San-Hyun Yoon

Selection of patients for natural cycle in vitro fertilization combined with in vitro maturation of immature oocytes

OBJECTIVE To determine a successful method of selecting patients for natural cycle IVF combined with in vitro maturation (IVM) of immature oocytes and to determine treatment efficacy. DESIGN Retrospective analysis of IVF treatments. SETTING Hospital IVF clinic. PATIENT(S) Women with infertility according to ovarian reserve and endocrine profile were selected for different treatments. INTERVENTIONS(S) According to screening criteria, infertile women were divided into three treatments: [1] natural cycle IVF combined with IVM (IVF/M), [2] IVM alone, and [3] controlled ovarian hyperstimulation (COH) with gonadotropin. MAIN OUTCOME MEASURE(S) The distribution of completed IVF-ET cycles in each treatment; clinical pregnancy and implantation rates in each treatment. RESULT(S) A total of 417 cycles were started, and 410 cycles (98.3%) were completed. Of 410 cycles, 151 (36.8%) were treated by natural cycle IVF/M, 63 (15.4%) underwent IVM alone, and 196 (47.8%) underwent COH. With increasing age fewer cycles can be treated by natural cycle IVF/M or IVM. Clinical pregnancy rates were 40.4% (61 of 151) for natural cycle IVF/M, 41.3% (26 of 63) for IVM alone, and 37.8% (74 of 196) for COH. There were no differences in implantation rate in the three groups (17.8% [82 of 462], 16.7% [35 of 210], and 20.1% [103 of 513]). CONCLUSION(S) Natural cycle IVF/M together with IVM-alone treatment can offer more than 50% of infertile women with an acceptable pregnancy and implantations rates.

Effect of gonadotrophin priming on in-vitro maturation of oocytes collected from women at risk of OHSS

This study examined the effects of human menopausal gonadotrophin (HMG) or human chorionic gonadotrophin (HCG) priming on cumulus-oocyte complex (COC) morphology, oocyte maturation and embryo development in patients undergoing in-vitro maturation (IVM) cycles. The patients were primed with nothing (group 1), low-dose HMG (group 2) or 10,000 IU HCG (group 3) before oocyte retrieval. COC with dispersed cumulus cell appearance was only observed in group 3. In addition, 11% of metaphase II stage oocytes at the time of retrieval were collected from group 3. Oocyte maturation in vitro in group 3 was faster than that in groups 1 and 2. The blastocyst development rate of residual embryos after embryo transfer in group 3 was significantly higher than that of groups 1 and 2 (P < 0.05). These results suggest that HCG priming may stimulate the COC, promote oocyte maturation, and improve developmental competence in IVM cycles.

Pregnancies Resulting from In Vitro Matured Oocytes Collected from Women with Regular Menstrual Cycle

Purpose: To demonstrate that human immature oocytes retrieved from women with regular menstrual cycles can undergo maturation and fertilization, and that the resulting embryos can establish pregnancies.Methods: Immature oocytes (n = 568) were retrieved from women with regular menstrual cycle. The intact immature oocytes (n = 506) were allowed to mature in YS medium supplemented with 70% human follicular fluid (hFF); the matured oocytes were fertilized with husband sperm. Two pronuclei oocytes were cocultured with cumulus cells in YS medium supplemented with 10% hFF until 2 or 3 days after insemination. The cleaved embryos were transferred in uteri.Results: Follicles were aspirated on Day 9.2 ± 5.3 of 63 natural cycles from 51 patients (mean age = 34.8 ± 4.0 years). The average number of retrieved immature oocytes was 9.0. The maturation rate was 74.3% (376/506). The two PN and cleavage rates were 72.6% (273/376) and 89.0% (243/273), respectively. Embryo transfer was achieved in 51 cycles and clinical pregnancy rate was 17.6% (9/51).Conclusions: The results suggest that in vitro matured oocytes can undergo fertilization and the resulting embryos may successfully lead to pregnancies. However, further research is needed to improve IVM technique to achieve success rate comparable to gonadotrophin stimulated cycles.

Alternative embryo transfer on day 3 or day 5 for reducing the risk of multiple gestations

Purpose: This study was carried out to reduce the possibility of high-order multiple gestations and the failure of embryo transfer by determining their replacement date based on the number and quality of 2-day embryos.Methods: All zygotes were cocultured with cumulus cells in 10 μl of YS medium containing 10% human follicular fluid (hFF) for 48 or 96 hr. In period I, all embryos were transferred on day 3 (1032 cycles). In period II, the embryos were transferred on either day 3 or day 5 by determining their replacement date based on the number and quality of 2-day embryos: there were 2701 patients in whom embryos were replaced on day 3 (in the case that the number of zygotes was less than eight and the number of good-quality embryos was less than three) and 1952 patients less than 40 years old in whom embryos were replaced on day 5 (in the case that the number of zygotes was eight or more and/or the number of good-quality embryos was three or more). On the other hand, patients who were 40 years old or more were alloted to day 3 transfer cycles, regardless of the number and quality of the 2-day embryos, due to the possibility of their not producing blastocyst-stage embryos in vitro.Results: The number of embryos transferred in period II was 2.9 ± 0.6, while that in period I was 3.7 ± 0.5. The multiple pregnancy rate was significantly decreased in period II (30.7%) compared to that (49.6%) in period I, while the pregnancy and implantation rates in period II (36.1 and 16.4%, respectively) were not lower than those (34.9 and 16.1%, respectively) in period I. The rate of triplet or more gestations was significantly minimized in period II (2.3%) compared to that in period I (26.5%).Conclusions: We propose that determination of the date on which embryos should be transferred based on the number and quality of embryos on day 2 may help to maintain an acceptable pregnancy rate, while minimizing embryo transfer failure and high-order multiple gestations.

Pregnancy resulting from transfer of repeat vitrified blastocysts produced by in-vitro matured oocytes in patient with polycystic ovary syndrome.

This report describes a live birth produced from repeat vitrification and thawing of blastocysts derived from in-vitro matured (IVM) oocytes in a woman with polycystic ovarian syndrome. Immature oocyte retrieval was performed on day 12 of her induced menstrual cycle. The patient was administered 10,000 IU of human chorionic gonadotrophin s. c. 36 h before immature oocyte retrieval. A total of 47 immature oocytes were collected. Following IVM of these immature oocytes, 76.6% (36/47) become mature (at metaphase II stage). Thirty oocytes (30/36, 86.1%) were normally fertilized following insemination by intracytoplasmic sperm injection. The fertilized zygotes (two-pronuclear stage) were co-cultured with cumulus cells in YS medium supplemented with 10% human follicular fluid. On day 5 after insemination, three blastocysts were transferred. Unfortunately, fresh embryo transfer did not result in pregnancy. The remaining 10 embryos developed to the expanded blastocyst stage. These remaining blastocysts were vitrified with electron microscope grids following artificial shrinkage. Three months later, three blastocysts were thawed due to a clinical error. Consequently, the embryos were revitrified. After a week, the three blastocysts were warmed again. Two of them developed to hatched blastocysts. Following transfer, a full-term pregnancy resulted in the delivery of healthy twins.

High implantation and pregnancy rates with transfer of human hatching day 6 blastocysts

Although improved outcomes of IVF after transfer of blastocysts has been reported (1, 2), it remains uncertain which blastocyst can develop into a viable fetus. At present, expansion of the blastocyst is the most practical selection criterion. However, if extended culture of embryos produces many expanded-stage blastocysts, it is not easy to decide which blastocysts to transfer (Fig. 1 A). In addition, some blastocysts fail to hatch out of the zona, despite evidence that the blastocoelic cavity had expanded and the zona pellucida had thinned (3).

Successful Birth After Transfer of Blastocysts Derived from Oocytes of Unstimulated Woman with Regular Menstrual Cycle After IVM Approach

AbstractPurpose: To report a delivery after transfer of blastocysts derived from eggs collected following in vivo HCG priming in a patient with regular menstrual cycles undergoing in vitro maturation (IVM) program. Methods: A woman had regular menstrual cycle and had experience of ovarian hyperstimulation syndrome (OHSS) during a previous conventional IVF-ET cycle. The patient was primed with 10,000 IU HCG 36 h before egg retrieval. After oocyte collection, the maturity of oocytes was evaluated and immature oocytes were cultured in IVM medium. The matured oocytes were fertilized with husband sperm, and normal fertilized eggs were cultured to blastocysts stage until embryo transfer in uteri. Results: Three MII-stage and 13 GV-stage oocytes were collected from the patient. Three mature oocytes were fertilized by conventional IVF. All three fertilized oocytes were developed to blastocysts. Immature oocytes were matured in vitro and insemination was carried out by ICSI. Out of eight fertilized zygotes, two developed to blastocyst stage. Transfer of three expanded blastocysts on Day 6 resulted in pregnancy in the patient and one healthy baby was born. Conclusions: This report provides an approach to treat infertile women with regular menstrual cycle and high risk of OHSS.

Successful pregnancy and delivery after ICSI with artificial oocyte activation by calcium ionophore in in-vitro matured oocytes: a case report

The achievement of a successful pregnancy and delivery after oocyte activation with calcium ionophore is reported in a couple having low fertilization rates after intracytoplasmic sperm injection (ICSI) of in-vitro matured oocytes. A couple, in which the wife had polycystic ovary syndrome and the husband had moderate oligoteratozoospermia, showed a low fertilization rate in a previous in-vitro maturation cycle (2/11 [18.2%]). The most likely cause of complete fertilization failure or low fertilization rates is failure of oocyte activation. Therefore, artificial oocyte activation by calcium ionophore was combined with ICSI to achieve viable fertilized oocytes. Oocytes were stimulated with calcium ionophore for 30 min after ICSI. The fertilization rate of oocytes activated with calcium ionophore (13/15 [86.7%] and 7/9 [77.8%]) was higher than that of the non-activated oocytes. In the latest cycle, three embryos derived from the activated oocytes were transferred into the uterus on day 3. Subsequently, two gestational sacs were identified on ultrasound. The patient delivered dizygotic twins (girl 2260 g and boy 2760 g) at 35 weeks and 6 days gestation by caesarean section. This result suggests that calcium ionophore could be useful for oocyte fertilization in couples with low fertilization rates after ICSI of in-vitro matured oocytes.

Optimization of a Dilution Method for Human Expanded Blastocysts Vitrified Using EM Grids After Artificial Shrinkage

Purpose: To verify a more effective dilution method that can be applied to human expanded blastocysts that are vitrified after artificial shrinkage. Methods: Surplus expanded blastocysts that remained after embryo transfer (ET) in in vitro fertilization (IVF) cycles, were cryopreserved. The blastocysts were vitrified on EM grids following artificial shrinkage. After thawing the blastocysts, cryoprotectants were diluted using either a 6- or 2-step method. We examined the survival rate and clinical outcome of blastocysts of 151 patients in our ET program after thawing. Results: The survival rate of blastocysts that were thawed using a 2-step method (91.6%, 239/261) was comparable with that of the 6-step method (89%, 186/209). The clinical pregnancy rate (45.9%, 39/85) and implantation rate (24.1%, 53/220) were slightly higher in the 2-step method than in the 6-step method (40.9%, 27/66; 19.4%, 33/170). Conclusions: Our data indicate that the 2-step dilution method could be a simpler and more effective protocol for human expanded blastocysts that are vitrified using EM-grid following artificial shrinkage.

Successful pregnancy after SrCl2 oocyte activation in couples with repeated low fertilization rates following calcium ionophore treatment

Abstract This report describes a successful pregnancy and delivery following oocyte activation with strontium chloride (SrCl2) in couples with repeated complete fertilization failure or low fertilization rates even after calcium ionophore treatment. Eight infertile couples who showed complete fertilization failure or low fertilization rates after conventional intracytoplasmic sperm injection (ICSI) and calcium ionophore treatment. When the results of fertilization were not satisfactory in the cycles, the oocytes were artificially activated by SrCl2 for the next attempts. Oocyte activation with SrCl2 significantly increased the fertilization rates, when compared with conventional ICSI or calcium ionophore treatment (61.7% vs. 20.0% or 25.3%, respectively). There was significant increase in the proportions of good-quality cleaved embryos (50.0% vs. 0% or 12.5%, respectively). The rate of surplus embryos that developed to blastocyst stage increased in SrCl2-treated oocytes, when compared with that in ICSI with or without calcium ionophore treatment (25.7% vs. 0% or 9.1%, respectively). Five successful pregnancies were attained after oocyte activation with SrCl2, of which eight healthy children were born. Physical and mental development of the children were normal from birth to 60 months. These results suggest that SrCl2 in treatment should be considered as an effective method for artificial oocyte activation (AOA) to improve fertilization rates and embryo quality in cases with complete fertilization failure or low fertilization rates after calcium ionophore treatment.