Effects of Sperm Pretreatment on Efficiency of ICSI-Mediated Gene Transfer in Pigs

Intracytoplasmic sperm injection (ICSI)-mediated gene transfer has been described as a technique to obtain transgenic offspring in mice. However, this approach has had limited success in domestic animals due to poor embryo development after ICSI. A first experiment was designed to improve embryo development comparing ICSI-mediated gene transfer with or without chemical activation (CA) in the ovine species. In the second experiment, ICSI-mediated gene transfer assisted by CA was used in porcine, feline, equine, and bovine species. Maturation and culture were done by standard procedures. Semen was collected by artificial vagina in ovine and bovine species. In pigs, ejaculates were obtained using the gloved-hand method, and in feline and equine species, sperm were obtained from epididymides. Samples were frozen by standard means. Thawed spermatozoa were washed twice in Na citrate at 2.8% with 100 µm EDTA at 495g for 5 min and resuspended in Na citrate with 0.5 µg of pCX-EGFP/million spermatozoa for 5 min at 0�C. The pCX-EGFP plasmid contained the egfp gene expressed under chimerical CMV-IE-chicken β-actin promoter control. Sperm cells were immediately injected into the metaphase II oocyte and CA was induced by incubation in TALP-HEPES with 5 µm ionomycin for 4 min, cultured in TCM199 for 3 h, and transferred to a droplet of 1.9 mm 6-dimethylaminopurine (DMAP) for 3 h. During the in vitro culture, exposure to blue light (488 nm) was performed to determine the percentage of green embryos, mosaic expression, and earliest stage of egfp expression. Fluorescence in situ hybridization analysis was performed labeling pCX-EGFP plasmid by nick translation for use as a probe. Statistical analysis was done by chi square. In ovine species, development to blastocyst stage (0/88 v. 3/86; P > 0.05) and number of green embryos (24/88 v. 39/86; P < 0.05) were greater with CA. The egfp expression in ovine embryos assisted by CA began at the 2- (7/39), 4- (9/39), or 8-cell (23/39). However, the expression in ovine embryos without CA occurred only at the 8-cell stage (24/24) stage. In porcine, bovine, feline, and equine species, green embryos were detected at a high proportion (33/55, 10/44, 9/35, and 5/17, respectively), and the percentage of fluorescent blastocysts was 2.3, 2.9, and 9.1% for ovine, feline, and bovine species, respectively. The egfp expression in porcine and feline embryos started at the 2-cell stage (36 and 22%, respectively), whereas it began in bovine and equine embryos at the 4-cell stage (9 and 40% respectively). All species showed a high frequency of mosaic expression (range 60-85%), and the preliminary FISH analysis demonstrated a variable number of integration events in porcine embryos. To our knowledge, this is the first report of exogenous DNA expression in feline and equine embryos. These results suggest that the CA accelerates and increases the pCX-EGFP expression in ovine embryos in agreement with previous studies that have shown earlier expression of genes for parthenogenetic and cloning embryos, both assisted by CA. In conclusion, ICSI-mediated gene transfer assisted by CA can be used to obtain exogenous gene-expressing embryos in domestic species with potential scientific and commercial interests.

IMSI is beneficial in cases of advanced maternal age: a prospective randomized study

Study question Is it possible to identify the optimal puncture position by image analysis where oocyte degeneration is reduced in ICSI procedure? Summary answer Visualizing the likelihood of unintentional membrane rupture (UMR) using image analysis can reduce oocyte degeneration and thereby generate more embryos available for treatment. What is known already It is known that the oocyte degeneration after both conventional-ICSI and Piezo-ICSI are often observed when UMR occurs during the puncturing process and the likelihood of UMR may depends on the location of the puncture site on the oolemma. Identifying the appropriate puncturing position may decrease the likelihood of membrane rupture and thus degeneration, however, there are no studies identifying the optimal puncture position during ICSI. Study design, size, duration We have developed the ICSI Position Detector (IPD), which can identify an area where rupture is likely to occur and visualize it on a video monitor. This study included 1,110 mature oocytes retrieved from 190 consenting patients. Matured oocytes were inseminated either by Conventional-ICSI (n = 437, average maternal age: 39.1±4.6 y.o.) or Piezo-ICSI (n = 673, average maternal age: 38.6±4.5 y.o.). ICSI was performed blindly, moving images were recorded and analyzed retrospectively during ICSI using IPD. Participants/materials, setting, methods Inseminated oocytes were subsequently divided into two groups according to IPD results, oocytes on which ICSI was performed at the position with a low chance of UMR (appropriate group), and with a high chance of UMR (non-appropriate group) by IPD. The rates of UMR, degeneration, fertilization (2PN), blastocyst formation, and good-quality blastocyst (Grade 3BB and above on day 5 by the Gardner scoring) were compared between the two groups of both conventional-ICSI and Piezo-ICSI. Main results and the role of chance The areas in which UMR is less likely to occur and more likely to occur are heterogeneously distributed. When ICSI was performed blindly, about half was from the non-appropriate position. In appropriate group of Piezo-ICSI, rates of UMR (4.3% vs. 17.4%, P < 0.001) and degeneration (1.0% vs. 5.7%, P < 0.001) were significantly lower than those of non-appropriate group, whereas rates of fertilization (88.0% vs. 78.4%, P < 0.01) and blastocyst formation (57.8% vs. 45.9%, P < 0.01) were significantly higher than those of non-appropriate group, respectively. The rate of good-quality blastocyst (28.8% vs. 24.9%) was in favor of appropriate group, but not significantly different. In appropriate group of Conventional-ICSI, rates of UMR (6.7% vs. 20.6%, P < 0.001) and degeneration (1.5% vs. 6.6%, P < 0.01) were significantly lower than those of non-appropriate group, whereas rate of fertilization (88.7% vs. 69.5, P < 0.001) was significantly higher than those of non-appropriate group, respectively. The rates of blastocyst formation (37.5% vs. 35.3%), good-quality blastocyst (21.3% vs. 18.7%) were all in favor of appropriate group, but not significantly different. Limitations, reasons for caution This is a single private fertility clinic study. Its reproducibility should be assessed in different laboratory conditions and the hands of different operators. Moreover, specific studies should be addressed on the effect of the other putative confounders under investigation (e.g. kind of ovulation trigger, patient demographics, culture environment, etc.). Wider implications of the findings This study demonstrated that the IPD is useful to identify the optimal puncture location site to prevent UMR on ICSI procedure, resulting in reducing UMR and degeneration of the oocytes and increasing culture results, thereby, generating more embryos available for transfer or cryopreservation. Trial registration number not applicable

Study question Does IMSI show beneficial effects on fertilization, blastocyst formation, euploid, clinical pregnancy, implantation, and miscarriage rates compared to conventional ICSI in the same patient group? Summary answer IMSI improved euploid, clinical pregnancy, and implantation rates and reduced miscarriage rate compared to the ICSI group. What is known already IMSI is performed under x6600 magnification to select morphologically normal spermatozoa, whereas conventional ICSI is x200-400. Clinical outcomes of IMSI have been reported to be better than those of ICSI. However, some studies have reported that the effectiveness of IMSI compared to ICSI is unclear. This study aims to examine fertilization, blastocyst formation, euploid, clinical pregnancy, implantation, and miscarriage rates between IMSI and ICSI in the same patients undergoing at least one ICSI-in vitro fertilization (IVF) cycle and one IMSI-IVF cycle. Study design, size, duration A retrospective cohort study was conducted involving 682 couples who underwent at least one ICSI-IVF cycle and one IMSI-IVF cycle from January 2021 to September 2024 in CHA Fertility Center Seoul station, Seoul, Korea. Patients who had ever undergone testicular sperm extraction were excluded from analyses to reduce the outlier effect. Participants/materials, setting, methods Sperm were selected under x400 and x6600 magnification in ICSI and IMSI groups, respectively. Trophectoderm biopsy was performed on day 5 or 6 of development. Preimplantation genetic testing was performed using next-generation sequencing. Clinical pregnancy and miscarriage were defined as the presence of the gestational sac after 6 weeks of gestation and pregnancy loss before 20 weeks of gestation, respectively. Statistical analyses were performed using the paired t-test and chi-square test. Main results and the role of chance The number of retrieved oocytes, inseminated oocytes, zygotes, blastocysts, biopsied embryos, and euploid embryos in the IMSI versus ICSI group were 9.08±7.48 vs. 9.00±6.77 (n = 682, p = 0.642), 6.97±5.73 vs. 6.71±5.10 (n = 682, p = 0.094), 5.31±4.45 vs. 5.15±4.04 (n = 682, p = 0.236), 1.78±1.94 vs. 1.65±1.42 (n = 682, p = 0.038), 1.76±1.95 vs. 1.39±1.37 (n = 430, p < 0.001), and 0.22±0.54 vs. 0.11±0.31 (n = 430, p < 0.001), respectively. The euploid rate (11.03±22.76% vs. 6.65±14.68%; n = 276, p = 0.002), implantation rate (47.0% (55/117) vs. 16.8% (35/208); p < 0.001) and clinical pregnancy rate (57.7% (45/78) vs. 23.9% (32/134); p < 0.001) were significantly higher in the IMSI group compared to ICSI. The miscarriage rate was significantly lower in the IMSI group than ICSI (13.3% (6/45) vs. 71.9% (23/32); p < 0.001). The study found that the IMSI group showed comparable fertilization rates (78.84±20.31% vs. 78.02±15.87%; n = 682, p = 0.374) and blastocyst formation rate (35.49±28.64% vs. 36.06±23.26%; n = 672, p = 0.638) to the ICSI group. Limitations, reasons for caution The live birth rate was not included in this study. Since IMSI is generally recommended for patients with male infertility, the clinical advantages of IMSI over ICSI remain unclear across all patients. Wider implications of the findings IMSI offers superior clinical outcomes compared to conventional ICSI, particularly in terms of euploid, implantation, clinical pregnancy, and miscarriage rates. These findings suggest that IMSI may be a more effective option for patients with poor sperm morphology. Trial registration number No

Physiology of early embryonic development is required for a further application of reproductive biotechnology in swamp buffalo. However nowadays a few studies were reported and basic knowledge is limited. This thesis composes of three parts as follows: EXP.1aimed to study the dynamics of early embryonic development, in terms of redistribution of cytoskeleton (microtubules, actin microfilaments) and chromatin configurations during the first cell cycle in swamp buffalo embryos. Swamp buffalo oocytes were matured and fertilized in vitro, presumptive zygotes and embryos were fixed at various time points post-IVF. Microtubules, microfilaments and chromatin were fluorescently labeled using monoclonal-α-tubulin, Phalloidin and DAPI, respectively. The redistribution pattern of cell cytoskeleton and chromosome of the zygotes and embryos was examined under an epifluorescent microscope. The results indicated that a dense network of microtubules or sperm aster in which radiating from the base of the decondensing sperm head plays a crucial role in the fertilization events about migration and apposition of male and female pronuclei in a normal fertilization process whereas microfilaments are considerably required for contractile ring formation during cleavage. Fertilization failure, at least in our current culture system, is predominantly caused by poor sperm penetration. However, partial digestion of ZP did not improve fertilization rate. EXP. 2 aimed to : 1) examine the efficiency of intracytoplasmic sperm injection (ICSI) technique, with or without chemical activation of in vitro matured buffalo oocytes, on sperm head decondensation; 2) compare the subsequent development of embryos following activation of ICSI (ICSI (+) activation group) and sham injection (Sham (+) activation group) oocytes (embryos obtained by in vitro fertilization of IVM oocytes served as a control group); and 3)clarify whether blastocysts were derived from syngamy or parthenogenesis, expression of Nnat, a paternally-expressed gene in blastocysts derived from IVF, ICSI and oocyte activation without sperm or sham injection was additionally examined using RT-PCR. Pronuclear formation rates in ICSI (+) activation and Sham (+) activation groups were higher than that of ICSI without activation (P<0.05). However, since 90.9% of presumptive zygotes in ICSI (+) activation group demonstrated pronuclear formation with an intact sperm head, we inferred that most were parthenotes. Neither developmental competence (morula and blastocyst formation rates) nor mean total cell number of blastocysts was significantly different among ICSI (+) activation, Sham (+) activation and IVF groups. Expression of Nnat mRNA was not detected in ICSI (+) activation blastocysts, indicating failure of male genome activation. EXP. 3 aimed to improve sperm head decondensation by pretreating spermwith various chemicals before ICSI. Sperm were treated with the following protocols; (1) 0.1% Triton-X 100 (TX), (2) 10 µM calcium ionophore (CaI), (3) freezing and thawing (FT) without any cryoprotectant, and (4) untreated control. In each treatmentsperm were then either treated or not with 5 mMdithiothreitol (DTT). Acrosome integrity and DNA fragmentation were evaluated in sperm before ICSI by staining of sperm with fluorescein isothiocyanate–labeled peanut agglutinin and TUNEL, respectively. Then in vitro matured oocytes were subjected to ICSI using sperm pretreated as described above. The results revealed significantly increased rates of acrosome-lost sperm cells after TX and CaI treatments, whereas FT treatment and no-treatment (control) significantly increased the proportion of acrosome-reacted sperm. DTT treatment had no significant effect on acrosome configuration of sperm. DNA fragmentation was not significant difference among treatments. At 18 h post-ICSI, female pronucleus (PN) formation was found only in activated oocytes. However, among all the activated ICSI oocytes, the majority of them contained intact sperm heads. Normal fertilization characterized by two PNs without intact sperm head was only observed in CaI and FT treatment and control groups when sperm were treated with DTT before ICSI. In conclusion, these results indicated that DTT treatment of sperm with reacted acrosome before ICSI together with an additional activation of the resultant ICSI oocytes are important for successful sperm head decondensation resulting in male pronuclear formation. This study is the first report to examine the redistribution of cytoskeleton (microtubules, actin microfilaments) and indicates the dynamic of early embryo development during the first cell cycle in swamp buffalo. The fundamental knowledge and techniques from our study can be used as a tool for further investigating the embryonic development. In addition, this study confirms for the first time about the failure of traditional ICSI technique in swamp buffalo oocytes. Sperm treatment before ICSI is nescessory for successful production of normally fertilized embryos.

Intracytoplasmic sperm injection (ICSI) has been intensively used to examine the early events of gamete activation, but few studies have been reported for swamp buffalo. The first objective (Exp. 1) was to compare the developmental competence of oocytes after ICSI using either live or dead frozen-thawed spermatozoa. Matured oocytes were fertilized by ICSI using live (n = 148) or dead (n = 151) spermatozoa, followed by chemical activation using calcium ionophore (A23187) and cyclohexamide (CHX) in SOF medium. In vitro fertilization (n = 149) served as thecontrol. Cleavage rate was recorded on Day 2 and blastocyst formation rate was evaluated on Day 7. The second objective (Exp. 2) was to examine the effects of ICSI and activation regime on the decondensation of buffalo spermatozoa. A total of 148 matured oocytes were subjected to ICSI. The sperm-injected oocytes (n = 87) were then activated using the activation protocol as described in Exp. 1. The ICSI oocytes without activation (n = 61) and sham-injected oocytes with activation (n = 35) were used as controls. Nuclear changes of presumptive zygotes were mor- phologically evaluated for pronuclear formation using 4′,6-diamidino-2-phenylindole procedure and epifluorescent microscopy at 18 h post-ICSI. Statistical differences were determined among the groups using chi-square test. In Exp. 1, the results showed that the percentages of cleavage and blastocyst formation rate were 79.7, 77.0, and 41.6% and 33.8, 30.5, and 14.8% in the live sperm, dead sperm, and IVF groups, respectively. Embryo development rates did not significantly differ between ICSI groups; however, these rates were significantly higher than in the IVF group (P < 0.05). In Exp. 2, the pronuclear formation rate was significantly higher in the ICSI with chemical (70.1%) and sham injection with chemical (60.6%) groups than in the ICSI without chemical group (3.2%; P < 0.01). However, most of the presumptive zygotes with pronuclear formation from the ICSI with chemical activation group showed only intact sperm heads instead of the full male pronuclear formation. Our study suggests that the chemical activation directly affected the female pronuclear formation and embryo development but that it was not associated with the male pronuclear formation. It is postulated that ICSI oocytes that developed to cleavage and blastocyst stages underwent parthenogenesis after chemical activation. This work was supported by TRF-MAG (MRG-WII515S056) and CHE-TRF Senior Research Fund (RTA5080010).

Circulating anti-mullerian hormone levels may predict blastocyst formation rates in donor IVF cycles

Lacking expression of paternally-expressed gene confirms the failure of syngamy after intracytoplasmic sperm injection in swamp buffalo (Bubalus bubalis)

Intracytoplasmic sperm injection (ICSI) of a nonmotile cell into the ooplasm for assisted fertilization is a highly specialized procedure for producing the next generation. The production of piglets by ICSI has succeeded when in vivo-matured oocytes have been used as recipients. Our objective was to generate viable piglets by using porcine oocytes matured in vitro and fertilized by ICSI after evaluating the efficacy of using donor spermatozoa in which the acrosome had been artificially removed by treatment with calcium ionophore A23187 (Ca-I). The rate of acrosomal loss in spermatozoa was increased significantly as the duration of treatment with 10 micro M Ca-I was prolonged for 30-120 min (Ca-I treated; 55.6-78.6%), whereas the rate was not different as the duration of incubation without Ca-I was prolonged for 30-120 min (control; 45.3-58.4%). On the sixth day of in vitro culture after injection of the sperm head and subsequent stimulation with an electrical pulse, the rates of blastocyst formation were not significantly different between the two groups: the rates for oocytes injected with Ca-I-treated sperm heads (incubated for 120 min) and for those injected with control sperm heads were 8.6% and 4.0%, respectively. The mean cell numbers of the blastocysts were not significantly different between the two groups (25.6 and 22.7, respectively). Within 2 h after the stimulation, the injected oocytes were transferred to estrous-synchronized recipients. The three recipients that received oocytes injected with Ca-I-treated sperm heads (77-150 oocytes per recipient) were not pregnant, whereas two of the four recipients given oocytes injected with control sperm heads (55-100 oocytes per recipient) were pregnant. One of these farrowed three (a male and two female) healthy piglets. The results demonstrate clearly that in vitro-matured oocytes injected with sperm heads are developmentally competent and can produce viable piglets. They also suggest that removal of the acrosome from the spermatozoon before injection does not affect the development of the blastocyst in vitro. This might not also improve the production of piglets in vivo.

Evaluation of mosaic frequency in transgenesis by sperm mediated gene transfer on the cultured prepubertal mouse testicular germ cells

The developmental competence of human metaphase I oocytes with delayed maturation in vitro

Production of female bovine embryos with sex-sorted sperm using intracytoplasmic sperm injection: Efficiency and in vitro developmental competence

Special Issue Subject Index Volume 1 (1974) to Volume 50 (1998)

Early preantral mouse follicles with a diameter of 110-160 microm were cultured in vitro for 10 or 12 days. Mature oocytes were retrieved following hCG, and fertilization was attempted either by in vitro fertilization (IVF) or intracytoplasmic sperm injection (ICSI). Two-cell and blastocyst formation rates and blastocyst cell numbers were compared between 10-day and 12-day in vitro-matured oocytes versus in vivo-matured oocytes. Uncleaved IVF oocytes were subjected to chromosome analysis. The 2-cell formation rate was significantly improved by ICSI compared with IVF both in 10-day (72.1% versus 56.1%; P = 0.03) and 12-day cultures (74.1% versus 54.5%; P = 0.028). Cytogenetic analysis of uncleaved MII oocytes following IVF showed that about 30% of MII oocytes showed no sign of sperm penetration. The blastocyst formation rate was significantly lower in 12-day versus 10-day cultures, whether fertilization was by IVF (40.7% versus 62.4%, P = 0.016) or by ICSI (32.5% versus 57.1%, P = 0.035). Blastocyst cell numbers from IVF and ICSI 10-day groups were similar and both significantly higher (P < 0.001) than from IVF 12-day cultures. All above expressed values were significantly higher for in vivo-matured oocytes. In conclusion, fertilization of oocytes from in vitro-matured mouse preantral follicles can be optimized with ICSI, giving significantly higher 2-cell formation rates than IVF. Blastocyst formation rate was not influenced by the technique of fertilization but rather by the extent of the in vitro culture period. Best results on preimplantation development of oocytes for in vitro-matured preantral follicles were obtained with ICSI on oocytes from 10-day in vitro cultures.

In pigs, the embryonic developmental ability after intracytoplasmic sperm injection (ICSI) is inferior to that resulting fromin vitro fertilization (IVF). We evaluated the timing of cell division up to blastocyst formation onembryonic development after ICSI using either whole sperm (w-ICSI) or the sperm head alone (h-ICSI) and IVF as a control. At10 h after ICSI or IVF, we selected only zygotes, and each of the zygotes/embryos was evaluated for cleavage every 24 h until168 h. We then observed a delay in the 1st and 2nd cleavages of h-ICSI embryos and also in blastocoele formation by w-ICSIembryos in comparison with IVF embryos. The rate of blastocyst formation and the quality of blastocysts in both ICSI groupswere inferior to those in the IVF group. In conclusion, the delay in cleavage of porcine ICSI embryos shows poorer embryonicdevelopment.