Cabergoline reduces the early onset of ovarian hyperstimulation syndrome: a prospective randomized study.

Prophylactic use of cabergoline has been associated with a decrease in the severity of ovarian hyperstimulation syndrome (OHSS). A prospective randomized study was designed to evaluate the potential of cabergoline to decrease the incidence of OHSS in high-risk patients undergoing assisted reproductive technology treatment; 166 patients with oestradiol concentrations over 4000 pg/ml on the day of human chorionic gonadotrophin (HCG) administration were evaluated. They all received 20 g routine preventive intravenous human albumin on the day of oocyte retrieval. They were then randomized into two groups: group A (n = 83) received 0.5 mg oral cabergoline per day for 3 weeks beginning on the day after oocyte retrieval, and group B (n = 83) received no medication. 'Early' OHSS was defined as being when the onset of the syndrome was initiated during the first 9 days after HCG administration, and 'late' OHSS was defined as being when the onset of the syndrome was initiated from 10 days after HCG administration. In group A, no patients progressed to 'early' OHSS and nine patients (10.8%) developed 'late' OHSS; in group B, 12 patients (15.0%) progressed to 'early' OHSS and three (3.8%) to 'late' OHSS. Although the risk of 'early' OHSS decreased significantly (P < 0.001), the risk of 'late' onset OHSS did not. The two groups presented no changes in pregnancy, implantation or miscarriages rates.

The role of sperm proteasomes during sperm aster formation and early zygote development: implications for fertilization failure in humans.

BACKGROUND Sperm aster organization during bovine and human fertilization requires a paternally-derived centriole that must first disengage from the sperm tail connecting-piece. We investigated the participation of the 26S proteasome in this process. METHODS Proteasome localization and enzymatic activity were studied in normal and pathological human spermatozoa by immunocytochemistry and enzyme-substrate assays. The role of proteasomes during bovine zygote development was investigated using a pharmacological proteasome-inhibitor, MG132, and with anti-proteasome antibodies delivered by Streptolysin O-permeabilization or with the Chariot reagent. Human zygotes discarded after ICSI failures (n = 28) were also examined. RESULTS Proteasomes were localized in the sperm acrosome and connecting-piece, as well as in the pronuclei of bovine and human zygotes. Proteasomal enzymatic activities were decreased in defective human spermatozoa. Disrupted sperm aster formation and pronuclear development were found after pharmacological and immunological block of proteasomes in human/bovine spermatozoa and oocytes, as well as in 28 discarded human post-ICSI fertilization failures. CONCLUSIONS Specific proteasome inhibition disrupts sperm aster formation and pronuclear development/apposition in bovine and human zygotes. Human spermatozoa with defective centriolar/pericentriolar structures have decreased proteasomal enzymatic activity. Release of a functional sperm centriole that acts as a zygote microtubule-organizing center probably relies on selective proteasomal proteolysis. These findings suggest an important role of sperm proteasomes in zygotic development.

In vitro differentiation of male mouse embryonic stem cells into both presumptive sperm cells and oocytes.

Pioneer work in male mouse embryonic stem (ES) cells differentiation into germ cells (GC) showed generations of male or female gametes in separate experiments, using genetically manipulated or preselected ES cells. In an attempt to produce both types of gametes from male mouse ES cells without any genetic manipulation or preselection, we induce the differentiation by retinoic acid (RA) within nonadherent embryoid bodies (EB). It seems that gamete-like cell formation occurs in the correct manner based on the expression of early and late GC-specific genes such as Oct-4, Mvh, Stella, Dazl, Piwil 2, Pdrd 1, Rex 14, Rnf 17, Bmp8b, Acrosin, Stra-8, Haprin, LH-R, Gdf9, Zp3, Zp2, Sycp1, and Sycp3. Immunofluorescence analysis of morphologically well-formed GC and presumptive gametes showed positive labeling for SSEA1, Oct-4, EMA-1, FE-J1, Dazl, Fragilis, Mvh, Acrosin, and acetylated alpha-tubulin. Conventional cytogenetic and FISH analysis indicated a chromosome reduction in ES-derived GC. Our data suggest that ES cells with XY chromosomes can produce under the same experimental conditions both types of presumptive gametes, and this production depends on their positional and temporal information within the EB context.

Location of the embryo-transfer catheter guide before the internal uterine os improves the outcome of in vitro fertilization.

The objective of this study was to assess whether positioning the embryo transfer (ET) catheter guide at the time of embryo expulsion, before or beyond the internal os, has an impact on IVF cycle outcome. We performed a retrospective study comparing IVF outcomes in relation to the ET guide position relative to the internal uterine os. We analyzed ultrasound-guided ETs in IVF-intracytoplasmic sperm injection (ICSI) cycles, performed with the tip of the ET catheter guide just before the internal os (group 1) and beyond the internal os (group 2). Implantation and pregnancy rates were significantly better in group 1 than in group 2, at 14.8% versus 11.8% and 57.3% versus 43.1, respectively. We conclude that passing the ET catheter guide beyond the internal os reduces implantation and pregnancy rates.

Embryos generated using testicular spermatozoa have higher developmental potential than those obtained using epididymal spermatozoa in men with obstructive azoospermia.

OBJECTIVE: To determine whether the injection of testicular spermatozoa results in more viable embryos (higher implantation rate) than injection of epididymal spermatozoa in cases of obstructive azoospermia. DESIGN: Retrospective analysis of 265 cases of testicular sperm aspiration (TESA) and percutaneous sperm aspiration (PESA), including 185 cases of obstructive azoospermia. SETTING: Private Infertility clinic. PATIENT(S): None, charts review. INTERVENTION(S): None, charts review. MAIN OUTCOME MEASURE(S): Clinical pregnancy rate (PR), implantation rate. RESULT(S): Although fertilization rates were higher in the PESA group, implantation rates were significantly better in the TESA group. There was also a trend to higher ongoing PR and lower miscarriage rates in TESA cases. CONCLUSION(S): In cases of obstructive azoospermia, embryos generated using testicular spermatozoa have higher developmental potential than those obtained using epididymal spermatozoa.

Interactions of the meiotic spindle with mitotic chromosomes in GV mouse oocytes.

During mitosis, a spindle checkpoint detects chromosome misalignment and halts the cell cycle progression. In meiosis of female germ cells, however, it is debatable whether such a checkpoint is present. This research employed a unique model in the mouse, mitotic chromosomes transferred to meiotic cytoplasts to investigate whether a meiotic oocyte's microtubule apparatus can effectively separate mitotic metaphase chromosomes, and whether a spindle checkpoint exists during its division. The intact germinal vesicle (GV) oocytes, enucleated GV cytoplasts, and enucleated GV cytoplasts at 15 h in-vitro maturation were transferred with a metaphase fibroblast cell. When mitotic chromosomes were transferred into enucleated or intact mouse GV oocytes, the first bipolar meiotic spindles were established and the reconstructed oocytes were able to extrude polar bodies. However, none of the reconstructed oocytes showed complete and accurate alignment of chromosomes, except the enucleated GV cytoplasts reconstructed after maturation. The spindle formation and polar body extrusion suggest that the first meiotic spindle was functional, and the chromosome misalignment did not prevent the onset of anaphase. The data indicate that a spindle checkpoint, providing surveillance of misaligned chromosomes, was overridden or compromised by the incompatibility between somatic chromosomes and meiotic spindles during the first meiotic division.

A prospective, randomized and blinded comparison between 10,000 IU urinary and 250 microg recombinant human chorionic gonadotropin for oocyte maturation in in vitro fertilization cycles.

PURPOSE: To compare the efficacy and safety of u-hCG with r-hCG in IVF cycles. METHODS: A prospective, investigator-blind, randomized, comparative study. Patients (n = 100) < or =35 years with IVF indication were randomly assigned on the day of hCG administration for oocyte maturation to receive either u-hCG (10,000 IU) or r-hCG (250 microg). RESULTS: No statistical differences were found between groups in relation to total number of oocytes retrieved, percentage of mature oocytes, number of injected oocytes, fertilization rates and number of embryos transferred. The data indicate a possible trend toward a higher incidence of pregnancy in the r-hCG group. Adverse events, predominantly injection-site reactions, were significantly more common in the u-hCG group. CONCLUSIONS: r-hCG is at least as effective for inducing final stages of oocyte maturation as 10,000 IU u-hCG and is also associated with significantly better patient tolerance and thus higher patient acceptability.

The optimal time for intracytoplasmic sperm injection in the human is from 37 to 41 hours after administration of human chorionic gonadotropin.

Fertilization rates increased continuously with the time elapsed after administration of hCG, reaching a peak of 84% when intracytoplasmic sperm injection (ICSI) was performed >41 hours after hCG administration. However, the highest implantation rate, 24%, was achieved when ICSI was performed 37-41 hours after hCG administration.

Evidence of parthenogenetic origin of ovarian teratoma: case report.

This case report represents one of the few documented cases of parthenote embryo retrieval from an IVF patient with a history of ovarian teratomas. A 29-year-old woman presented at our centre with a history of primary infertility for 6 years due to male factor. She had undergone left oophorectomy 4 years before due to an ovarian teratoma. An ultrasound scan performed during basal evaluation revealed two complex images in the right ovary suggesting teratomas, measuring 2.5 x 2.4 and 1.7 x 1.3 cm. A significant extent of sonographically normal ovarian parenchyma was present, and the patient underwent the long leuprolide acetate protocol of ovarian stimulation with recombinant FSH for an IVF-ICSI cycle. She had 13 metaphase II (MII), four metaphase I (MI), two germinal vesicle (GV) oocytes and one 4-cell embryo retrieved. Eight out of nine injected oocytes were fertilized normally while one was unfertilized. Embryo transfer was carried out 72 h after retrieval. The 4-cell (parthenote) embryo recovered at oocyte retrieval continued to cleave in culture, developing into a 7-cell embryo by the next day. The embryo was morphologically normal, presenting an evident nucleus in each blastomere. Fluorescent in situ hybridization (FISH) returned two signals for the X chromosome in each blastomere that was analysed. Of the eight normally fertilized embryos, three were transferred, resulting in a normal singleton pregnancy and the birth of a healthy baby.

Impact of subserosal and intramural uterine fibroids that do not distort the endometrial cavity on the outcome of in vitro fertilization-intracytoplasmic sperm injection.

OBJECTIVE: To further evaluate the effects of intramural and subserosal uterine fibroids on the outcome of IVF-ET, when there is no compression of the endometrial cavity. DESIGN: Retrospective, matched-control study from January 2000 to October 2001. SETTING: Private IVF center. PATIENT(S): Two hundred forty-five women with subserosal and/or intramural fibroids that did not compress the uterine cavity (fibroid group) and 245 women with no evidence of fibroids anywhere in the uterus (control group). INTERVENTION(S): In vitro fertilization-intracytoplasmic sperm injection (IVF-ICSI) cycles. MAIN OUTCOME MEASURE(S): The type of fibroid (intramural, subserosal), number, size (cm), and location of intramural leiomyomas (fundal, corpus) were recorded. Outcomes of IVF-ICSI cycles were compared between the two groups. RESULT(S): There was no correlation between location and number of uterine fibroids and the outcomes of IVF-ICSI. Patients with subserosal or intramural fibroids <4 cm had IVF-ICSI outcomes (pregnancy, implantation, and abortion rates) similar to those of controls. Patients with intramural fibroids >4.0 cm had lower pregnancy rates than patients with intramural fibroids 4 cm and that such patients be submitted to treatment before they are enrolled in IVF-ICSI cycles. Whether or not women with fibroids > 4 cm would benefit from fibroid treatment remains to be determined.

Nuclear and microtubule dynamics of G2/M somatic nuclei during haploidization in germinal vesicle-stage mouse oocytes.

During the haploidization process, it is expected that diploid chromosomes of somatic cells will be reduced to haploid for the generation of artificial gametes. In the present study, we aimed to use enucleated mouse oocytes at the germinal vesicle-stage (G2/M) as recipients for somatic cells that are also synchronized to the G2/M stage for haploidization. The reconstructed oocytes were then induced to undergo meiosis in vitro and observed for their nuclear morphology and microtubule network formation at various expected stages of the meiotic division. Following in vitro maturation, more than half (62/119, 52.1%) of the reconstructed oocytes completed the first round of meiosis-like division, as evidenced by the extrusion of pseudopolar bodies (PBs). However, accelerated PB extrusion, approximately 3-4 h earlier than that by control oocytes occurred. Furthermore, abnormally large pseudo-PBs, as large as four times the normal PB sizes, were observed. During the process of in vitro maturation at both the expected stages of metaphase I (MI) and metaphase II (MII), condensed chromosomes were observed in 38.7% and 55.2% of oocytes, respectively. However, two other types of nuclear configurations were also observed: 1) uneven distribution of chromatin and 2) an interphase-like nucleus, indicating deficiencies in chromosome condensation. Following oocyte activation, more than half (21/33, 63.6%) of the reconstructed oocytes with pseudo-PBs formed separated pseudopronuclei (PN), suggesting formation of functional spindles. The formation of bipolar spindle-like microtubule network at both the expected MI and MII stages during in vitro maturation was confirmed by immunohistochemistry. In summary, this study demonstrated that a high proportion of G2/M somatic nuclei appear to undergo meiosis-like division, in two successive steps, forming a pseudo-PB and two separate pseudo-PN upon in vitro maturation and activation treatment. Moreover, the enucleated geminal vesicle cytoplast retained its capacity for meiotic division following the introduction of a somatic G2/M nucleus.

Toward pre-conceptual genetic analysis of human spermatozoa.

Nuclei of mature mammalian spermatozoa are extraordinarily resistant to chemical and thermal injury. Additionally, decondensation of spermatozoa DNA can be accompanied by little or no visual changes of the sperm head. This study tested whether human spermatozoa could be recovered following several cycles of primer extension preamplification (PEP) and used to achieve fertilization and subsequent development of human oocytes. An attempt was also made to amplify PEP buffer after spermatozoon removal. The results demonstrate that the sperm head can be successfully recovered following treatment with KOH or proteinase K followed by one to four cycles of PEP. It is also shown that following this treatment, the spermatozoa can be injected into the oocytes and will transform into a pronucleus if the oocyte is activated by sperm cytosolic fraction. In some cases, it was also possible to obtain polymerase chain reaction signals using a buffer after sperm cells were removed following several cycles of PEP. Although sperm participation in development was confirmed by fluorescence in-situ hybridization, light microscopy revealed some degree of damage to spermatozoal chromosomes. It is concluded that pre-conceptual analysis of sperm cells may be possible, but more research is necessary to determine the optimal conditions that would preserve sperm DNA integrity while allowing accurate diagnoses.

Uterine cavity findings and hysteroscopic interventions in patients undergoing in vitro fertilization-embryo transfer who repeatedly cannot conceive.

OBJECTIVE: To assess hysteroscopic findings in patients undergoing IVF-ET who repeatedly failed to conceive despite transfer of good-quality embryos. DESIGN: Prospective, observational study. SETTING: Clinical research unit for reproductive medicine in a private clinic. PATIENT(S): Fifty-five patients with a normal uterine cavity on hysterosalpingography before the initial IVF-ET cycle and two previous failed IVF-ET attempts despite transfer of a minimum of two good-quality embryos on each occasion. INTERVENTION(S): Standard transvaginal ultrasonography and diagnostic and therapeutic hysteroscopy. MAIN OUTCOME MEASURE(S): Endometrial findings on transvaginal ultrasonography and hysteroscopy and outcome of the cycles after surgical hysteroscopy and antibiotic therapy. RESULT(S): Twenty-five (45%) patients had abnormal endometrial findings and underwent treatment to correct the lesions. All patients underwent a third IVF-ET cycle. Pregnancy (50% vs. 20%) and implantation (19% vs. 5.5%) rates were significantly higher in patients who were treated for uterine abnormalities than in patients who had normal uterine cavities on hysteroscopy. CONCLUSION(S): The incidence of pathologic findings on hysteroscopy is high in patients with repeated failures of IVF-ET. Evaluation of endometrial integrity by hysteroscopy is highly valuable and should be applied to all such cases.

Pronuclear morphology evaluation with subsequent evaluation of embryo morphology significantly increases implantation rates.

OBJECTIVE: To elucidate the relative predictive value of implantation markers at different stages of preimplantation development. DESIGN: Correlation of pronuclear morphology with embryo morphology and implantation rates in retrospective and prospective analysis of in vitro fertilization/intracytoplasmic sperm injection (IVF-ICSI) treatment cycles. SETTING: Private infertility center. PATIENT(S): A total of 441 couples undergoing infertility treatment. INTERVENTION(S): None. MAIN OUTCOME MEASURE(S): Size of pronuclei and distance between them, the number and polarization of nucleolus precursor bodies (NPB) at the one-cell stage, embryo cleavage and fragmentation rates on days 2 and 3, and pregnancy and implantation rates. RESULT(S): Polarization of the NPB in both pronuclei had a statistically significant correlation with normal membrane breakage during ICSI (40%, compared with 33% easy, and 31% difficult membrane breakage) and also with faster cleavage and lower fragmentation rates of embryos. Sixty-one percent of implanting embryos had polarization of the NPB in both pronuclei compared with 37% for all embryos. Larger distance between pronuclei and their unequal size had a statistically significant correlation with slower cleavage and inferior embryo quality. Embryo selection based on only pronuclear morphology or on only day-3 embryo morphology yielded implantation rates of 15.1% and 12.1%, respectively. Embryo selection based on sequential evaluation of both pronuclear morphology and embryo morphology on day 3 resulted in a 21.1% implantation rate. CONCLUSION(S): Polarization of NPB in both pronuclei is as reliable marker of implantation as embryo morphology on day 3. However, pronuclear morphology assessment improves embryo selection only when it is combined with embryo morphology evaluation on day 3.

Heterotopic triplet pregnancy: report and video of a case of a ruptured tubal implantation with living embryo concurrent with an intrauterine twin gestation.

This report presents a case of triplet heterotopic gestation after intracytoplasmic sperm injection (ICSI)-IVF treatment, with a left ruptured ectopic tubal implantation with a living embryo and successful outcome of the concurrent intrauterine twin gestation. A couple whose infertility was caused by oligoasthenozoospermia was referred for ICSI treatment. Three good quality embryos were transferred at the request of the patient. Early gestational control was performed by ultrasound at weeks 5 and 7 of gestation. The patient reported to the centre during week 7 with severe abdominal pain and with signs of peritoneal irritation. Transvaginal ultrasound revealed an extra-uterine ruptured implantantion. During the concomitantly performed laparoscopic procedure, a living embryo was observed after opening the extra-uterine embryonic sac. Heartbeat activity was present and lasted for 5 min after surgical resection of the tubal implantation. The patient was discharged from hospital without complications. The intrauterine twin gestation was not affected and two healthy infants were born at week 38 of gestation. Heterotopic pregnancy should be ruled out in patients submitted to IVF-embryo transfer, although no predisposing factors are present in some cases. Precise diagnosis may be delayed due to some important characteristics of the IVF-embryo transfer treatment. Nevertheless, this condition should be diagnosed by ultrasound before tubal rupture to avoid obvious complications. Laparoscopy remains the gold standard for diagnosis and treatment in these cases. The presentation of the heterotopic pregnancy was recorded on video and may be viewed on the internet at www.rbmonline.com/Article/710.

Novel use of laser to assist ICSI for patients with fragile oocytes: a case report.

An inadvertent consequence of intracytoplasmic sperm injection (ICSI) is the degeneration of some of the microinjected oocytes. Most patients may not suffer any disadvantage through losing oocyte(s) during micromanipulation; however, in some circumstances, this can result in a reduction of the chances for pregnancy. This study reports a clinical pregnancy obtained by a novel approach using laser-assisted micro-opening of the zona pellucida prior to ICSI to secure a non-traumatic microinjection that avoids degeneration of oocytes. A total of 12 oocytes were obtained from the 36 year old patient in her third IVF treatment cycle, following two previously failed attempts where very high degeneration rates of oocytes after ICSI were recorded, together with suboptimal embryo quality. Five of the 11 matured (MII) oocytes were submitted to conventional ICSI and the other six MII oocytes first underwent laser-assisted opening of the zona pellucida (5-7 microm hole size was created with a 1.48 microm diode laser) before microinjection (LA-ICSI). Three of the five conventionally microinjected oocytes degenerated while one oocyte fertilized normally and developed to a good quality embryo. After the LA-ICSI procedure, one of the six oocytes degenerated and four oocytes fertilized normally; of these, two developed to excellent quality embryos, one to a good quality embryo and one to a poor quality embryo. The three best embryos (LA-ICSI group) were transferred to the patient on day 3. Rising serum human chorionic gonadotrophin concentrations were measured 12 days after transfer and on week 7 two implantation sites were detected, together with regular heart activity. The results of the present report suggest that laser-assisted ICSI may provide a safer approach to non-traumatic microinjection of oocytes than conventional ICSI, thereby minimizing the risk of degeneration and possibly also improving embryo quality. Therefore, it is suggested that laser-assisted ICSI might be applied in all cases associated with difficult zona pellucida penetration or/and fragile oolemma, or where patients have very few oocytes available, to improve the chances for pregnancy.

Relationship between time period after vasectomy and the reproductive capacity of sperm obtained by epididymal aspiration.

BACKGROUND: It is not well defined whether the elapsed time after vasectomy has any influence on the outcome of IVF-ICSI using epididymal sperm. We analysed retrospectively the results of 151 ICSI cycles in which sperm of vasectomized men were used at different time periods after vasectomy. METHODS: Oocytes were obtained after a desensitizing ovarian stimulation protocol using GnRH agonist in association with recombinant FSH and HCG. Sperm were retrieved by percutaneous epididymal sperm aspiration. The cycles were split into three groups: < or =10 years after vasectomy (group 1, n = 47), 11-19 years after vasectomy (group 2, n = 79), and > or =20 years after vasectomy (group 3, n = 25). RESULTS: As might be expected, the mean age of men differed in the three groups (group 3 > group 2 > group 1), and the mean age of the women was also significantly higher in group 3 than in groups 1 and 2, although no differences were described between groups 2 and 3. All other laboratory and clinical parameters were similar in the three groups. Ongoing pregnancy and implantation rates (34, 25, 8% and 22, 15, 6% respectively) decreased significantly from group 1 to group 3. CONCLUSION: Pregnancy and implantation rates after ICSI with sperm from vasectomized men are negatively correlated with the time interval from vasectomy, which cannot be explained purely by male or female ageing.

Pharmacological concentrations of follicle-stimulating hormone and testosterone improve the efficacy of in vitro germ cell differentiation in men with maturation arrest.

OBJECTIVE: To examine whether in vitro differentiation of germ cells from men with maturation arrest is improved by augmenting FSH and T concentrations above the values effective in samples from men with normal spermatogenesis. DESIGN: Prospective, controlled in vitro study. SETTING: Private assisted reproduction centers and a university department. PATIENT(S): Men with meiotic or postmeiotic maturation arrest. INTERVENTION(S): Testicular spermatid extraction, in vitro culture of testicular biopsy samples, intraoocyte injection of elongated spermatids, embryo culture and transfer. MAIN OUTCOME MEASURE(S): Progression of in vitro germ cell differentiation, fertilization, and pregnancy outcomes with in vitro cultured germ cells. RESULT(S): In some cases of meiotic and postmeiotic maturation arrest, more advanced germ cell stages were achieved by in vitro culture in the presence of 500 IU/L FSH as compared with 50 IU/L FSH. The beneficial effect of 500 IU/L FSH was further potentiated by a simultaneous increase of T concentration from 1 to 10 microM. Fertilizations with germ cells recovered after incubation with these pharmacological hormone concentrations gave rise to viable embryos and the births of five healthy babies. CONCLUSION(S): Pharmacological concentrations of FSH and T are beneficial for in vitro maturation of germ cells from some men with in vivo maturation arrest.

ICSI and day 5 embryo transfers: higher implantation rates and lower rate of multiple pregnancy with prolonged culture.

This retrospective review study, carried out in a private IVF clinic, compared pregnancy and implantation rates with day 3 versus day 5 embryo transfers in a selected group of patients. Participants were patients who failed to achieve pregnancy in at least one previous attempt with embryo transfer on days 2 or 3, and had more than five oocytes fertilized. A total of 296 patients who had undergone day 3 (group A) transfers were compared with 154 who had undergone day 5 transfers (group B). Interventions were intracytoplasmic sperm injection (ICSI), day 3 and day 5 embryo transfer. Outcome measures were pregnancy, implantation, multiple gestation and blastocyst formation rates. Overall, 86.4% of embryos were at the six- to eight-cell stage at 72 h and 30% developed to blastocyst by day 5. The mean number of embryos transferred was 4.0 on day 3 and 3.0 on day 5. Pregnancy and implantation rates were 34.8 and 11.5% in group A, versus 45.3 and 18.5% in group B. Multiple gestation rate was 47.1% in group A and 28.5% in group B. Prolonging embryo culture in vitro to day 5 improved embryo selection and implantation rates. A significant decrease in high order gestations was achieved by reducing the number of embryos transferred, without compromising the pregnancy rates.