Transfer of IVF-contaminated blastocysts with removal of the zona pellucida resulted in live births.

PURPOSE: Bacterial contamination may cause loss or damage to cultured oocytes or embryos, resulting in cancelation or delaying of a fresh embryo transfer. While live births have been reported following the transfer of embryos contaminated with yeast, very little information is available on how to handle embryos with bacterial contamination. We report two cases of successful pregnancy in patients with bacterial contamination of embryo culture dishes. METHODS: We retrospectively reviewed 878 oocyte retrievals performed between January 2011 and December 2014. Bacterial contamination was recorded in two split IVF/ICSI cases, where contamination occurred in embryo culture drops containing embryos from conventional insemination but not from ICSI on day 3. RESULTS: To minimize the adverse effects of bacterial contamination on transfer outcomes, we removed the zona pellucida of contaminated frozen blastocysts and successfully obtained clinical pregnancies following transfer of zona-free blastocysts that were previously contaminated during IVF culture. CONCLUSIONS: Removal of the zona pellucida is an appropriate approach to handle blastocysts contaminated with bacteria during in vitro culture.

Gap junction blockade induces apoptosis in human endometrial stromal cells.

One of the most dynamic adult human tissues is the endometrium. Through coordinated, cyclical proliferation, differentiation, leukocyte recruitment, apoptosis, and desquamation, the uterine lining is expanded and shed monthly, unless pregnancy is established. Errors in these steps potentially cause endometrial dysfunction, abnormal uterine bleeding, failed embryonic implantation, infertility, or endometrial carcinoma. Our prior studies showed that gap junctions comprised of Gap junction alpha-1 (GJA1) protein, also known as connexin 43 (CX43), subunits are critical to endometrial stromal cell differentiation. The current studies were undertaken to explore the mechanism of endometrial dysfunction when gap junction intercellular communication (GJIC) is disrupted. Gap junction blockade by two distinct GJIC inhibitors, 18α-glycyrrhetinic acid (AGA) and octanol (OcOH), suppressed proliferation and induced apoptosis in endometrial stromal cells, as manifested by reduced biomarkers of cell viability, increased TUNEL staining, caspase-3 activation, sub-G1 chromosomal DNA complement, as well as shortened telomere length. Unexpectedly, we also observed that the chemical inhibitors blocked CX43 gene expression. Moreover, when endometrial stromal cells were induced to undergo hormonal decidualization, following a 7-day exposure to 10 nM 17ß-estradiol + 100 nM progesterone + 0.5 mM dibutyryl cAMP, characteristic epithelioid changes in cell shape and secretion of prolactin were blunted in the presence of AGA or OcOH, recapitulating effects of RNA interference of CX43. Our findings indicate that endometrial stromal cell proliferation and maintenance of decidualized endometrial function are GJIC-dependent, and that disruption of gap junctions induces endometrial stromal cell apoptosis. These observations may have important implications for several common clinical endometrial pathologies.

Selection of first in vitro fertilization cycle stimulation protocol for good prognosis patients: gonadotropin releasing hormone antagonist versus agonist protocols.

OBJECTIVE: The objective of this study was to compare outcome parameters in patients anticipated to have a good response to stimulation based upon baseline characteristics using either a gonadotropin releasing hormone (GnRH) agonist or antagonist protocol in their first in vitro fertilization (IVF) cycle. STUDY DESIGN: A retrospective chart review of all first-time IVF cycles performed during the time period 2005 through 2007 in an academic teaching center. Patients <40 years of age with a normal baseline follicle stimulating hormone (<10 mIU/mL) and normal antral follicle counts (> or = 3 in each ovary) were included. All patients studied were undergoing their first IVF cycle. The main outcome measures were clinical pregnancy and live birth rates. RESULTS: Included in the study were 755 patients undergoing a GnRH agonist protocol and 378 patients undergoing a GnRH antagonist cycle. Implantation rates (39.4% vs. 39.5%), cancellation rates (22.4% vs. 19.2%), clinical pregnancy rates (43.6% vs. 48.6%) and live birth rates (34.9% vs. 40.1%) were similar between GnRH antagonist and GnRH agonist protocol groups, respectively. CONCLUSION: Clinical pregnancy and live birth rates are similar in good responders utilizing either a GnRH agonist or antagonist during their first cycle of IVF.

Diagnosis of gestational diabetes mellitus: is it time for a new critical value?

OBJECTIVE: To identify the value for the 1-hour glucose tolerance test (GTT) that would maintain 100%, 90% and 75% sensitivity for identifying abnormal 3-hour GTT results in prenatal patients from an East Coast, urban, university hospital setting. STUDY DESIGN: Two hundred forty-two women who underwent the 3-hour GTT during pregnancy between January 1, 2004, and February 1, 2005, at a university hospital laboratory and private laboratories were included. The preceding 1-hour GTT results were obtained from these women, and a receiver operating characteristic (ROC) curve was constructed to identify a 1-hour GTT cutoff value that would maintain 100%, 90% and 75% sensitivity. A subgroup analysis was performed of patients of Asian ethnicity. This study was approved by the institutional review board. RESULTS: To maintain 100% sensitivity of the 1-hour GTT in predicting an abnormal 3-hour GTT, the 1-hour GTT cutoff value could be raised to 144 mg/dL in our population. For 90% and 75% sensitivities, the values were 150 and 156 mg/dL, respectively. There was no clinically significant difference in ROC curve evaluation between Asian and non-Asian groups. CONCLUSION: Raising the current level of 135 mg/dL for a 1-hour GTT to potentially decrease the need for the 3-hour GTT should be considered if larger patient series yield findings similar to those in our population.

Endometrial Stromal Decidualization Responds Reversibly to Hormone Stimulation and Withdrawal.

Human endometrial stromal decidualization is required for embryo receptivity, angiogenesis, and placentation. Previous studies from our laboratories established that connexin (Cx)-43 critically regulates endometrial stromal cell (ESC) differentiation, whereas gap junction blockade prevents it. The current study evaluated the plasticity of ESC morphology and Cx43 expression, as well as other biochemical markers of cell differentiation, in response to decidualizing hormones. Primary human ESC cultures were exposed to 10 nM estradiol, 100 nM progesterone, and 0.5 mM cAMP for up to 14 days, followed by hormone withdrawal for 14 days, mimicking a biphasic ovulatory cycle. Reversible differentiation was documented by characteristic changes in cell shape. Cx43 was reversibly up- and down-regulated after the estradiol, progesterone, and cAMP treatment and withdrawal, respectively, paralleled by fluctuations in prolactin, vascular endothelial growth factor, IL-11, and glycodelin secretion. Markers of mesenchymal-epithelial transition (MET), and its counterpart epithelial-mesenchymal transition, followed reciprocal patterns corresponding to the morphological changes. Incubation in the presence of 18α-glycyrrhetinic acid, an inhibitor of gap junctions, partially reversed the expression of decidualization and MET markers. In the absence of hormones, Cx43 overexpression promoted increases in vascular endothelial growth factor and IL-11 secretion, up-regulated MET markers, and reduced N-cadherin, an epithelial-mesenchymal transition marker. The combined results support the hypothesis that Cx43-containing gap junctions and endocrine factors cooperate to regulate selected biomarkers of stromal decidualization and MET and suggest roles for both phenomena in endometrial preparation for embryonic receptivity.

The first case described: monozygotic twin sisters with the fragile X premutation but with a different phenotype for premature ovarian failure.

OBJECTIVE: To describe the first case of monozygotic twin sisters with fragile X premutation and discordance for premature ovarian failure (POF). DESIGN: A descriptive case study. SETTING: Academic center. PATIENT(S): Monozygotic twin sisters with fragile X premutation and discordance for POF. INTERVENTION(S): Serum laboratory testing, fragile X premutation screening, zygosity testing, X-inactivation ratio and Southern blot studies. MAIN OUTCOME MEASURE(S): Incidence of POF in this twin cohort. RESULT(S): Zygosity analysis using polymerase chain reaction of 15 polymorphic markers via capillary gel electrophoresis in these patients confirmed their monozygosity. X-inactivation studies were performed using the human androgen receptor (HUMARA) gene and revealed similar X-inactivation ratios for both the patient and her sister (11:89 and 12:88, respectively) from peripheral serum samples. Southern blot evaluation of the proband and her sister revealed a similar methylation pattern in which the premutation allele was unmethylated much more than the normal allele. The contribution of the premutation on the active allele as determined by Southern blot analysis was consistent between sisters. CONCLUSION(S): The inactivation ratio studies and subsequent Southern blot analysis do not show differences between the patients; therefore, we are unable to identify a causative mechanism for the identical sisters' discordant phenotypes. It is possible that the inactivation ratios observed from the peripheral blood specimens obtained from the sisters do not represent the allele expression and skewing present at the level of the ovary.

Chronic endometritis is a frequent finding in women with recurrent implantation failure after in vitro fertilization.

OBJECTIVE: To determine the role of endometrial sampling for identification and treatment of chronic endometritis (CE) in patients undergoing IVF-ET who repeatedly failed to conceive despite the transfer of good-quality embryos. DESIGN: Retrospective chart review. SETTING: University-based tertiary fertility center. PATIENT(S): Thirty-three patients with recurrent implantation failure (RIF) who underwent endometrial sampling and subsequent ET were analyzed based on immunohistochemically confirmed CE: CE present on biopsy (group 1; n = 10) and CE absent on biopsy (group 2; n = 23). Patients with RIF undergoing IVF cycles during the same time period who did not have endometrial sampling were used as controls (group 3; n = 485). INTERVENTION(S): Endometrial sampling for CE and subsequent antibiotic treatment in affected patients followed by another IVF-ET cycle. RESULT(S): Chronic endometritis was identified in 30.3% of patients with RIF. Group 1 had lower implantation rates (11.5%) in the IVF cycle following treatment than did group 2 and group 3 (32.7% and 20.3%, respectively). Clinical pregnancy and ongoing pregnancy rates were similar across groups. CONCLUSION(S): Recurrent implantation failure warrants investigation of CE as a contributing factor. Women demonstrating CE on endometrial sampling have lower implantation rates in a subsequent IVF-ET cycle; however, there were no differences in subsequent clinical pregnancy or ongoing pregnancy rates after successful antibiotic treatment.

Progesterone activates a progesterone receptor membrane component 1-dependent mechanism that promotes human granulosa/luteal cell survival but not progesterone secretion.

CONTEXT: Progesterone (P4) promotes its own secretion and the survival of human granulosa/luteal (GL) cells. OBJECTIVE: The objective of these studies was to determine whether progesterone receptor membrane component-1 (PGRMC1) mediates P4's actions. DESIGN: In vitro studies were conducted on GL cells from women undergoing in vitro fertilization and GL5 cells, which are derived from GL cells. SETTING AND PATIENTS: GL cells were obtained from women undergoing fertility treatment at a university-based clinic and used for in vitro studies. MAIN OUTCOME MEASURES: PCR, Western blot, and immunocytochemistry were used to detect various progestin binding proteins. (3)H-P4 binding kinetics were assessed on partially purified PGRMC1. Apoptosis was determined after culture by either TUNEL or DAPI staining. P4 was measured by an ELISA assay. PGRMC1 was depleted using small interfering RNA. RESULTS: GL and GL5 cells expressed several P4 binding proteins including the nuclear progesterone receptor (PGR), progestin/adipoQ receptors (PAQR 7, 8, and 5) and PGRMC1. Ligand binding studies revealed that both P4 and the progestin, R5020, bound PGRMC1 with an EC(50) of approximately 10 nm. Interestingly, P4 inhibited apoptosis at concentrations in the 10 nm range, whereas R5020 stimulated P4 secretion at concentrations of at lease 16 mum. Depleting PGRMC1 attenuated P4's antiapoptotic action but failed to influence R5020-induced P4 secretion. CONCLUSIONS: These studies conclusively demonstrate that in human GL cells PGRMC1 functions as a receptor through which P4 activates a signal cascade that prevents apoptosis. In contrast, PGRMC1 does not mediate P4's ability to acutely promote its own secretion.