Vanessa N. Weitzman

Meiotic Arrest in Human Oocytes Is Maintained by a Gs Signaling Pathway

Abstract In mammalian oocytes, the maintenance of meiotic prophase I arrest prior to the surge of LH that stimulates meiotic maturation depends on a high level of cAMP within the oocyte. In mouse and rat, the cAMP is generated in the oocyte, and this requires the activity of a constitutively active, Gs–linked receptor, GPR3 or GPR12, respectively. To examine if human oocyte meiotic arrest depends on a similar pathway, we used RT-PCR and Western blotting to look at whether human oocytes express the same components for maintaining arrest as rodent oocytes. RNA encoding GPR3, but not GPR12, was expressed. RNA encoding adenylate cyclase type 3, which is the major adenylate cyclase required for maintaining meiotic arrest in the mouse oocyte, was also expressed, as was Gαs protein. To determine if this pathway is functional in the human oocyte, we examined the effect of injecting a function-blocking antibody against Gαs on meiotic resumption. This antibody stimulated meiotic resumption of human oocytes that were maintained at the prophase I stage using a phosphodiesterase inhibitor. These results demonstrate that human oocytes maintain meiotic arrest prior to the LH surge using a signaling pathway similar to that of rodent oocytes.

Maturation, Fertilization, and the Structure and Function of the Endoplasmic Reticulum in Cryopreserved Mouse Oocytes

Abstract Oocyte cryopreservation is a promising technology that could benefit women undergoing assisted reproduction. Most studies examining the effects of cryopreservation on fertilization and developmental competence have been done using metaphase II-stage oocytes, while fewer studies have focused on freezing oocytes at the germinal vesicle (GV) stage, followed by in vitro maturation. Herein, we examined the effects of vitrifying GV-stage mouse oocytes on cytoplasmic structure and on the ability to undergo cytoplasmic changes necessary for proper fertilization and early embryonic development. We examined the endoplasmic reticulum (ER) as one indicator of cytoplasmic structure, as well as the ability of oocytes to develop Ca2+ release mechanisms following vitrification and in vitro maturation. Vitrified GV-stage oocytes matured in culture to metaphase II at a rate comparable to that of controls. These oocytes had the capacity to release Ca2+ following injection of inositol 1,4,5-trisphosphate, demonstrating that Ca2+ release mechanisms developed during meiotic maturation. The ER remained intact during the vitrification procedure as assessed using the lipophilic fluorescent dye DiI. However, the reorganization of the ER that occurs during in vivo maturation was impaired in oocytes that were vitrified before oocyte maturation. These results show that vitrification of GV-stage oocytes does not affect nuclear maturation or the continuity of the ER, but normal cytoplasmic maturation as assessed by the reorganization of the ER is disrupted. Deficiencies in factors that are responsible for proper ER reorganization during oocyte maturation could contribute to the low developmental potential previously reported in vitrified in vitro-matured oocytes.

Predictive value of embryo grading for embryos with known outcomes

OBJECTIVE To compare pronuclear morphology (Z-score), day 3 embryo grade, and day 3 cell number in the prediction of successful implantation rates (IRs), including cycles in which all or none of the embryos implanted. DESIGN Retrospective analysis. SETTING University-based IVF center. PATIENT(S) Four hundred twenty-six fresh IVF day 3 transfers of 852 embryos in women <36 years of age from January 2000 to December 2003 in whom all or none of the embryos implanted. MAIN OUTCOME MEASURE(S) Evaluation of Z-scores, embryo morphology, cell number, and IR. RESULT(S) Day 3 parameters were more predictive than Z-scores. When early parameters were poor (Z-score) but late parameters were both good, the IR was 38%, compared with 4% when the Z-score was good but the late parameters were poor. CONCLUSION(S) Embryo grading systems are useful in the prediction of embryo implantation. In particular, cell number and embryo grade are more predictive than Z-scores. Therefore, late parameters have a better prognostic value than Z-scores when selecting embryos for transfer.

Comparison of luteal estradiol patch and gonadotropin-releasing hormone antagonist suppression protocol before gonadotropin stimulation versus microdose gonadotropin-releasing hormone agonist protocol for patients with a history of poor in vitro fertilization outcomes

OBJECTIVE To compare IVF outcomes in poor-responder patients undergoing stimulation after luteal phase E(2) patch/GnRH antagonist (LPG) protocol versus microdose GnRH agonist protocol. DESIGN Retrospective analysis. SETTING University-based IVF center. PATIENT(S) Forty-five women undergoing ovarian stimulation for IVF using the LPG protocol were compared with 76 women stimulated with the microdose GnRH agonist protocol from May 2005 to April 2006. MAIN OUTCOME MEASURE(S) Cancellation rate, number of oocytes retrieved, and clinical pregnancy rates. RESULT(S) The mean number of oocytes (9.1 +/- 4.1 vs. 8.9 +/- 4.3) and mature oocytes (6.7 +/- 3.5 vs. 6.8 +/- 3.1) retrieved were similar, as were the fertilization rates (70.0% +/- 24.2% vs. 69.9% +/- 21.5%) and the number of embryos transferred (2.5 +/- 1.1 vs. 2.7 +/- 1.3). The cancellation rate was not significantly different between the groups (13/45, 28.9% vs. 23/76, 30.3%). Likewise, there were no significant differences among the implantation rate (15.0% vs. 12.5%), clinical pregnancy rate (43.3% vs. 45.1%), and ongoing pregnancy rate per transfer (33.3% vs. 26.0%) between both groups. CONCLUSION(S) This study demonstrates that the use of an E(2) patch and a GnRH antagonist during the preceding luteal phase in patients with a history of failed cycles can provide similar IVF outcomes when compared with the microdose GnRH agonist protocol.

Prognostic value of beta-human chorionic gonadotropin is dependent on day of embryo transfer during in vitro fertilization

OBJECTIVE To determine threshold β-hCG levels predictive of an ongoing pregnancy (OP), live birth (LB), and multiple gestation (MG) in IVF cycles resulting from day-3 (D3) vs. day-5 (D5) embryo transfers (ET), to compare IVF cycle characteristics and pregnancy outcomes in D3 vs. D5 ET groups, and to assess the degree to which maternal characteristics and cycle parameters were predictive of higher β-hCG levels. DESIGN Retrospective analysis. SETTING Infertility center. PATIENT(S) Women who had ET performed for IVF cycles between July 2004 and January 2010. INTERVENTION(S) Embryo transfer performed on either D3 or D5 after oocyte fertilization. MAIN OUTCOME MEASURE(S) Beta-hCG on day 15 after oocyte fertilization. RESULT(S) Beta-hCG levels were significantly higher with D5 ET compared with D3 ETs (D3: 103.6 ± 4.4 IU/L vs. D5: 198.0 ± 10.6 IU/L), and a multivariate analysis demonstrated that D5 ET was a significant predictor of higher β-hCG levels. The β-hCG thresholds predictive of OP were 78 IU/L and 160 IU/L for D3 and D5 ET, which predicted OP in 96% and 91% of cases, respectively. Similarly, for LB, the β-hCG thresholds were 94 IU/L (79% positive predictive value [PPV]) and 160 IU/L (88% PPV), and for MG were 250 IU/L (18% PPV) and 316 IU/L (34% PPV), respectively. CONCLUSION(S) Initial β-hCG levels are dependent on the day of ET and are a reliable and highly predictive tool for OP outcomes.

In vitro fertilization outcomes in patients experiencing a premature rise in luteinizing hormone during a gonadotropin-releasing hormone antagonist cycle

Patients undergoing controlled ovarian hyperstimulation and pituitary suppression with a GnRH antagonist who experienced a transient premature rise in LH were compared with those who did not have an early surge. Those experiencing a premature LH surge had equivalent clinical and ongoing pregnancy rates per ET.