Normal Fertilization Rate Research Articles

P-039 Male age is associated with sperm DNA integrity: Selection of high DNA integrity sperm by microfluidics sorting is critical to clinical outcomes in older patients

Abstract Study question Does sperm DNA integrity affect clinical outcomes of ICSI? Summary answer Use of high DNA integrity sperm selected by microfluidics sperm sorting results in lower miscarriage rates in the patients of 39-years old and more. What is known already High sperm DNA damage is associated with decreased normal fertilization, embryo development and pregnancy rates, and an increased miscarriage rate. On the other hand, oocytes from older women have decreased pregnancy rate, and increased miscarriage rate because of possibility of low ability to repair sperm with DNA fragmentation, and dramatical increases of aneuploidy as women age. A microfluidic sperm selection chamber (MSS, ZyMōt™; DxNow) is a device designed to collect sperm with higher chromatin integrity than density gradient centrifugation (DGC). Study design, size, duration Sperm analysis was performed by sperm chromatin dispersion (SCD) test and comet assay in the same sample of 15 cases between October 2020 and February 2021. ICSI outcomes by DGC and MSS were compared with blastocyst development, and pregnancy rates in vitrified-thawed single blastocyst transfers cycle for 518 cases between August 2018 and May 2021. Participants/materials, setting, methods SCD test was optimized as a rapid procedure, with sperm showing a halo deemed normal, and those without a halo abnormal. Comet assay results were analyzed using CometScore 2.0, with comparison of %Tail DNA. ICSI outcomes were analyzed using multiple logistics regressions of male and female ages. Main results and the role of chance We found a positive correlation between male age and sperm DNA fragmentation rates in raw semen using SCD test (r = 0.70) and Comet assay (r = 0.42). Higher DNA integrity sperm could select using MSS than DGC. In this study with ICSI outcomes, 170 of 318 (53.5%) blastocyst transfers resulted in pregnancy, and 49 (28.8%) subsequently miscarried. The data were classified according to less than or more than 39 years old of male age detected by multiple logistics regressions. In patients with ≥39 years of male age, the female age was significantly higher and blastocyst and pregnancy rates were significantly lower, and the miscarriage rate was significantly higher than <39 years of male age. Since sperm DNA fragmentation increased in accordance with male age, we compared MSS and DGC in the patients with male age ≥39 years. There was no significant difference in blastocyst, pregnancy, and miscarriage rates in female age <39 years. While in ≥ 39 years of female age, blastocyst and pregnancy rates in MSS were not significantly different from DGC, but the miscarriage rate in MSS was significantly lower than in DGC (27.3 vs. 57.1%). Limitations, reasons for caution The sample size for each study was small. Analysis of sperm DNA fragmentation and samples in ICSI outcomes were not the same. The retrospective nature of ICSI outcomes in this study does not allow controlling of unknown confounders. Wider implications of the findings Sperm DNA fragmentation depended on male age affected fertility outcomes. However, when male age is higher, masking the effect of male age by female age. In this study, we found out the improvement of ICSI outcome by using high DNA integrity sperm selected by MSS in both ≥39 years. Trial registration number Not applicable

P-238 Treatment options to overcome impaired fertilization and embryonic development caused by an oocyte-related deficiency in the Patl2-/- mouse model

Abstract Study question Can assisted oocyte activation (AOA) or spindle transfer (ST) overcome the lower fertilization and blastocyst rates observed in the Patl2-/- mouse model? Summary answer ST, but not AOA, has the potential to restore both normal fertilization and blastocyst rates in a mouse model with an oocyte-related deficiency. What is known already PATL2 is an RNA-transcriptional repressor involved in the regulation of maternal mRNAs during oocyte maturation. Mutations in PATL2 have been detected in patients with oocyte maturation arrest, reduced fertilization and embryo developmental arrest. Consequently, these patients are mostly referred to oocyte donation. Both AOA, inducing artificially calcium oscillations by calcium ionophores, and ST, replacing bad-quality oocyte cytoplasm with healthy cytoplasm, have been proposed to treat oocyte-related infertility. However, there is a lack of studies showing which oocyte-related infertility indications may benefit from which specific treatment. The Patl2-/- mouse model offers a unique opportunity to study the efficiency of these treatments. Study design, size, duration An experimental study using C57BL/6NTac-Patl2tm1a mice was conducted between April to December 2021. Breeding of heterozygous C57BL/6NTac-Patl2tm1a mice resulted in a total of 156 pups, from which 22 were homozygous C57BL/6NTac-Patl2tm1a ( Patl2-/-) females. Metaphase II (MII) oocytes were collected from Patl2-/- (test group) and Patl2+/+ (control group) females and used to evaluate different oocyte quality markers, as well as the efficiency of AOA and ST treatments to improve the oocyte-related subfertility observed in Patl2-/- mice. Participants/materials, setting, methods Four- to 12-week-old mice were subjected to ovarian hyperstimulation and oocyte collection, where oocyte maturation was assessed. Afterwards, MII oocytes were used to evaluate oocyte diameter, spindle abnormalities, activation rate (AR) and blastocyst rate (BR) after PIEZO-ICSI and calcium releasing capacity after SrCl2 exposure. Finally, Patl2-/- MII oocytes were treated with AOA, by direct exposure to SrCl2, or ST treatment, by transferring the Patl2-/- spindle to Patl2+/+ cytoplasm (previously enucleated) followed by SrCl2 exposure. Main results and the role of chance No difference in the number of cumulus-oocyte complexes (COCs) and mature MII oocytes was observed between 6 Patl2-/- mice (128 MII/159 COCs) and 5 Patl2+/+ mice (108 MII/132 COCs). However, Patl2-/- MII oocytes showed a significantly lower cytoplasm diameter (68,11 ± 2,25, n = 79) than Patl2+/+ oocytes (75,19 ± 2,09, n = 77, p-value<0,001), suggesting that Patl2 affects oocyte growth but does not seem to affect oocyte maturation. Nuclear maturity was not compromised either, as a similar percentage of oocytes with normal spindle was observed in Patl2-/- (48,4%, 15/31) and Patl2+/+ MII oocytes (50%, 18/36). Activation and blastocyst rates after PIEZO-ICSI were lower in Patl2-/- (AR = 45,5%, 5/11 and BR = 20,0%, 1/5) than in Patl2+/+(AR = 75,0%, 21/28 and BR = 52,4%, 11/21). In addition, total calcium released during oocyte activation after SrCl2 exposure was not different between Patl2-/- (AxF=6,88 AU, n = 30) and Patl2+/+ MII oocytes (AxF=5,04 AU, n = 28, p-value=0,087). In line with these results, AOA treatment did not improve activation and blastocyst rates in Patl2-/- oocytes (AR = 66,7%, 44/66 and BR = 45,5%, 20/44) compared to Patl2+/+ oocytes (AR = 96,5%, 110/114 and BR = 79,1%, 87/110, p-value<0,001). Nonetheless, when performing ST using Patl2-/- as spindle donors and Patl2+/+ as cytoplasm recipients, activation (8/8, 100,0%) and blastocyst rate (6/8, 75,0%) were restored to normal values. Limitations, reasons for caution This study describes preliminary results and is limited by its small sample size. In addition, we did not identify an increased number of immature oocytes and spindle abnormalities in Patl2-/- female mice in contrast to previously published reports. Wider implications of the findings ST could be offered to treat PATL2- related female-infertility caused by a cytoplasmic deficiency, to increase both fertilization and blastocyst rates. AOA would not be beneficial for these patients as calcium releasing capacity of Patl2-/- oocytes does not seem to be affected, suggesting a deficiency independent from calcium releasing machinery. Trial registration number Not applicable

P-093 A comparison of the effects of IMSI and HOST for sperm selection on morphokinetic parameters and ploidy status in sibling embryos

Abstract Study question Are hypo-osmotic swelling test (HOST) and intracytoplasmic morphologically selected sperm injection (IMSI) both effective alternative methods for sperm selection? Summary answer Similar results were obtained in embryo morphokinetics between HOST and IMSI. What is known already HOST is a simple, low-cost and rapid test based on the swelling of the tail of the sperm in a hypoosmotic environment. Less DNA fragmentation, membrane integrity and aneuploidy rates have been found in sperm selected by the HOST method. It also allows identification of viable sperm in a totally immotile sperm sample. IMSI, on the other hand, is a method for identifying the morphologically best sperm using high-magnification microscope. In studies, IMSI has been associated with high fertilization rates. However, it is a time-consuming method that requires expensive equipment. Study design, size, duration Time-Lapse Monitoring (TLM)-incubated sibling embryos of 25 male factor patients who applied for ART treatment to Istanbul, Sisli Memorial ART and Genetics Center in 2021 were included into the study. Sibling embryos were grouped according to their sperm selection method. In the IMSI group 170 embryos, in the HOST group 173 embryos were evaluated retrospectively in terms of the morphokinetic parameters, the rates of blastulation, Top- Quality (TQ) and Good Quality (GQ) embryos and euploidy. Participants/materials, setting, methods Blastocyst grading was performed according to Gardner’s classification. Blastocysts 3AA,4AA,4AB,4BA,4BB,5AA,5AB,5BA,5BB were accepted as TQ+GQ. Blastulation rates was determined including the embryos at least 2 expansion. PGT-A results were obtained by Next Generation Sequencing(NGS) method. The comparison of the TLM parameters and clinical features of the patients were performed using Student’s t-test. Chi-square test was used for comparing the rates of fertilization, blastulation, TQ+GQ embryo and euploidy in each group. Main results and the role of chance No statistical difference was observed between IMSI and HOST groups in terms of morphokinetic parameters (tPN, tPNf, t2, t3, t4, t5, t6, t7, t8, t9, tSC, tM, tSB, tB, tEB) (p > 0.05). There were higher rates of normal fertilization in the HOST group (90.8%) than in the IMSI group (82.4%) and of TQ+GQ embryo (HOST: 50%, IMSI: 47.3%). However, these diffrences were not statistically significant (p > 0.05). There was statistically lower abnormal fertilization rate was found in HOST group (2.3% vs 7.1%, p = 0.038). When PGT-A results were compared (HOST:19 embryos, IMSI: 19), slightly higher results were observed in IMSI group than HOST group ( 63%, 58% respectively) (p > 0.05). Limitations, reasons for caution This was a retrospective of a small number of cases. Wider implications of the findings The similarity of the outcome in these initial findings studying a small number of cases, suggest that this study should be extended. Trial registration number None

P-148 Denudation employing human recombinant hyaluronidase enzyme does not affect fertilization and blastocyst formation rates in sibling oocytes from patients undergoing ART: A prospective randomized study

Abstract Study question Does human recombinant hyaluronidase (Cumulase®) improve fertilization and blastocyst formation rates in sibling human oocytes compared to bovine-derived hyaluronidase? Summary answer The use of Cumulase® for oocyte denudation does not appear to affect fertilization or usable blastocyst formation rate in patients undergoing intracytoplasmic sperm injection (ICSI). What is known already Bovine-derived hyaluronidase has been widely employed to denude the cumulus-oocyte complexes (COCs) prior to ICSI or oocyte vitrification. Nonetheless, the recent concern for its safety, due to its animal origin, has led to the development of alternatives ascertaining the enzyme’s purity. It has been reported that human recombinant hyaluronidase is not equally effective as its bovine-derived analogue, leading to a longer time required for complete denudation coupled by the subsequent toxicity risk for oocytes. This may result to lower fertilization and blastocyst formation rates. Although there are some studies on IVF outcomes with recombinant hyaluronidase, its use remains rather limited. Study design, size, duration This randomized double-blinded clinical trial was conducted at Citmer Reproductive Medicine, in Puebla and Monterrey, Mexico from February to December 2021. The impact of denudation on sibling oocytes from 103 patients employing bovine-derived hyaluronidase (InVitroCare®) or human recombinant hyaluronidase (ICSI Cumulase® (rHuPH20), CooperSurgical®) was evaluated. A total of 1237 oocytes were assigned as follows: The Hyaluronidase Group included 626 oocytes, while the Cumulase® Group included 611 oocytes. Preimplantation Genetic Testing was performed in 18 cycles. Participants/materials, setting, methods After retrieval, COCs were incubated for 2-4 h and placed in microdropets with 10 IU/mL bovine-derived hyaluronidase or Cumulase® in culture media GlobalTotal® (LifeGlobal®) for granulosa cells removal. COCs were gently pipetted up and down employing 20 μL and 150 μm tips. Metaphase II oocytes were injected and incubated for 17-20 hours in 8% CO2, 20% O2, 37°C. Normally fertilized zygotes were cultured to blastocyst stage and embryo development was evaluated. Seventy-nine blastocysts were biopsied. Main results and the role of chance The mean age of the patients was 32.12±1.11 years old. A total of 506 and 493 metaphase II oocytes from Groups 1 and 2 respectively were subjected to ICSI. Normal fertilization rates for oocytes treated with Hyaluronidase and Cumulase® were 71.34% (n = 361) and 72.41% (n = 357) respectively, with no statistically significant differences between the two groups (p = 0.35). Usable blastocysts rate (selected for embryo transfer or blastocyst cryopreservation) did not differ with any statistical difference between the bovine-derived hyaluronidase (n = 170, 47.09%) and the Cumulase® treated oocytes (n = 158, 44.25%), (p = 0.44). Preimplantation genetic testing for aneuploidies (PGT-A) was performed in 18 cycles. Seventy-nine blastocysts were biopsied and tested. Fourty-seven embryos from the bovine-derived hyaluronidase group were biopsied, presenting with a euploidy rate of 51.06% (n = 24), compared to 32 embryos from the recombinant hyaluronidase group with a euploidy rate of 50% (n = 16). Results indicated no statistically significant difference (p = 0.92). These data support that human recombinant hyaluronidase presents with similar efficacy to bovine-derived hyaluronidase. in terms of normal fertilization, embryo development as well as euploidy rates. Limitations, reasons for caution The small sample size regarding the euploidy outcome stands as a limitation of the study. Further studies should focus on the effect of recombinant hyaluronidase on genetic anomalies on blastocysts. Wider implications of the findings Fertilization, usable blastocyst and euploidy rates did not differ when employing bovine-derived or human recombinant hyaluronidase, rendering the latter a safe and efficient alternative. However, it is important to assess long-term effects, as only a limited number of studies report on perinatal and neonatal outcomes Trial registration number 25-12-21MTYPUECIT

Comparative Clinical Study of Percutaneous Epididymal Sperm Aspiration and Testicular Biopsy in the Outcome of ICSI-Assisted Fertility Treatment in Patients with Obstructive Azoospermia.

ObjectiveTo compare and contrast the effects of percutaneous epididymal sperm aspiration (PESA) and testicular sperm aspiration (TESA) on the outcome of intracytoplasmic sperm injection (ICSI)-assisted fertility treatment in patients with obstructive azoospermia.MethodsPatients with obstructive azoospermia with an age distribution of 20–36 years admitted to the male department of the Reproductive Center of the Second Affiliated Hospital of South China University (Hengyang Nanhua Xing Hui Reproductive Health Hospital) from December 2018 to December 2020 were used in this study. One group was set up as the PESA group to perform PESA, and the other group was set up as the TESA group to perform percutaneous testicular biopsy for sperm extraction. Patients who were unsuccessful in PESA continued to undergo TESA, and if sperm were retrieved, they were classified as the TESA group. General information on male patients and their partners was collected and compared in patients from different sperm source groups. Embryo development (normal fertilization rate, high-quality embryo rate, and high-quality blastocyst rate) and pregnancy outcome (clinical pregnancy rate, miscarriage rate, and ectopic pregnancy rate) were compared between the two groups.ResultsFinally, there were 26 patients in the PESA group and 31 patients in the TESA group. There were no significant differences in terms of age, years of infertility, testosterone level, (FSH) follicle-stimulating hormone level, and testicular volume between the male patients in the PESA and TESA groups of two different sperm sources, and no significant differences were found in the general conditions of the female patients in terms of age, number of eggs obtained, number of sinus follicles, basal FSH value, and basal E2 value (p > 0.05). The rate of high-quality blastocysts in the TESA group was significantly higher than that in the PESA group (p < 0.05); the differences in clinical normal fertilization rate, high-quality embryo rate, clinical pregnancy rate, miscarriage rate, and ectopic pregnancy rate between the two groups were not statistically significant (p > 0.05).ConclusionICSI with different sources of sperm in patients with male factor infertility alone, which had no significant effect on embryo development, embryo implantation rate, clinical pregnancy rate, and miscarriage rate, resulting in better clinical outcomes.

Effects of Oocyte Cytoplasmic Central Granulation on Embryonic Development, Blastocyst Formation, and Pregnancy Outcome in Assisted Reproductive Technology and Its Mechanism.

To investigate whether oocyte centrally located cytoplasmic granulation (CLCG) affects embryonic development, blastocyst formation, and pregnancy outcomes in assisted reproductive technology, fifty patients with CLCG in all oocytes were selected as the CLCG group. Then, 150 patients with no abnormal oocyte morphology were randomly recruited as the control group. Both groups underwent laparoscopy, hysterosalpingography (HSG), vaginal ultrasound, and male semen examination. The down-regulation regimen was selected regarding the patient's ovarian reserve. The egg maturation rate, normal fertilization rate, cleavage rate, available embryo rate, and high-quality embryo rate in the CLCG group were greatly inferior to those in the control group, with remarkable differences (P< 0.05). CLCG grading of oocytes after 30 cycles in the CLCG group indicated that there were 36 cases in the mild group and 14 cases in the severe group. The egg maturation rate in the mild group was lower than that in the severe group, with a notable difference (P< 0.05). The fertilization rate, cleavage rate, available embryo rate, and high-quality embryo rate in the mild group were higher than those in the severe group, with a notable difference (P< 0.05). Compared with the control group, there was no considerable difference in the implantation rate, clinical pregnancy rate, and abortion rate between the CLCG group (P> 0.05). In summary, oocyte CLCG may affect fertilization, embryonic development, and blastocyst formation, but not pregnancy outcomes.

Early Cervical Lesions Affecting Ovarian Reserve and Reproductive Outcomes of Females in Assisted Reproductive Cycles.

To estimate the effects of early cervical lesions (ECL) on female reproductive function and IVF/ICSI cycle outcomes, a retrospective cohort study involving 111 infertile women from 2014 to 2019 was performed. Thirty-seven women with a history of ECL and seventy-four controls, undergoing IVF/ICSI cycles, were included in the ECL group and comparison group respectively. Demographic characteristics, ovarian reserve, and IVF/ICSI cycle outcomes of both groups were collected. Basal serum FSH level, AMH level, AFC, number of oocytes retrieved and matured, normal fertilization rate, embryo available rate, blastocyst formation rate, implantation rate, pregnancy rate, and cumulative live birth rate (CLBR) were assessed and compared. We observed that while both groups were similar concerning baseline features, significantly more women in the ECL group were diagnosed as poor ovarian response (POR), compared with those in the comparison group (27.0% vs. 10.8%, P=0.003). The pregnancy rate and LBR for a complete cycle were both significantly lower in the ECL group (38.5% vs. 58.8%, P=0.021; 28.9% vs. 48.2%, P=0.025, respectively). The conservative and optimal CLBRs for up to four complete cycles in the ECL group were also lower than those in the comparison group (40.5% vs. 55.4%, P=0.140; 45.9% vs. 67.6%, P=0.028). Longer time intervals (over one year) between ECL diagnosis/treatment and assisted reproductive technology (ART) cycle start negatively affected the pregnancy rate and LBR. In conclusion, female patients with ECL history seemingly have a lower ovarian reserve, reduced pregnancy rate, and decreased live birth rate (LBR), compared with age-matched women undergoing IVF/ICSI.

Investigating the impact of SARS-CoV-2 infection on basic semen parameters and in vitro fertilization/intracytoplasmic sperm injection outcomes: a retrospective cohort study

BackgroundThis study aimed to evaluate the influences of SARS-CoV-2 infection on semen parameters and investigate the impact of the infection on in vitro fertilization (IVF) outcomes.MethodsThis retrospective study enrolled couples undergoing IVF cycles between May 2020 and February 2021 at Tongji Hospital, Wuhan. Baseline characteristics were matched using propensity score matching. Participants were categorized into an unexposed group (SARS-COV-2 negative) and exposed group (SARS-COV-2 positive) based on a history of SARS-CoV-2 infection, and the populations were 148 and 50 after matching, respectively. IVF data were compared between the matched cohorts. Moreover, semen parameters were compared before and after infection among the infected males. The main measures were semen parameters and IVF outcomes, including laboratory and clinical outcomes.ResultsGenerally, the concentration and motility of sperm did not significantly differ before and after infection. Infected males seemed to have fewer sperm with normal morphology, while all values were above the limits. Notably, the blastocyst formation rate and available blastocyst rate in the exposed group were lower than those in the control group, despite similar mature oocytes rates, normal fertilization rates, cleavage rates, and high-quality embryo rates. Moreover, no significant differences were exhibited between the matched cohorts regarding the implantation rate, biochemical pregnancy rate, clinical pregnancy rate, or early miscarriage rate.ConclusionsThe results of this retrospective cohort study suggested that the semen quality and the chance of pregnancy in terms of IVF outcomes were comparable between the males with a history of SARS-CoV-2 infection and controls, although a decreased blastocyst formation rate and available blastocyst rate was observed in the exposed group, which needs to be reinforced by a multicenter long-term investigation with a larger sample size.

In vitro maturation of human oocytes maintaining good development potential for rescue intracytoplasmic sperm injection with fresh sperm.

BACKGROUNDThe outcomes of the use of commercial in vitro maturation (IVM) medium to culture immature oocytes obtained from conventional ovulation induction, followed by rescue intracytoplasmic sperm injection (RICSI), are not ideal. It is thus difficult to widely adopt this approach in clinical practice. Therefore, it is necessary to explore methods for improving the clinical outcome of IVM.AIMTo study the effect of sperm on the developmental potential of in vitro-matured oocytes in conventional culture.METHODSThis was a retrospective study of patients whose immature oocytes were harvested from conventional oocyte stimulation cycles and underwent ICSI at our hospital between June 2018 and August 2020. RICSI was performed using sperm collected on the day of oocyte harvest (old) and sperm collected on the day of RICSI (fresh) and oocytes matured in vitro after 24 h of culture in conventional medium. The rates of in vitro oocyte maturation, normal fertilization, normal cleavage, day-3 top-quality embryos, and useful blastocyst formation were compared between the two groups.RESULTSIn total, 102 germinal vesicle (GV)-stage immature oocytes were cultured in the old sperm group. In the fresh sperm group, 122 GV-stage immature oocytes were collected and cultured in vitro for 24 h. There were no significant differences in the general conditions of males and females between the two groups (P > 0.05). The oocyte maturation, normal fertilization, and normal cleavage rates of the old and fresh groups were 51.0% vs 55.7%, 61.5% vs 64.7%, and 93.8% vs 93.2%, respectively. None of the rates differed significantly (P > 0.05) between the two groups. However, the day-3 top-quality embryo and useful blastocyst rates of the old and fresh sperm groups were 16.6% vs 63.4%; 6.67% vs 34.6%, respectively. The day-3 top-quality embryos and useful blastocyst rates of the old sperm group were significantly lower than those of the fresh group (P < 0.05). CONCLUSION In vitro maturation with conventional culture medium combined with the use of fresh sperm collected on the day of RICSI is an easy-to-implement strategy for patients whose oocytes are completely or mostly immature.

Addition of MitoTEMPO to the maturation medium improves in vitro maturation competence of bovine oocytes.

The effects of MitoTEMPO, a mitochondria-targeted antioxidant, and its non-targeted parent, TEMPO, on bovine oocyte maturation competence have not been determined so far. Hence, our study was aimed to investigate the effects of supplementing maturation medium with different concentrations of MitoTEMPO (0.00, 0.10, 1.00 and 10.00 µM) or TEMPO (0.00, 5.00, 10.00 and 15.00 mM) on in vitro maturation (IVM) and fertilization (IVF) of bovine oocytes. The oocytes after IVM and IVF were evaluated for the signs of nuclear maturation and normal fertilization. The average number of spermatozoa penetrated per oocyte and the level of intracellular reactive oxygen species (ROS) were also evaluated. The results showed that percentages of bovine oocytes reached the metaphase II stage of meiosis were significantly higher in the 1.00 µM MitoTEMPO group compared to the control group (without antioxidant supplementation). The normal fertilization rate also tended to be greater in this group than the control group. In comparison with the control group, the medium supplementation with 1.00 µM MitoTEMPO led to a significant decrease in the intracellular ROS level. The average number of spermatozoa penetrated per oocyte was not significantly different among the antioxidant-treated and the non-treated groups. The TEMPO addition to the maturation medium affected neither the rate of maturation/fertilization nor the level of intracellular ROS in bovine oocytes. Based on these results, we concluded that MitoTEMPO at a concentration of 1.00 µM had beneficial effects on the quality and fertilization potential of bovine oocytes.

Influence of the sperm DNA fragmentation index on the outcome of rescue ICSI and the clinical value of rescue ICSI.

As a remedy for the failure of in vitro fertilization (IVF), rescue intracytoplasmic sperm injection (R-ICSI) has been widely carried out, but it has failed to significantly improve the fertilization rate and clinical pregnancy rate. Sperm DNA fragmentation index (DFI) was highly correlated with pregnancy outcome of artificial assisted reproduction. This study aims to investigate the effect of the sperm DFI on the outcome of R-ICSI and the clinical value of R-ICSI. This retrospective analysis was conducted among 140 infertile couples receiving R-ICSI in from January 2014 to December 2019. The subjects were assigned into a total fertilization failure (TFF)+low DFI group (R-ICSI after TFF and DFI<30%) (n=63), a TFF+high DFI group (R-ICSI after TFF and DFI≥30%) (n=16), a partial fertilization failure (PFF)+low DFI group (R-ICSI after PFF and DFI<30%) (n=52), a PFF+high DFI group (R-ICSI after PFF and DFI≥30%) (n=9). All transferred embryos were come from R-ICSI. The general clinical data [infertility duration, male age, female age, basal serum level of follicle stimulating hormone (FSH), basal serum level of luteinizing hormone (LH), antral follicle count, endometrial thickness of human chorionic gonadotropin (HCG) day, and eggs] and R-ICSI cycle outcomes (fertilization rate, normal fertilization rate, cleavage rate, good embryo rate, implantation rate, clinical pregnancy rate and live birth rate) were analyzed. In addition, the effect of R-ICSI on the fertilization outcome of conventional IVF total fertilization failure and partial fertilization failure was explored. There was no significant difference in the general clinical data and R-ICSI cycle outcome between the TFF+low DFI group and the TFF+high DFI group (all P>0.05). There was no significant difference in the general clinical data between the PFF+low DFI group and the PFF+high DFI group (all P>0.05). The fertilization rate and normal fertilization rate in the PFF+low DFI group were significantly higher than those in the PFF+high DFI group (85.40% vs 72.41%, 71.90% vs 58.62%, respectively; both P<0.05). However, there was no significant difference in cleavage rate, good embryo rate, implantation rate, clinical pregnancy rate, and live birth rate between the 2 groups (all P>0.05). The R-ICSI cycle of TFF: A total of 79 fresh cycles, 57 fresh transplant cycles, a total of 761 unfertilized oocytes, and 584 M II oocytes were treated with R-ICSI, the fertilization rate was 83.22%, the normal fertilization rate was 75.51%, the cleavage rate was 98.15%, the good embryo rate was 40.74%, the implantation rate was 30.56%, and the clinical pregnancy rate was 43.86%; 29 live births were obtained. The R-ICSI cycle of PFF: A total of 61 fresh cycles, 31 fresh transplant cycles, a total of 721 unfertilized oocytes, and 546 M II oocytes were treated with R-ICSI; the fertilization rate was 83.33%, the normal fertilization rate was 69.78%, the cleavage rate was 97.36%, the good embryo rate was 44.39%, the implantation rate was 25.42%, and the clinical pregnancy rate was 45.16%; 12 live births were obtained. In the case of partial fertilization failure of IVF, the sperm DFI affects the fertilization rate and normal fertilization rate of R-ICSI; whether it is a TFF of IVF or PFF of IVF, ICSI can be used as an effective remedy way.

Intracytoplasmic Sperm Injection Improves Normal Fertilization Rate and Clinical Pregnancy Rate in Male Infertility.

The purpose of this study was to investigate the effect of intracytoplasmic sperm injection (ICSI) on the normal fertilization rate of oocytes and the clinical pregnancy rate of cycles in male infertility. Eighty cases of male infertility patients attending our hospital from March 2020 to March 2021 were selected and divided into observation group and control group using the random number table method, with 40 cases in each group. The control group was treated with in vitro fertilization (IVF), and the observation group was treated with ICSI. The normal fertilization rate of oocytes and the clinical pregnancy rate of the cycle were compared between the two groups, and the adverse pregnancy outcome and obstetric and perinatal complications were compared between the two groups The rate of normal fertilization and clinical pregnancy in the cycle was higher in the observation group (P < 0.05). The differences were not statistically significant (P > 0.05) when comparing adverse pregnancy outcomes between the two groups. The difference was not statistically significant (P > 0.05) when comparing obstetrics (5.41%, 10.34%) and perinatal complications (8.33%, 14.81%) between the two groups. ICSI in male infertility is significantly effective in improving the rate of normal oocyte fertilization and the clinical pregnancy rate of the cycle. It also has a low impact on adverse pregnancy outcomes and obstetric and perinatal complications and has a high safety profile.

Influence of Women´s Residence Region on Assisted Reproduction Treatments - Experience of a Tertiary Center in Northern Portugal.

ObjectiveData on the possible influence of women´s region of residence, within the same country, on the outcomes of medically assisted reproduction cycles are scarce. This study aims to assess the impact of the women's region of residence on the results of in-vitro fertilization cycles.MethodsWe evaluated in-vitro fertilization cycles between 2010 and 2017, performed in a northern Portugal assisted reproduction center. We defined two groups: Douro Litoral (group 1; n=783), and Trás-os-Montes and Alto Douro (group 2; n=178). We analyzed demographics and cycle-related variables, and we calculated the rates for embryo transfer cycles. We used the Mann-Whitney and Chi-square tests and p<0.05 was considered statistically significant.ResultsWe included 961 cycles. The region of residence had no effect on the following variables: women´s age; body mass index; or duration of infertility (p>0.05). Group 2 had a statistical significant lower number of previous cycles than group 1 (1.3±0.5 and 1.5±0.7; p=0.005). In the sub-analysis of cycles with embryo transfer (n=781), group 1 obtained had rates of normal fertilization (62.5% vs. 57.5%; p=0.04), miscarriage rate (30.0 vs. 10.9%; p=0.007), and lower implantation rates compared to group 2 (33.3% vs. 50.0%; p<0.001).ConclusionsWomen from the region of Trás-os-Montes e Alto Douro had a lower number of previous cycles, compared to those from the Douro Litoral, despite the absence of statistical significant differences in terms of age or infertility duration. These findings reinforce the need to contemplate the sociodemographic and socioeconomic variables in this context.

THE EFFECTIVITY OF CYSTEAMINE SUPPLEMENTATION ON IMPROVING THE IN VITRO FERTILIZATION RATE OF SHEEP OOCYTES

The purpose of this study was to evaluate the effectiveness of cysteamine supplementation on the maturation medium and/or in vitro fertilization medium with regards to improving the normal fertilization rate of sheep oocytes, which are characterized by the formation of two pronuclei. Grade A and B oocytes were matured in medium-199 with 0.3% bovine serum albumin (BSA), 10 IU/mL follicle stimulating hormone (FSH), 10 IU/mL human chorionic gonadotrophin (hCG), and 50 μg/mL gentamicin added for 24 hours in a 5% CO2 incubator at 39° C. The treatment group was divided into the following groups: a control group with no cysteamine (P1), a group with 100 μM cysteamine in the maturation medium (P2), a group with 100 μM cysteamine in the fertilization medium (P3), and a group with 100 μM cysteamine both in the maturation and fertilization medium (P4). The fertilization was carried out by incubating sperm-oocytes for 12 hours and then staining them with 1% aceto-orcein to observe the formation of a pronucleus. Normal fertilization rates obtained by each treatment group were 56.5% (P1), 57.1% (P2), 57.8% (P3), and 59.9% (P4) with no significant difference (P0.05) between groups. It was concluded that 100 μM cysteamine supplementation in both the maturation medium and fertilization medium was not able to increase the normal fertilization rate of sheep oocytes.

Usefulness of expanding the indications of early rescue intracytoplasmic sperm injection.

PurposeEarly rescue intracytoplasmic sperm injection (ICSI) is often performed in cases in which not even a single oocyte has extruded a second polar body 6 h after insemination. We evaluated the usefulness of expanding the indications of early rescue ICSI to cases in which <80% of oocytes have extruded second polar bodies 6 h after insemination.MethodsEarly rescue ICSI was performed on oocytes that were denuded 2.5 h post‐insemination and whose extrusion of the second polar bodies had been examined 6 h post‐insemination with a PolScope.ResultsIn vitro fertilization was performed on 24 496 oocytes of 4944 cycles, and 1438 cycles had <80% rate of the second polar body extrusion. Rescue ICSI was performed on 3933 oocytes. Three pronuclei (3PN) incidence of rescue ICSI was 3.0% in oocytes with ≥50% rate of the second polar body extrusion. With respect to the second polar body extrusion rate, no differences were observed in normal fertilization, blastocyst development, implantation, miscarriage, or live birth rates for rescue ICSI.ConclusionBy expanding the indications of early rescue ICSI using the PolScope to cases in which <80% of oocytes have extruded the second polar bodies, many fertilized oocytes can be obtained without considerably increasing the 3PN rate.

Analysis of maturation dynamics and developmental competence of in vitro matured oocytes under time-lapse monitoring

BackgroundTo improve the developmental competence of in vitro cultured oocytes, extensive literature focused on maturation rate improvement with different additives in culture medium, while studies investigating the maturation dynamics of oocytes during in vitro maturation (IVM) and the influencing factors on oocyte viability are scarce.MethodsThe study involved a retrospective observation by time-lapse monitoring of the IVM process of 157 donated GV oocytes from 59 infertile couples receiving ICSI in 2019, in Tongji Hospital, Wuhan, China. The GV oocytes derived from controlled ovarian hyperstimulation (COH) cycles underwent rescue IVM (R-IVM), and the maturation dynamics, including GVBD time (GV-MI), time from GVBD to maturation (MI-MII), maturation time (GV-MII), and MII arrest duration (MII-ICSI), were recorded by time-lapse monitoring. The matured oocytes were inseminated at different MII arrest points and subsequent embryo developments were assessed. The effects of baseline clinical characteristics, oocyte diameters, and maturation dynamics on the developmental competence of the oocytes were also analyzed.ResultsTotally, 157 GV oocytes were collected. GVBD happened in 111 oocytes, with a median GV-MI duration of 3.7 h. The median MI-MII duration was 15.6 h and the median GV-MII duration was 19.5 h. The maturation rate reached 56.7% at 24 h and 66.9% at 48 h, and the clinical factors, including patient age, FSH level, AMH level, ovarian stimulation protocol, and serum estradiol and progesterone levels on hCG trigger day, showed no effects on the 24-h maturation rate. The normal fertilization rate of oocytes resuming meiosis within 8 h and matured within 24 h was significantly higher than that of oocytes resuming meiosis after 8 h and matured after 24 h. Furthermore, among those oocytes matured within 24 h, the high-quality embryo formation rate of oocytes resuming meiosis within 4.5 h and matured within 19 h was significantly higher. All stated time was measured from the start point of IVM. Additionally, for oocytes from patients with serum progesterone levels less than 1 ng/ml on hCG trigger day, the high-quality embryo formation rate was significantly increased.ConclusionR-IVM technology could increase the available embryos for patients in routine COH cycles, but excessive culture beyond 24 h is not recommended. GV-MI duration of the oocyte, recorded by time-lapse system, and serum progesterone levels of patients on hCG trigger day can significantly affect the developmental potential of the IVM oocytes.

Efficacy of artificial oocyte activation in patients with embryo developmental problems: a sibling oocyte control study.

To explore whether artificial oocyte activation (AOA) can improve embryo developmental potentiality and pregnancy outcomes for patients with a history of embryo developmental problem. This was a retrospective study and candidate patients with embryo development problems were collected. A total of 1422 MII eggs from the enrolled 140 patients were randomized divided equally into 2 groups, half for the AOA group (AOA), and the rest of sibling mature eggs for the control group (non-AOA). The patients were further divided into two subgroups: (1) the rate of good-quality day 3 embryos was 0% (group 1, n = 66); (2) the rate of good-quality day 3 embryos ranged from 1 to 30% (group 2, n = 74). In the early embryonic growth, there were no significant differences in the outcomes of AOA and non-AOA groups in terms of normal fertilization rates, cleavage rates, day 3 good-quality embryo rates and available blastocyst rates (72.7% vs. 79.3%, 97.4% vs. 98.0%, 20.1% vs. 19.7%, 6.6% vs. 8.4% in group 1, respectively; 77.7% vs. 81.9%, 98.1% vs. 97.0%, 25.8% vs. 22.1%, 9.6% vs. 9.3% in group 2, respectively). In the late embryonic growth, no significant differences were found in biochemical and clinical pregnancy rates, implantation rates, miscarriage rates, and live-birth rates (50.0% vs. 45.2%, 45.2% vs. 40.5%, 37.3% vs. 31.3%, 10.5% vs. 11.8%, 40.5% vs. 35.7%, respectively) between two groups. In addition, neonatal outcomes were similar in both the groups as well. Our study demonstrated that the AOA using ionomycin 1h after ICSI did not bring benefits to the early or late development of embryos derived from patients with a history of embryo developmental problems.

Effect of early rescue ICSI and split IVF-ICSI in preventing low fertilization rate during the first ART cycle: A real-world retrospective cohort study.

PurposeTo determine the utility of short gamete coincubation in in vitro fertilization (IVF‐S) combined with early rescue intracytoplasmic sperm injection (R‐ICSI) and split IVF‐ICSI in preventing low fertilization based on a retrospective cohort study.MethodsCouples with a high risk of low IVF fertilization during the first ART cycle underwent IVF‐S with R‐ICSI or split IVF‐ICSI. Fertilization rate, embryo quality, and clinical outcomes were measured.ResultsAfter propensity score matching, we included 188 couples in the IVF‐S with R‐ICSI group as Group 1 and 720 in the split IVF‐ICSI group as Group 2. Normal fertilization rates were similar; however, Group 1 had a higher multiple pronuclei rate (10.42% vs. 4.50%, p < 0.001) but a higher embryo utilization rate (59.84% vs. 53.60%, p < 0.001). The groups were similar in the rates of high‐quality embryos, embryo implantation, clinical pregnancy, and live birth. Low IVF fertilization rate was 4.79% and 9.03% in Group 1 and Group 2, respectively, with similar fertilization rate and embryo development.ConclusionIVF‐S with early R‐ICSI and split IVF‐ICSI were effective strategies in preventing low fertilization rate. IVF‐S with early R‐ICSI could become the preferred approach because of its advantages—higher embryo utilization rate, fewer ICSI procedures, similar clinical pregnancy rate, and live birth rate.

Magnetic-activated cell sorting of nonapoptotic spermatozoa with a high DNA fragmentation index improves the live birth rate and decreases transfer cycles of IVF/ICSI.

The present study aimed to evaluate the clinical outcomes of magnetic-activated cell sorting (MACS) in sperm preparation for male subjects with a sperm DNA fragmentation index (DFI) ≥30%. A total of 86 patients who had undergone their first long-term long protocol were selected. The protocol involved in vitro fertilization (IVF) and intracytoplasmic sperm injection (ICSI) cycles, and the patients were divided into the MACS or control groups. The MACS group included sperm samples analyzed with MACS that were combined with density gradient centrifugation (DGC) and the swim-up (SU) technique (n = 39), and the control group included sperm samples prepared using standard techniques (DGC and SU; n = 41). No differences were noted with regard to basic clinical characteristics, number of oocytes retrieved, normal fertilization rate, cleavage rate, or transplantable embryo rate between the two groups in IVF/ICSI. In addition, the clinical pregnancy and implantation rates of the first embryo transfer cycles indicated no significant differences between the two groups. However, there was a tendency to improve the live birth rate (LBR) of the first embryo transfer cycle (63.2% vs 53.9%) and the cumulative LBR (79.5% vs 70.7%) in the MACS group compared with the control group. Moreover, the number of transferred embryos (mean ± standard deviation [s.d.]: 1.7 ± 0.7 vs 2.3 ± 1.6) and the transfer number of each retrieved cycle (mean ± s.d.: 1.2 ± 0.5 vs 1.6 ± 0.8) were significantly lower in the MACS group than those in the control group. Thus, the selection of nonapoptotic spermatozoa by MACS for higher sperm DFI could improve assisted reproductive clinical outcomes.

Protective Mechanism of Luteinizing Hormone and Follicle-Stimulating Hormone Against Nicotine-Induced Damage of Mouse Early Folliculogenesis.

Previous studies have shown that nicotine could impair the germ cell cyst breakdown and the primordial follicle assembly by autophagy. In this paper, we discovered that luteinizing hormone (LH) and follicle-stimulating hormone (FSH) could counteract the damage caused by nicotine of mouse germ cell cyst breakdown. The neonatal mice were separately intraperitoneally injected with nicotine, nicotine plus LH, nicotine plus FSH, and saline (control) for 4 days. Compared with the nicotine group, the quality of oocytes and the number of follicles were remarkably increased in the nicotine plus LH group or nicotine plus FSH group. LH and FSH could alleviate nicotine-induced oocyte autophagy by different pathways. LH reduced the nicotine-induced autophagy by restoring the phosphorylation level of adenosine 5′-monophosphate-activated protein kinase α-1, while FSH by downregulating the phosphorylation level of Forkhead box class O 1. In addition, in a subsequent study of 6-week mice in different treated groups, we found that LH and FSH supplementation significantly improved normal maturation rates, fertilization rates, and embryo’s developmental potential of oocytes in oocytes exposed to nicotine. Taken together, these results suggested that LH and FSH could counteract the damage caused by nicotine and finally ensure normal germ cell cyst breakdown and early embryo development.