Amin A. Milki

Incidence of monozygotic twinning with blastocyst transfer compared to cleavage-stage transfer

OBJECTIVE To evaluate the incidence of monozygotic twinning (MZT) in pregnancies conceived after blastocyst transfer compared to cleavage-stage transfer. DESIGN Retrospective study. SETTING University IVF program. PATIENT(S) All IVF patients with viable pregnancies conceived during a 4-year period. INTERVENTION(S) Blastocyst transfer or day 3 ET. MAIN OUTCOME MEASURE(S) Incidence of MZT assessed by transvaginal ultrasound. RESULT(S) There were 11 incidences of MZT in 197 viable pregnancies (5.6%) with blastocyst transfer compared to 7 of 357 viable pregnancies (2%) with day 3 ET. In 10 of 18 pregnancies, MZT was observed in the setting of a higher order multiple gestation (6 of 11 for blastocyst transfer and 4 of 7 for day 3 ET). In the day 3 ET group, assisted hatching or intracytoplasmic sperm injection (ICSI) did not increase MZT (4 of 213, 1.9%) compared to cycles without zona breaching (3 of 144, 2.1%). Similarly, in the blastocyst-transfer group, ICSI did not increase the incidence of MZT (4 of 74, 5.5% for ICSI and 7 of 123, 5.7% for non-ICSI IVF). CONCLUSION(S) Compared to day 3 ET, blastocyst transfer appears to significantly increase the incidence of gestations with MZT. This information should be taken into account when counseling patients about the pros and cons of extended culture.

Comparison of blastocyst transfer with day 3 embryo transfer in similar patient populations

OBJECTIVE To compare implantation and pregnancy rates (PRs) achieved with blastocyst transfer (BT) and day 3 ET in similar patient populations. DESIGN Retrospective analysis. SETTING Academic infertility center. PATIENT(S) One hundred consecutive patients <40 years undergoing IVF, each with more than three eight-cell embryos on day 3. INTERVENTION(S) Patients used their own eggs for IVF or IVF and intracytoplasmic sperm injection. Embryos were cultured in P1 medium (Irvine Scientific, Santa Ana, CA) until day 3, when they were either transferred or, in the case of embryos for BT, incubated in Blastocyst Medium (Irvine Scientific), followed by transferring on day 5. MAIN OUTCOME MEASURE(S) Implantation and PRs. RESULT(S) There were no statistically significant differences in patient age, FSH level, or number of oocytes or zygotes. The BT group had fewer embryos transferred (mean, 2.4) compared with the day 3-ET group (mean, 4.6). The viable PR (cardiac activity at 6-7 weeks was considered indicative of a viable pregnancy) was higher with BT (68%, 34/50) than with day 3 ET (46%, 23/50). The implantation rate was increased with BT (47%, 56 sacs/120 embryos) compared with day 3 ET (20%, 46 sacs/231 embryos). CONCLUSION(S) The BT group in our study had higher implantation and PRs compared with the day 3-ET group. Better embryo selection, improved embryo-uterine synchrony, and decreased cervical mucus on day 5 may have accounted for the enhanced outcome. Our data support the use of BT to limit the number of embryos transferred while improving PRs.

Blastocyst-ET and Monozygotic Twinning

AbstractPurpose: To examine the rate of monozygotic twinning associatedwith blastocyst transfer using commercially available,cell-free culture systems with unmanipulated blastocysts. Methods: A retrospective analysis was conducted in multipleprivate and academic infertility centers throughout theUnited States, of 199 pregnant patients following in vitrofertilization (IVF) blastocyst embryo transfer (ET). Humanembryos obtained through standard IVF stimulation protocolswere cultured in commercially available, cell-free mediasystems and transferred as blastocysts. The main outcomemeasure was the rate of monozygotic twinning. Results: A total of 199 blastocyst-ET pregnancies wereachieved during the study period at the fertility centersexamined. Monozygotic twinning was noted in 10/199 (5;pc)of these pregnancies. All were monochorionic diamnionic. Conclusions: Monozygotic twinning previously has beenreported following IVF, especially in relation to assistedhatching. While blastocyst transfer has been available formany years using coculture, there have been no publishedmulticenter reports of monozygotic twinning associated withunmanipulated blastocysts. In a multicenter analysis, a definiteincrease in monozygotic twinning was seen followingblastocyst-ET. We believe this phenomenon is real and thatthis information should be considered when counselingpatients for treatment.

Two-blastocyst transfer has similar pregnancy rates and a decreased multiple gestation rate compared with three-blastocyst transfer

OBJECTIVE To examine the effect of the number of blastocysts transferred on pregnancy and multiple gestation rates. DESIGN Retrospective study. SETTING Academic infertility center. PATIENT(S) Patients < 40 years undergoing IVF, with FSH levels of < 15 mIU/mL and more than three eight-cell embryos. INTERVENTION(S) Embryos were cultured in P1 until day 3 and then transferred to blastocyst medium. A maximum of three blastocysts were transferred. MAIN OUTCOME MEASURE(S) Pregnancy, multiple gestation, and implantation rates. RESULT(S) All 55 patients developed blastocysts and underwent ET. Twenty-four patients had three embryos transferred and 29 patients had two embryos transferred. Two patients had only one embryo each for transfer. There was no difference in the viable pregnancy rate between the two-blastocyst transfer and three-blastocyst transfer groups (62% vs. 58%). In the two-blastocyst transfer group, 39% of pregnancies were multiple gestations (all twin gestations), compared with 79% of pregnancies in the three-blastocyst transfer group (50% twin gestations, 29% triplet gestations). The implantation rate was 47% in both groups. CONCLUSION(S) A commercially available, sequential culture system is highly effective for producing viable blastocysts. Two-blastocyst transfer eliminated the risk of triplets while maintaining the same high success rates seen with three-blastocyst ET.

Is infertility a risk factor for female sexual dysfunction? A case-control study

OBJECTIVE To determine the impact of infertility on female sexual function. DESIGN A case-control study. SETTING Academic infertility and gynecology practices. PATIENT(S) One hundred nineteen women with infertility and 99 healthy female controls without infertility between the ages of 18 and 45 years were included in this study. INTERVENTION(S) Anonymous survey and Female Sexual Function Index. MAIN OUTCOME MEASURE(S) Female Sexual Function Index scores, frequency of sexual intercourse and masturbation, and sex-life satisfaction. RESULT(S) Twenty-five percent of our control group had Female Sexual Function Index scores that put them at risk for sexual dysfunction (<26.55), whereas 40% of our patients with infertility met this criterion. Compared with the control group, the patients with infertility had significantly lower scores in the desire and arousal domains and lower frequency of intercourse and masturbation. The patients with infertility retrospectively reported a sex-life satisfaction score that was similar to that of the controls before their diagnosis, whereas their current sex-life satisfaction scores were significantly lower than those of the controls. CONCLUSION(S) Women with a diagnosis of infertility were found to be at higher risk for sexual dysfunction on the basis of their Female Sexual Function Index scores compared with women without infertility. The interaction of sexual function and infertility is complex and deserves further study.

Etiology of recurrent pregnancy loss in women over the age of 35 years

OBJECTIVE To determine the rate of embryonic chromosomal abnormalities, thrombophilias, and uterine anomalies in women over the age of 35 years with recurrent pregnancy loss (RPL). DESIGN Retrospective cohort study. SETTING Academic reproductive endocrinology and infertility clinic. PATIENT(S) Women>or=35 years old with >or=3 first trimester miscarriages. INTERVENTION(S) None. MAIN OUTCOME MEASURE(S) Age, number of prior losses, cytogenetic testing of the products of conception (POC), uterine cavity evaluation, parental karyotype, TSH, and antiphospholipd antibody (APA) and thrombophilia testing. Aneuploidy in the POC in women with RPL was compared with sporadic miscarriages (<or=2 losses) in women>or=35 years. RESULT(S) Among 43 RPL patients, there were 50 miscarriages in which cytogenetic analysis was performed. In the RPL group, the incidence of chromosomal abnormalities in the POC was 78% (39 out of 50) compared with a 70% incidence (98 out of 140) in the sporadic losses. Thrombophilia results in the RPL patients were normal in 38 patients, four patients had APA syndrome, and one had protein C deficiency. Forty out of 43 had normal uterine cavities. Both TSH and parental karyotypes were normal in all of the patients tested. When the evaluation of RPL included karyotype of the POC, only 18% remained without explanation. However, without fetal cytogenetics, 80% of miscarriages would have been unexplained. CONCLUSION(S) In older patients with RPL, fetal chromosomal abnormalities are responsible for the majority of miscarriages. Other causes were present in only 20% of cases.

Accuracy of day 3 criteria for selecting the best embryos

OBJECTIVE To assess the accuracy of day 3 morphologic criteria in identifying the best embryos. DESIGN Prospective observational study. SETTING University IVF program. PATIENT(S) One hundred cycles in women desiring blastocyst transfer who had > or =3 eight-cell embryos on day 3. INTERVENTION(S) On day 3, the embryologist chose the two embryos that would have been transferred that day. On day 5, embryos were examined to determine the best and second-best blastocysts. MAIN OUTCOME MEASURE(S) Accuracy of day 3 picks as measured in culture on day 5, outcome of nontransferred picks, and cryopreservation rate. RESULT(S) All cycles reached the blastocyst stage and 73% had cryopreservation. The mean number of blastocysts was 4.8 (3.2 on day 5 and 1.6 on day 6). Neither pick was chosen in 39% of cycles; one pick was transferred in 38%; and both picks were transferred in 23%. Of 116 nontransferred picks, 51 were frozen and 65 arrested, with both picks arresting in 9 cycles. The single best blastocyst was chosen from the picks in 39% of cycles. CONCLUSION(S) Morphologic criteria for cleavage-stage embryo selection may fall short when the transfer is limited to two embryos. Culture to blastocyst is warranted in this population to avoid high-order multiples and still be able to choose the two embryos with the highest implantation potential.

Expression of vascular endothelial growth factor mRNA in human preimplantation embryos derived from tripronuclear zygotes

OBJECTIVE To detect the expression of vascular endothelial growth factor (VEGF) mRNA and/or secretion of VEGF protein by human preimplantation embryos. DESIGN Human preimplantation embryos not suitable for uterine transfer were examined for beta-actin and VEGF mRNA expression. Culture media from normally fertilized and developing preimplantation embryos were assessed for VEGF protein secretion. SETTING Clinics and academic research laboratories at the Departments of Obstetrics and Gynecology at the Stanford University, Palo Alto, California and the Heinrich-Heine-University, Düsseldorf, Germany. PATIENT(S) Couples undergoing IVF by intracytoplasmic sperm injection for various reasons. INTERVENTION(S) Six unfertilized oocytes and 33 pathologically fertilized (tripronucleic, 3PN) preimplantation embryos were examined for VEGF mRNA expression, and 16 embryos were examined for VEGF protein secretion. MAIN OUTCOME MEASURE(S) Embryonic expression of VEGF mRNA and VEGF protein as determined by reverse transcription (RT)/nested polymerase chain reaction (PCR) and ELISA. RESULT(S) VEGF mRNA and protein could not be detected in unfertilized oocytes. However, 30/33 preimplantation embryos did express VEGF mRNA (11/12 10-to-16-cell embryos, 3/4 morulae, 11/12 early blastocysts, 5/5 hatched blastocysts). The VEGF protein level was below the sensitivity of the ELISA. CONCLUSION(S) Production of VEGF may give the embryo the ability to induce neoangiogenesis at the implantation site, thus creating an environment necessary for its survival.

Tissue sampling technique affects accuracy of karyotype from missed abortions.

AbstractPurpose: To determine if careful specimen selection and washing of tissue from first trimester missed abortion products of conception specimens increases the sensitivity of routine cytogenetics in detecting aneuploidy. Methods: Retrospective review of cytogenetics results from tissue from dilation and curettage for missed abortion in a university fertility practice between 1998 and 2001. A technique of careful selection and washing of the specimen was implemented in July 1999. Results from before (n = 15) and after (n = 41) this change were compared. Cytogenetics reports from other physicians using the same laboratory were used for comparison (n = 59). Results: The percentage of 46XX results was significantly decreased in the test group when compared to historical and community controls: 29% vs. 73% and 56% respectively. The percentage of aneuploid results was significantly higher in the test group at 61% vs. 7% and 36% in the historical and community controls respectively. Conclusion: Thorough separation and cleaning of villi prior to sending missed abortion specimens significantly increases sensitivity of conventional cytogenetics for detecting aneuploidy by decreasing maternal contamination.

Poor Prognosis with In Vitro Fertilization in Indian Women Compared to Caucasian Women Despite Similar Embryo Quality

Background Disease prevalence and response to medical therapy may differ among patients of diverse ethnicities. Poor outcomes with in vitro fertilization (IVF) treatment have been previously shown in Indian women compared to Caucasian women, and some evidence suggests that poor embryo quality may be a cause for the discrepancy. In our center, only patients with the highest quality cleavage stage embryos are considered eligible for extending embryo culture to the blastocyst stage. We compared live birth rates (LBR) between Indian and Caucasian women after blastocyst transfer to investigate whether differences in IVF outcomes between these ethnicities would persist in patients who transferred similar quality embryos. Methodology/Principal Findings In this retrospective cohort analysis, we compared IVF outcome between 145 Caucasians and 80 Indians who had a blastocyst transfer between January 1, 2005 and June 31, 2007 in our university center. Indians were younger than Caucasians by 2.7 years (34.03 vs. 36.71, P = 0.03), were more likely to have an agonist down regulation protocol (68% vs. 43%, P<0.01), and were more likely to have polycystic ovarian syndrome (PCOS), although not significant, (24% vs. 14%, P = 0.06). Sixty eight percent of Indian patients had the highest quality embryos (4AB blastocyst or better) transferred compared to 71% of the Caucasians (P = 0.2). LBR was significantly lower in the Indians compared to the Caucasians (24% vs. 41%, P<0.01) with an odds ratio of 0.63, (95%CI 0.46–0.86). Controlling for age, stimulation protocol and PCOS showed persistently lower LBR with an adjusted odds ratio of 0.56, (95%CI 0.40–0.79) in the multivariate analysis. Conclusions/Significance Despite younger age and similar embryo quality, Indians had a significantly lower LBR than Caucasians. In this preliminary study, poor prognosis after IVF for Indian ethnicity persisted despite limiting analysis to patients with high quality embryos transferred. Further investigation into explanations for ethnic differences in reproduction is needed.