiol valerate (progynova 2 mg; Bayer) once daily starting 10days before expected menses. Ovarian stimulation was started one day after the last estradiol dose, in a dose ranging from 225 to 375 IU of hMG. While in group B , 57 patients received stimulation in a dose of 225 to 375 IU of hMG starting on cycle day 2 without estradiol pretreatment. Main results and the role of chance The daily dose of hMG was significantly lower in The study group (P = 0.048), the stimulation days were significantly higher in the control group (P = 0.049), the total number of hMG ampoules was significantly higher in the control group (P = 0.041), and the implantation rate was higher in the study group (P = 0.045) , also the pregnancy rate (positive pregnancy test) was higher in the study group (p = 0.044) but there was no significant statistical difference detected as regards the clinical pregnancy rate between both groups (P = 0.180) , the serum estradiol on day of hCG ( p = 0.055), serum progesterone level on hCG day (P = 0.71) and the endometrial thickness (p = 0.780) . Also there is no significant statistical difference between the two groups as regarding number of follicles on day of hCG. (P = 0.803), the number of mature oocytes (P = 0.703) and no significant difference in the number of good quality embryos (p = 0.532) and the ratio of mature oocytes to number of follicles on day of hCG (P = 0.835), with no significant statistical difference between both groups. Limitations, reasons for caution The limitation of this study is the small number of patients as we included only 114 patients. Wider implications of the findings Luteal estradiol in antagonist cycles improved the outcomes of ICSI procedure by reducing the number of gonadotropin ampoules used for COH and reduced duration of stimulation, also the implantation rate is improved by luteal estradiol administration. Trial registration number NCT05197374

Perinatal outcome of babies born after using a simplified IVF culture system versus ICSI with sibling oocytes: a prospective cohort study

Research questionIs there a difference in perinatal outcome in the same patient cohort for babies conceived following randomization of sibling oocytes allocated to a simplified IVF culture system (SCS) or intracytoplasmic sperm injection (ICSI) followed by conventional culturing? DesignThe study compared the perinatal outcomes of 367 babies born from 1 January 2013 until 31 December 2020 after using split SCS and ICSI insemination of sibling oocytes in a selected group of normo-responsive women, excluding cases of severe male infertility. Primary outcome measures were preterm birth (PTB; <37 weeks’ gestation), low birthweight (LBW; <2.5 kg) and small for gestational age (SGA) as a primary outcome parameter while secondary outcome measures included mean birthweight, mean gestational age, extreme prematurity (<32 weeks), very low birthweight (<1.5 kg), perinatal mortality, multiple pregnancy and Caesarean section rate. ResultsA total of 105 and 103 singleton babies were born after fresh embryo transfer (FRET) and 71 and 50 singletons after frozen embryo transfer (FET) in the SCS and ICSI groups, respectively. For babies born after FRET, the LBW rate was 2.9% (3/105) for SCS and 7.8% (8/103) for ICSI (P = 0.10). LBW occurred in 4.2% (3/71) and 0% (0/50) of babies born after the transfer of cryopreserved-thawed SCS and ICSI embryos, respectively (P = 0.14). The rate of PTB was 3.8% and 6.8% for SCS and ICSI in FRET cycles (P = 0.33), and 8.5% and 6.0% for SCS and ICSI in FET cycles (P = 0.62). One congenital malformation was found in the SCS FET group. ConclusionThere was no difference in perinatal outcome for singleton and twin babies born after SCS and ICSI.