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PubMed1994

Assisted fertilization with intracytoplasmic sperm injection.

OBJECTIVE: To evaluate the significance of intracytoplasmic sperm injection in severe male factor infertility and previous failed fertilization. DESIGN: Prospective observational study. SETTING: Private infertility clinic, London. SUBJECTS: Sixty-nine patients with a long-standing history of infertility of which 48 had previous failed fertilization, 15 had < 1 million progressive motile sperm per ejaculate, and the remaining 6 had obstructive azoospermia. INTERVENTION: Assisted fertilization with primary intracytoplasmic sperm injection was carried out in 69 IVF. OUTCOME: Normal (two pronuclei [2PN]) fertilization and pregnancy rates. RESULTS: A total of 967, oocytes were collected and 785 were subsequently microinjected. Normal fertilization (2PN) occurred in 410 oocytes (52%) and 90.5% of those cleaved. Sixty-four patients underwent ET, with a total of 181 embryos transferred. Twenty-five patients conceived with a pregnancy rate of 39% per transfer. The implantation rate was 16% and the total pregnancy loss rate 24%. Failed fertilization after intracytoplasmic sperm injection occurred in four cases. CONCLUSIONS: Intracytoplasmic sperm injection is increasingly becoming the treatment of choice in infertile couples where assisted fertilization is indicated. The high fertilization and pregnancy rates observed with this technique, together with a low risk of abnormalities, has revolutionized treatment of male factor infertility.

Adult↗
PubMed2006

Elective cryopreservation of embryos at the pronucleate stage in women at risk of ovarian hyperstimulation syndrome may affect the overall pregnancy rate.

The aim of this retrospective study was to assess the pregnancy outcome and incidence of severe ovarian hyperstimulation syndrome (OHSS) in patients at risk of developing OHSS who had embryo cryopreservation at pronucleate stage and deferred embryo transfer in a university-affiliated teaching hospital over a 5-year period. Elective embryo cryopreservation and deferred transfer in patients at risk of OHSS does not compromise the cumulative pregnancy rate per patient and also results in a low overall incidence of severe OHSS.

Cryopreservation↗
PubMed1989

[In vitro fertilization program. Preliminary results].

Three infertile couples were submitted to in vitro fertilization and uterine embryo transfer (IVF+ET) and 7 to in vitro fertilization and pronuclear stage tubal transfer (IVF+PROST). In order to programmed menstruation Norethisterone, 10 mg daily, were administered during the cycle preceding the one of controlled ovarian hyperstimulation. In order to inhibit endogenous production of FSH and LH, leuprolide acetate, a Gn-RH agonist, was injected subcutaneously 1 mg daily during 6 days and 0.5 mg fowardly from the luteal phase of the cycle proceeding the one of hyperstimulation until the day of HCG administration. To achieve superovulation pure FSH (Metrodine), HMG (Pergonal) and HCG (Endocorion) were used. Oocyte retrieval was performed through transvaginal puncture under ultrasonographic control. For oocyte and embryo identification and classification, spermatozoa separation and capacitation and gamete insemination and incubation procedures habitual techniques were employed. Pronuclear embryo tubal transfer was performed through a laparoscope 17 hours after insemination and embryo transfer to the uterine cavity after 48 hours. Nine of 10 patients responded to gonadotrophin hyperstimulation and were submitted to ovarian puncture. 69 oocytes (7, per patient) were obtained, 59 (81.15% of which were mature. 74.55% of the inseminated oocytes fertilized. Two patients got pregnant: one, submitted to IVF+PROST, presently has a multiple pregnancy with triplets and the second, submitted to IVF+UT, had a missed abortion at 8 weeks of pregnancy.

Adult↗
PubMedDate not supplied

Counselling on cryopreservation of pronucleated oocytes.

The German Embryo Protection Law permits only the cryopreservation of supernumerary pronuclear stage eggs. Data are needed to counsel patients about their individual benefits from this procedure. All fresh embryo transfer cycles performed from January 1994 until December 1998 in which supernumerary pronucleate eggs were cryopreserved (n = 557) were analysed retrospectively, together with data from all subsequent cycles involving transfers of frozen-thawed pronucleate eggs (n = 420) from January 1994 until June 2001. The additional cumulative pregnancy rate per fresh cycle was 11.5%. This rate depended on the number of embryos per transfer, i.e. 1.9, 8.2 and 13.0% respectively when one, two or three embryos were transferred (P < 0.05). A strong correlation was found between the numbers of cryopreserved pronucleate eggs and pregnancy rates, of 9.3, 10.5 and 17.1% when 1-3, 4-6, or at least 7 pronucleate eggs were available respectively. Additional benefit in terms of this rate from cryopreservation for a patient with and without a pregnancy in the fresh embryo transfer cycles was 5.3 and 12.7% respectively. It is concluded that higher pregnancy rates in cycles involving cryopreserved eggs for patients who did not become pregnant in fresh transfer cycles reveals the disadvantage of the German Embryo Protection Law, which does not allow embryo selection. Therefore, cryopreservation of pronucleate eggs with a higher developmental potential is possible. The total pregnancy rate could be raised from 28.0 to 35.5% per fresh transfer cycle.

Age Factors↗
PubMed1998

Uterine leiomyomas reduce the efficacy of assisted reproduction cycles: results of a matched follow-up study.

A matched follow-up study design was used to test the hypothesis that pregnancy rates following assisted reproduction procedures do not differ between women with or without intramural or sub-serosal uterine leiomyomas. Women undergoing their first in-vitro fertilization (IVF)-embryo transfer or zygote intra-Fallopian transfer (ZIFT) cycle between January 1993 and June 1995 were included. Cases (women with leiomyomas) were matched 1:1 with the next consecutive control (women without leiomyomas) according to age, number of embryos transferred, embryo grade, and the route of embryo transfer (uterine or Fallopian). Assisted reproduction cycles were performed in an identical manner, independent of the presence or absence of uterine leiomyomas. The main outcomes measured were clinical pregnancy and delivery rates. A total of 182 cycles was evaluated. Of the 91 assisted reproduction cycles performed in the leiomyoma group, there were 34 clinical pregnancies (37%) and 30 deliveries (33%). Of the 91 assisted reproduction cycles in the control group, there were 48 clinical pregnancies (53%) and 44 deliveries (48%). The Mantel-Haenszel estimate of relative risk indicated that the presence of a uterine leiomyoma significantly reduced the chance for a clinical pregnancy or delivery. These findings suggest that leiomyomas are associated with a reduction in the efficacy of assisted reproduction cycles.

Adult↗
PubMed1994

Evaluation of two gonadotropin-releasing hormone (GnRH) analogues (leuprolide and buserelin) in short and long protocols for assisted reproduction techniques.

PURPOSE: The administration of two GnRH analogues (buserelin and leuprolide acetate) in long and short protocols was evaluated prospectively. In the long protocol, the analogues were given from day 1 of the cycle for at least 14 days and then hMG administration was started, while in the short protocol the analogues were initiated from cycle day 1, adding hMG from day 3. The patients were divided into four groups according to the protocol used: Group I, buserelin-long; Group II, buserelin-short; Group III, leuprolide-long; and Group IV, leuprolide-short. Serum E2 levels on the day of hCG injection and the number of follicles observed, oocytes retrieved, and embryos obtained, as well as implantation rates, were significantly higher (P < 0.001) in the long protocols than in the short ones. RESULTS: Pregnancy rates were similar in all groups, although a trend for better results was observed in buserelin-long compared to buserelin-short. There were no differences in the results achieved with buserelin or leuprolide. CONCLUSION: The administration of GnRH analogues (buserelin and leuprolide acetate) in long protocols induced a more intense ovarian response and was associated with significantly higher implantation rates and also a trend for higher pregnancy rates, although this difference was not statistically significant.

Adult↗
PubMed2006

Investigation and treatment of repeated implantation failure following IVF-ET.

Pregnancy rate following one cycle of IVF and ET can be as high as 60%. But even in the very successful units, some couples fail repeatedly. The causes for repeated implantation failure (RIF) may be because of reduced endometrial receptivity, embryonic defects or multifactorial causes. Various uterine pathologies, such as thin endometrium, altered expression of adhesive molecules and immunological factors, may decrease endometrial receptivity, whereas genetic abnormalities of the male or female, sperm defects, embryonic aneuploidy or zona hardening are among the embryonic reasons for failure of implantation. Endometriosis and hydrosalpinges may adversely influence both. In this mini review, we discuss the suggested methods for evaluation and treatment of RIF: repeated hysteroscopy, myomectomy, endometrial stimulation, immunotherapy, preimplantation genetic screening (PGS), assisted hatching, zygote intra-Fallopian transfer (ZIFT), co-culture, blastocyst transfer, cytoplasmic transfer, tailoring stimulation protocols and salpingectomy for hydrosalpinges.

Embryo Implantation↗
PubMed2002

Nuclear and microtubule reorganization in nuclear-transferred bovine embryos.

We studied the nuclear and microtubule dynamics in nonactivated and pre-activated chromatin-removed oocytes following transfer of nuclei from bovine fibroblast cells. Immediately after fusion between membranes of oocytes and fibroblasts, a microtubule aster containing a gamma-tubulin spot was seen near the transferred nucleus in most oocytes regardless of activation conditions. Most fibroblast nuclei transferred into nonactivated oocytes underwent premature chromosome condensation (PCC) and divided into two masses of chromosomes. In contrast, fibroblast nuclei in pre-activated oocytes rarely underwent PCC, but formed a swelled pronuclear-like structure. Under nonactivation condition, microtubular spindles surrounded condensed chromosomes during the division of two nuclear structures. Gamma-tubulins were detected in the vicinity of condensed chromosomes, suggesting transient spindle formation. Two pronuclear-like structures near the microtubular aster containing gamma-tubulin spot(s) later formed a syngamy-like nuclear structure. While 20% of reconstructed oocytes under nonactivated conditions developed to morulae and blastocysts, only 4% of reconstructed oocytes under pre-activated conditions developed to morulae and blastocysts. These results suggest introduction of a foreign centrosome during somatic cell nuclear transfer, which probably plays a role in nuclear remodeling and subsequent development.

Animals↗
PubMed2001

Molar pregnancy with a coexistent fetus after intracytoplasmic sperm injection. A case report.

BACKGROUND: Normal fertilization is usually considered to have occurred when two pronculei (2PN) and two polar bodies are observed. Exceptions are the single pronucleated zygote resulting from asynchronous pronuclei. CASE: A 29-year-old woman entered a program of intracytoplasmic sperm injection and embryo transfer because of her husband's oligoasthenoteratozoospermia. Two cleavage-stage embryos (four blastomeres, grade 1 and 2) were obtained from one fertilized oocyte containing distinct 2PN and the other a single pronucleus (1PN). At 15 weeks' gestation the patient developed severe preeclampsia requiring termination of the pregnancy. Histopathologic examination and DNA ploidy by image analysis were consistent with a twin pregnancy combining a complete hydatidiform mole and normal pregnancy. CONCLUSION: We hypothesize that this 1PN was at the origin of the hydatidiform mole. This case highlights the danger of transferring an embryo having 1PN.

Abortion, Therapeutic↗
PubMed2000

Prospective randomized study of two cryopreservation policies avoiding embryo selection: the pronucleate stage leads to a higher cumulative delivery rate than the early cleavage stage.

OBJECTIVE: To compare the cumulative live birth rates obtained after cryopreservation of either pronucleate (PN) zygotes or early-cleavage (EC) embryos. DESIGN: Prospective randomized study. SETTING: University hospital. PATIENT(S): Three hundred eighty-two patients, involved in an IVF/ICSI program from January 1993 to December 1995, who had their supernumerary embryos cryopreserved either at the PN (group I) or EC (group II) stage. For 89 patients, cryopreservation of EC embryos was canceled because of poor embryo development (group III). Frozen-thawed embryo transfers performed up to December 1998 were considered. MAIN OUTCOME MEASURE(S): Age, oocytes, zygotes, cryopreserved and transferred embryos, damage after thawing, cumulative embryo scores, implantation, and cumulative live birth rates. RESULT(S): The clinical pregnancy and live birth rates were similar in all groups after fresh embryo transfers. Significantly higher implantation (10.5% vs. 5.9%) and pregnancy rates (19.5% vs. 10.9%; P< or = .02 per transfer after cryopreserved embryo transfers were obtained in group I versus group II, leading to higher cumulative pregnancy (55.5% vs. 38.6%; P < or = .002 and live birth rates (46.9% vs. 27.7%; P< or = .0001. CONCLUSION(S): The transfer of a maximum of three unselected embryos and freezing of all supernumerary PN zygotes can be safely done with significantly higher cumulative pregnancy chances than cryopreserving at a later EC stage.

Adult↗
PubMed2004

Regulation of histone H3 lysine 9 methylation in oocytes and early pre-implantation embryos.

Epigenetic modifications of the genome, such as covalent modification of histone residues, ensure appropriate gene activation during pre-implantation development, and are probably involved in the asymmetric reprogramming of the parental genomes after fertilization. We investigated the methylation patterns of histone H3 at lysine 9 (H3/K9), and the regulatory mechanism involved in the asymmetric remodeling of parental genomes during early preimplantation development in mice. Immunocytochemistry with an antibody that specifically recognizes methylated H3/K9 showed a very weak or absent methylation signal in the male pronucleus, whereas a distinct methylation signal was detected in the female pronucleus. This asymmetric H3/K9 methylation pattern in the different parental genomes persisted until the two-cell stage. However, de novo methylation of H3/K9 occurred and the asymmetry was lost during the four-cell stage. The unmethylated male pronucleus underwent de novo methylation when it was transferred into enucleated GV- or MII-stage oocytes, which suggests that histone H3 methylase is active before fertilization, but not afterwards, and that the asymmetric methylation pattern is generated by this change in methylase activity in the cytoplasm after fertilization. Thus, histone H3 is methylated only in the maternal chromosomes, which are present in the oocytes before fertilization, and is not methylated in the paternal chromosomes, which are absent. The maintenance of asymmetric H3/K9 methylation patterns in early embryos is an active process that depends on protein synthesis and zygotic transcription, as de novo methylation in the male pronucleus occurred when either protein synthesis or gene expression was inhibited by cycloheximide or alpha-amanitin, respectively. In addition, corresponding de novo methylation of H3/K9 and DNA occurred when the male pronucleus was transferred to an enucleated GV oocyte. Our results suggest that H3/K9 methylation is an epigenetic marker of parental genome origin during early preimplantation development.

Animals↗
PubMed2002

Production of transgenic mice from vitrified pronuclear-stage embryos.

Cryopreservation of pronuclear-stage embryos would be useful for transgenic technology and genome preservation purposes. We compared a novel vitrification technique (solid surface vitrification, SSV) with another vitrification method in straws for cryosurvival and to generate transgenic progeny from cryopreserved mouse zygotes following microinjection. The SSV solution consisted of 35% ethylene glycol (EG), 5% polyvinyl-pyrrolidone (PVP), and 0.4 M trehalose in M2 supplemented with 4 mg/ml BSA; the in straw vitrification solution was 7 M EG in M2 plus BSA. In experiment I, we compared the effect of the vitrification solutions alone, without cooling. No reduction was detected in survival and cleavage rates. In experiment II, SSV yielded a significantly higher percentage of morphologically normal zygotes (96%) that also cleaved at significantly higher rates (80%) when compared to that following "in straw" vitrification (68 and 66%, respectively). Cleavage rate in the non-vitrified control group (93%) was significantly higher than that of both vitrified groups. Following embryo transfer, there was no difference in the rate of pups obtained from the SSV, "in straw" vitrified, and control groups (97/457, 21%; 15/75, 20% and 56/209, 27%, respectively). In experiment III, SSV vitrified and fresh embryos were used for pronuclear DNA injection. Survival rate of vitrified embryos after microinjection was reduced compared to nonvitrified ones (64 vs. 72%, respectively; P < 0.05); however, development to two-cell stage was not different (76 vs. 72%, respectively). Following embryo transfer of vitrified vs. fresh microinjected embryos, in both cases 10% live pups were generated, including transgenic pups. The results demonstrated that the efficiency of generating transgenic pups from SSV vitrified pronuclear zygotes is comparable to that from fresh embryos.

Animals↗
PubMed1995

[Cryopreservation of human oocytes in the pronuclear stage with an automated "open system"].

In the period from June 1990 to February 1993, 718 excess pronucleated oocytes from 185 cycles were cryopreserved by using the "open freezing system" in our IVF-programme at the Department of Gynaecology and Hospital of the University of Bonn. 1.5 mol propanediol and 0.1 mol sucrose were used as a cryoprotectant. During this time 287, pronuclear oocytes were thawed; of these 245 survived the freezing process and were classified as intact. The survival rate in this study was 85.4%, though if only one intact pronuclear oocyte was transferred, no pregnancy could be achieved, whereas if two or three pronuclear oocytes were transferred, seven to ten pregnancies resulted, respectively (23.3% and 23.8% pregnancy rates). Eight healthy children have been born to date from four other ongoing pregnancies, one of them is a twin pregnancy.

Cell Survival↗
PubMed1999

The probability of abnormal preimplantation development can be predicted by a single static observation on pronuclear stage morphology.

This retrospective study was undertaken to determine whether further developmental progression of two-pronucleated (2PN) zygotes can be predicted by a single, non-invasive examination of pronuclei, with the use of criteria based on the number and distribution of nucleolar precursor bodies in each pronucleus. The normal range of pronuclear variability was defined by analysis of zygotes giving rise to embryos transferred in 100%-implantation cycles (pattern 0). Morphological patterns differing from pattern 0 were classified as patterns 1-5. The frequency of developmental arrest of pattern 0 zygotes was only 8.5% as compared with 31.6, 21.9, 30.0, 20.5 and 24. 1% for patterns 1-5 respectively. Relationships of pronuclear patterns with blastomere multinucleation and cleaving embryo morphology were also noted. Clinical pregnancy was achieved in 22 of 44 (50%) treatment cycles in which at least one pattern 0 embryo was transferred, but only in two of 23 (9%) cycles in which only pattern 1-5 embryos were transferred. These data present new evaluation criteria which can be used to predict the developmental fate of human embryos as early as the pronuclear stage, without requiring repeated observations or an exact timing of pronuclear zygote inspection. Further prospective study is needed for clinical validation of these criteria.

Blastocyst↗
PubMed1992

[Trial of in vitro fertilization via epididymal duct aspirated sperm].

We made a trial of our method by which sperm are collected directly from epididymal duct, then subjected to in vitro fertilization. The subjects were a total of 22 cases comprising 14 of obstructed azoospermia and 8 of severe oligoasthenozoospermia, on which the surgery was performed 26 times. Using the microscope, an epididymal duct was directly punctured to make a suction of the fluid or was slightly incised to make a suction of the outflow. Sperms thus collected were preincubated for 4 to 6 hours, then subjected to in vitro fertilization. Condition of collected sperms; 32 x 10(4)-15300 x 10(4) sperms were obtained in real number. Sperm motility rate was 0-82%, averaging 25.0%. Sperm forward motility were fairly good in 18 of 26 test cases. Sperm condition was so poor as to make the insemination impossible in 4 test cases (16%). Fertility rate for cases of obstructed azoospermia was 53% (8/15 test cases) and 63% (55/87 eggs) in the test cases and the number of eggs, respectively, compared with 33% (2/6 test cases) and 5.4% (2/37 eggs), respectively for cases of oligo-asthenozoospermia. Pregnancy; Successful pregnancy was obtained in 2 of the cases of obstructed azoospermia, in each of which sperms were collected from the head of the epididymis, then passed into the maternal body by zygote intra fallopian tubal transfer (ZIFT); with the one case resulting in uneventful delivery in November 1991 and the other in a miscarriage. The above results suggested the present method would become an epoch-making approach in the treatment of severe male infertility.

Epididymis↗
PubMed1999

Production of transgenic rats using cryopreserved pronuclear-stage zygotes.

We investigated the application of cryopreserved pronuclear-stage zygotes for the production of transgenic rats. Most of the pronuclear-stage zygotes cryopreserved by conventional two-step freezing or vitrification appeared morphologically normal, but the proportion of frozen zygotes that developed into fetuses following transfer (59.7-60.2%) was higher than that of vitrified zygotes (5.5-22.1%). When the frozen-thawed zygotes were used for DNA microinjection, 97.5% survived after DNA microinjection and 25.1% of the transferred zygotes developed into fetuses. These proportions were comparable to those of the fresh control zygotes (97.0 and 30.0%, respectively). The integration efficiency of the exogenous DNA into fetuses was similar between the frozen group (3.3% per injected zygote) and the control group (3.5%). These results indicate that pronuclear-stage rat zygotes can be successfully cryopreserved by conventional two-step freezing for production of transgenic rats.

Animals↗