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Uroplakin (UP) tetraspanins and their associated proteins are major mammalian urothelial differentiation products that form unique two-dimensional crystals of 16-nm particles (“urothelial plaques”) covering the apical urothelial surface. Although uroplakins are highly expressed only in mammalian urothelium and are often referred to as being urothelium specific, they are also expressed in several mouse nonurothelial cell types in stomach, kidney, prostate, epididymis, testis/sperms, and ovary/oocytes. In oocytes, uroplakins colocalize with CD9 on cell-surface and multivesicular body-derived exosomes, and the cytoplasmic tail of UPIIIa undergoes a conserved fertilization-dependent, Fyn-mediated tyrosine phosphorylation that also occurs in Xenopus laevis eggs. Uroplakin knockout and antibody blocking reduce mouse eggs’ fertilization rate in in vitro fertilization assays, and UPII/IIIa double-knockout mice have a smaller litter size. Phylogenetic analyses showed that uroplakin sequences underwent significant mammal-specific changes. These results suggest that, by mediating signal transduction and modulating membrane stability that do not require two-dimensional-crystal formation, uroplakins can perform conserved and more ancestral fertilization functions in mouse and frog eggs. Uroplakins acquired the ability to form two-dimensional-crystalline plaques during mammalian divergence, enabling them to perform additional functions, including umbrella cell enlargement and the formation of permeability and mechanical barriers, to protect/modify the apical surface of the modern-day mammalian urothelium.

In this case-control study of euthyroid first-cycle IVF patients ≥38 years old with singleton baby, miscarriage, biochemical pregnancy, and no pregnancy outcomes from 2005–2008, we assayed frozen serum for autoimmune thyroid disease (AITD) and thyroid function at cycle start, trigger, and 4 and 5 weeks' gestation. AITD prevalence in older infertile women was similar across clinical outcomes, and although AITD was associated with a higher baseline TSH, TSH remained within acceptable ranges, suggesting that T 4 supplementation may not affect maternal outcomes in older euthyroid AITD patients through 5 weeks gestation.

To explore the long- and short-term trends in LH-suppression protocol use and patient profile characteristics.Descriptive study, retrospective cohort.Large, university-based IVF center.Four thousand five hundred one fresh IVF cycles categorized by use of GnRH antagonist, luteal GnRH agonist, and follicular microdose GnRH agonist.None.Frequency of use of LH-suppression protocol, patient and cycle characteristics, and outcomes at 10-year (1996-2005), 5-year (2001-5), and 3-year intervals (2004-6).In both the40 andor=40 age groups, GnRH antagonist use increased from 2001 to 2005, while luteal GnRH agonist and microdose use decreased. The most recent luteal agonist patients were better responders and had higher implantation, clinical pregnancy, and delivery rates. Antagonist patients in the40 andor=40 age groups had a better response in 2005 than in 2001 with higher clinical pregnancy rates. Microdose patients responded worse in 2005 than in 2001, although pregnancy rates did not change significantly. Such trends were echoed from 2004 to 2006.The target population for GnRH antagonist has broadened to include younger, normal responders in addition to the traditional poor responder. Luteal agonist and microdose protocols are chosen less frequently and remain targeted toward good and poor responders, respectively.

Although several early IVF successes were achieved after transferring fully formed blastocysts, the majority of embryos replaced worldwide over the past 30 years have been at the cleavage stage. The programme at this study centre has previously found that delaying an embryo transfer to day 5 can reduce the chance for a high-order multiple gestation without compromising the pregnancy rate because fewer embryos can be replaced. To evaluate the impact of transfer day and embryonic stage at cryopreservation on cycle outcome, 6069 fresh and 706 frozen transfers from 2000-2006 performed at this study centre were retrospectively analysed. Approximately half of the fresh transfers were performed on day 3, with a shift to day-5 transfer over the study period with no change in cryopreservation incidence. Implantation, clinical pregnancy and live birth rates were significantly higher following day-5 transfer. When frozen-thawed embryos (2-cell to day-6 blastocysts) were transferred, acceptable pregnancy and live birth rates were achieved at all stages but thawed embryos transferred as day-5 blastocysts generated consistently higher clinical pregnancy and live birth rates. Transfer of embryos frozen on day 6 had the highest miscarriage and lowest live birth rates. Barring government regulation, an IVF programme's day for cryopreservation generally depends on its management of and success with fresh embryo transfer.

To reevaluate clinical management of isolated teratozoospermia, in couples initiating IVF.Retrospective analysis of fertility indices in 535 cycles.A large, university-based fertility center.Consecutive couples (n = 495) who had a semen analysis using Kruger/Tyberberg strict criteria at our center within 12 months before undergoing their first and/or second IVF cycle in 2002-2004 with2 million postwash, motile sperm on the day of egg retrieval.Eggs were fertilized either by conventional IVF or ICSI. Semen analysis and gamete/embryo manipulation was standardized in all cases.Fertilization, fertilization failure, pregnancy, and live birth rates.There was no statistical difference in fertilization, fertilization failure, pregnancy, and live birth rates in the first or second IVF cycle when comparing couples with isolated teratozoospermia (5% normal morphology) to those with a normal semen analysis. Furthermore, no improvement in these outcomes was noted when ICSI was used to treat these teratozoospermic couples.Because isolated teratozoospermia generally does not impact on the major indices of IVF, these patients need not be subjected to the unnecessary cost and potential risks of ICSI. Future studies, however, should focus on different sperm morphologic and biochemical parameters to determine if they are important for clinical management in IVF.

To assess whether the use of extended embryo culture can reduce the incidence of high-order multiple gestations, a retrospective analysis of 7,418 fresh ETs performed in a university-based IVF clinic from 1997-2003 was conducted, comparing program results before and after institution of a protocol to select patients for extended culture of in vitro fertilized embryos. The incidence of triplet pregnancies was significantly reduced in patients at highest risk for high-order multiple gestations, i.e., those at

To assess the rates of select obstetric outcomes in oocyte donation (OD) recipients aged35 years andor =40 years and compare them to similarly aged IVF patients.Retrospective anonymous questionnaire study.University-based IVF center.Live-birth outcome was experienced by 199 OD recipients and 488 IVF patients35 oror =40 years.The OD or IVF cycles.Response rate, pregnancy outcome, and complications.Response rate was 60%. The OD recipients had significantly higher rates of pregnancy-induced hypertension (PIH) than their IVF counterparts. The OD35 years had the highest rate (42%), followed by ODor =40 years (26%), IVFor =40 years (14%), and IVF patients35 years (12%). In twin pregnancies, the rates of PIH remained higher in the OD groups: OD35 years (56%), ODor =40 years (36%), IVFor =40 years (25%), and IVF35 years (22%). Twin pregnancy rate was lowest in IVF patientsor =40 years (19%) and a lower preterm delivery rate (16%) reflected this difference. The cesarean section rates were 50% for singleton and 78% for twin deliveries in the OD patients35 year; in OD patientsor =40 years, the rates were 75% and 84%, respectively.The OD recipients are at higher risk for untoward obstetric outcomes than their IVF counterparts. Young OD recipients reported the highest rate of PIH, warranting further investigation into an association between early loss of ovarian function and PIH.

Cell allocation and subsequent lineage commitment in the human embryo may be established as early as in the unfertilized oocyte. This phenomenon might be the result of subtle differences of gene expression and protein distribution. To assess whether gene expression profiling by reverse transcription–polymerase chain reaction could be a suitable tool for the detection of cell allocation and lineage commitment, the expression pattern of the putative inner cell mass marker gene Oct-4 and the trophectodermal marker genes β-HCG and β-LH were correlated in individual blastomeres of preimplantation human embryos. In 2- to 5-cell stage embryos, expression of β-HCG and Oct-4 mRNA was negatively correlated in all blastomeres with statistical significance, suggesting that cell allocation can be assessed by those markers at early stages. In 7- to 10-cell stage embryos, expression of β-LH and Oct-4 mRNA was negatively correlated in some blastomeres without statistical significance, suggesting that more experiments are necessary to decide if lineage commitment can be assessed in some cells by those markers at later stages.

BACKGROUND: Cytoplasmic defects are thought to cause aneuploidies in oocytes and embryos and oocyte ‘reconstruction’ by germinal vesicle (GV) transfer may circumvent such defects. In mice ‘reconstructed’ oocytes undergo meiosis and fertilize normally, but early embryonic development is compromised if their ooplasm matured in vitro. This study employs sequential MII spindle and/or pronucleus (PN) transfer to assess the embryonic potential of MII nuclei that form following GV transfer. METHODS AND RESULTS: Mouse embryos generated by these procedures were transferred to the oviducts of pseudopregnant mice to monitor pregnancy outcome. Following GV transfer, the resultant metaphase II (MII) nuclei were activated either in situ or transferred and activated in ooplasts from in-vivo matured oocytes. When exchanged with the female PN of a fertilized zygote, only the PNs that developed in in-vivo matured ooplasts generated live offspring. Viable offspring also resulted when MII nuclei were transferred to in-vivo matured ooplasts and fertilized by insemination with sperm or by artificial activation and male PN transfer. Significantly, the offspring displayed normal fertility as adults. CONCLUSION: This report of live births following GV transfer in mice illustrates the importance of the maturational history of the ooplasm at PN formation for normal embryonic and fetal development.

In human embryos, blastomeres differentiate into trophectoderm (TE) cells and inner cell mass (ICM) cells of blastocysts. Although morphologically indistinguishable, blastomeres at early cleavage stages are likely to undergo changes on a molecular level that make them destined to become ICM or TE cells. While the transcription factor Oct-4 might serve as a marker for totipotent ICM cells, human chorionic gonadotrophin might be used as the equivalent for TE cells. This study reports a reverse transcription-polymerase chain reaction procedure to assess human beta-HCG mRNA concentrations as well as ploidy in individual blastomeres from normally and abnormally fertilized human embryos. beta-HCG mRNA was detected in both euploid and aneuploid cells and in oocytes. Surprisingly, beta-LH mRNA was also detected in some euploid blastomeres. In regard to preimplantation genetic diagnosis, assessment of expression levels of beta-HCG and Oct-4 mRNA in individual biopsied cells might serve as a tool to identify embryogenic blastomeres in combination with testing for chromosome and single gene abnormalities.

Oct-4, a decisive factor that maintains totipotency in murine embryonic and germ cells, is exclusively expressed in such cells. In mice, different levels of oct-4 expression in blastomeres predict development towards inner cell mass (ICM) (high oct-4) or trophectoderm (TE) (low oct-4). To address whether the mouse model also applies to human embryos, the cytoplasm of individual human blastomeres from normally and abnormally fertilized embryos was tested for Oct-4 expression by reverse transcription-polymerase chain reaction (RT-PCR). The nuclei of the same blastomeres were subjected to fluorescence in-situ hybridization (FISH) to determine ploidy. A significant difference in Oct-4 mRNA levels was revealed between blastomeres. The distribution of blastomeres with high Oct-4 levels varied according to the cleavage stage of the embryo: the more blastomeres, the lower the percentage with high Oct-4 levels. Aneuploid blastomeres did not exhibit lower Oct-4 mRNA levels than diploid ones. Thus, differential Oct-4 expression in individual human blastomeres appears to direct cells towards the ICM or TE lineages without regard to chromosomal status. Oct-4 might be used as a marker in preimplantation genetic diagnosis to identify embryogenic blastomeres.

The expression of the transcription factor Oct-4 is thought to be one of the decisive factors that maintain totipotency in embryonic and germ cells. In mice, oct-4 is exclusively expressed in germ cells and totipotent cells of the embryo. In humans, Oct-4 is expressed in germ cells, embryonic stem cells and whole embryos at various stages of development. However, there is limited information about the distribution of Oct-4 expression in human embryos. In an attempt to address this issue, the inner cell mass (ICM) and trophectoderm (TE) of 17 human blastocysts were separated and Oct-4 mRNA expression individually assessed by reverse transcription-polymerase chain reaction (RT-PCR). In discarded blastocysts that developed from two pronuclear zygotes, the mean Oct-4 expression was 31 times higher in totipotent ICM cells than in differentiated TE cells. This finding suggests that, in accordance with data from the mouse, Oct-4 is highly expressed in human ICM cells as opposed to TE cells; this in turn supports the hypothesis that Oct-4 plays a similar role to maintain totipotency in these two species.

The Herlitz type of junctional epidermolysis bullosa (H-JEB) is a severe inherited bullous disease which leads to the early demise of the affected newborn. Mutations in the genes encoding the 3 polypeptides of the anchoring filament protein laminin 5 underlie this condition. We studied 2 families with affected children who previously died from H-JEB. Mutation screening using heteroduplex analysis and direct sequencing of the PCR products revealed a previously described hotspot mutation in LAMB3 (R635X), and a novel delayed termination codon in LAMB3 in the first proband. In the second proband, we found a novel initiation codon mutation in LAMB3, and a novel 2 bp deletion in LAMB3. For preimplantation genetic diagnosis (PGD) in these families, we developed nested multiplex PCR assays, amplifying the mutations and informative intragenic polymorphisms in the probands. Single embryonic cells were biopsied from 8-cell embryos using standard techniques, and subjected to the multiplex PCR assay followed by restriction enzyme digestion. Embryos found not to carry either mutation were transferred to the mothers, and a pregnancy was established in the second family as evidenced by the elevated level of HCG, although the pregnancy did not persist. This study illustrates the feasibility of PGD for an inherited skin disorder for the first time.

Objective: To investigate the effects of two virucidal compounds, MAP30 (Momordica anti–human immunodeficiency virus [HIV] protein; molecular weight, 30 kd) and GAP31 (Gelonium anti-HIV protein; molecular weight, 31 kd), obtained from Momordica charantia and Gelonium multiflorum , respectively, on the motility and vitality of human spermatocytes. Design: Prospective, controlled study. Setting: New York University School of Medicine. Patient(s): Ten healthy men undergoing evaluation for infertility provided 10 semen specimens. Intervention(s): Human sperm were treated with the anti-HIV agents, MAP30 and GAP31. Nonoxynol-9, a commonly used spermicide, and phosphate-buffered saline were used as the positive and negative controls, respectively. Main Outcome Measure(s): The motility and vitality of human spermatocytes treated with MAP30 and GAP31 at doses that inhibit HIV-1 and herpes simplex virus. Result(s): MAP30 and GAP31 did not inhibit the motility or vitality of human sperm cells over a dose range of 100–0.1 μg/mL, whereas nonoxynol-9 demonstrated spermicidal action on all 10 samples over the same dose range. Conclusion(s): The antiviral agents, MAP30 and GAP31, were not toxic to human sperm cells at the doses at which they inhibit HIV-1 and herpes simplex virus. They had no effect on the motility of spermatozoa, even at a dose of 1,000 times the maximum effective concentration. These results indicate that MAP30 and GAP31 may be useful as nonspermicidal protection against sexually transmitted diseases.

We evaluated the maturational competence of mouse oocytes reconstructed by the transfer and electrofusion of germinal vesicles (GV) into anuclear cytoplasts of GV stage oocytes (both auto- and hetero-transfers), metaphase II stage oocytes or zygotes. Following in-vitro culture, the maturation rates of the reconstructed oocytes to metaphase II did not significantly differ between auto- and hetero-transfers (40/70 versus 95/144 respectively); these rates also did not differ from those of control oocytes (57/97) which were matured in vitro without micromanipulation and electrofusion. In contrast, when a GV was transferred into an enucleated metaphase II oocyte or a zygote, only a few reconstructed oocytes underwent germinal vesicle breakdown (5/30 and 2/21 respectively); moreover, none reached metaphase II stage. Cytogenetic and immunofluorescence analyses were conducted on hetero-GV oocytes that extruded a first polar body. Each oocyte showed two groups of chromosomes, one in the cytoplast and one in the polar body, as well as a bipolar spindle with twenty univalent chromosomes. Our findings suggest that oocytes reconstructed by GV transfer into a cytoplast of the same developmental stage mature normally in vitro through metaphase II. Such oocytes may be a useful research model to elucidate the cytoplasmic and nuclear mechanisms regulating meiosis and the relationships between meiotic errors and age-related changes in the oocyte.

Objective: To assess the impact of ET difficulty on IVF outcome and to optimize the ET procedure. Design: Retrospective analysis of IVF outcome by ET catheter type and ET difficulty. Prospective treatment and follow-up of patients with a history of extremely difficult cervical passage. Setting: Large university-based IVF program. Patient(s): All patients Intervention(s): Surgical correction of cervical stenosis. Main Outcome Measure(s): Pregnancy and embryo implantation rates. Result(s): Only 0.6% of ETs were "extremely difficult." Pregnancy rates were not statistically significantly different among ETs graded easy, moderate, and difficult. In contrast, no pregnancies occurred in the rare "extremely difficult" ET group. Eight patients with a history of extremely difficult cervical passage underwent surgical correction of their cervical stenosis. Twelve postoperative IVF-ET in these women resulted in eight clinical pregnancies, six of which were multiple gestations. The embryo implantation rate of these cycles was 42.2%. Conclusion(s): Patients with a history of extremely difficult ET may benefit from hysteroscopic evaluation and possible modification of their cervical canal before a future IVF attempt.

Objective: To describe different techniques of testicular tissue culture and their effect on sperm motility, mainly in cases of totally immotile spermatozoa, and to compare the effect of in vitro culture with that of motility stimulants. Design: Prospective study. Setting: University teaching hospital. Patient(s): Ten patients undergoing testicular biopsy for diagnostic purposes or for intracytoplasmic sperm injection. Intervention(s): Dissected testicular biopsy samples and tissue blocks were cultured at 37°C for up to 96 hours. Immediately after dissection, immotile testicular spermatozoa were incubated for 30 minutes in pentoxifylline and 2-deoxyadenosine. Main Outcome Measure(s): Sperm motility and vitality. Result(s): Overall, dissected samples showed improved sperm motility, which peaked within 48 hours of culture. Unlike motility, vitality declined linearly, from 56.3% ± 19% at initial assessment to 18.8% ± 11% at 96 hours. Five samples had initially immotile spermatozoa, of which four acquired motility at 48 hours. In vitro culture showed results comparable with those of incubation with pentoxifylline and 2-deoxyadenosine. Culture of tissue blocks did not improve motility or vitality compared with dissected tissue. Conclusion(s): The motility of testicular spermatozoa was enhanced or initiated after in vitro culture. Testicular biopsy culture may be an alternative to the use of motility stimulants to obtain motile spermatozoa for intracytoplasmic sperm injection, particularly when oocytes are not immediately available.

Objective: To look for correlations between acridine orange (AO) staining and semen parameters before and after sperm separation procedures and to assess whether the AO test predicts fertilization or pregnancy outcomes after standard IVF and intracytoplasmic sperm injection. Design: Prospective study that simultaneously assesses sperm morphology and nuclear protein maturity on a cell-by-cell basis before and after preparative procedures. Setting: University teaching hospital. Patient(s): Men (n = 140) undergoing diagnostic semen analysis. Main Outcome Measure(s): Acridine orange fluorescence of sperm nuclei, semen parameters, IVF outcome. Result(s): In unprocessed samples, 90% of sperm with normal heads displayed green fluorescence (mature nuclear protein); significantly lower percentages of green fluorescence were observed in sperm with abnormal heads. The percentage of mature normal sperm in the specimen correlated with motility. Sperm maturity after swim-up or Percoll gradient was significantly improved for sperm with normal or abnormal heads. The percentage of mature normal sperm correlated with motility after either Percoll or swim-up. Neither the percentages of mature nuclei nor mature normal nuclei correlated with fertilization or pregnancy outcome. Conclusion(s): Nuclear protein maturation correlates with sperm motility and morphology. Because morphologically normal and motile sperm are more mature, separation procedures should generate a population of sperm with the highest fertilization capacity. Acridine orange staining, however, did not predict fertilization efficiency or pregnancy outcome in IVF cycles.

Purpose:Single-cell nested polymerase chain reaction (PCR) and Dde1 endonuclease digestion were used to detect the presence of a Marfan's syndrome mutation in human preimplantation embryos derived from in vitro fertilization (IVF). These procedures were conducted to eliminate the possibility of transmission of the affected allele from the father to his offspring. The mutation on chromosome 15 is transmitted as an autosomal dominant trait, and the chance of having a child affected with the disease is 50%.

Although round spermatids have been studied extensively using staining techniques and electron microscopy, little information is available about their appearance in living conditions. We describe a method of collecting and identifying round spermatids from ejaculates and testicular biopsies. The validity of the selection procedure was confirmed by fluorescence in-situ hybridization. Based on cell size, morphological characteristics of nucleus and cytoplasm, and on the nucleus/cytoplasm ratio, we harvested a population of cells that was 84% haploid. This procedure can be applied to select spermatids for clinical or research purposes.

OBJECTIVE: Our purpose was to evaluate whether tamoxifen has estrogenic endometrial effects as defined by histologic study or alterations in steroid hormone receptor expression. STUDY DESIGN: Nineteen postmenopausal tamoxifen-treated breast cancer patients who also had endometrial sampling were identified from files in the Department of Obstetrics and Gynecology. To examine the subgroup of 15 polyps, age-matched, non–hormonally treated patients with polyps ( n = 8) or atrophic endometria ( n = 5) served as comparison groups. Proliferative ( n = 3) and secretory ( n = 5) endometria served as procedural controls. Immunohistochemical studies for steroid receptors (estrogen, progesterone) were performed. RESULTS: Glandular cell progesterone receptor was significantly increased and stromal cell estrogen receptor was significantly decreased in tamoxifen-treated versus atrophic endometria. Progesterone receptor staining was not significantly different in tamoxifen-treated versus control polyps, although staining was high in both groups. Stromal cell estrogen receptor staining was significantly reduced in tamoxifen-treated versus control polyps, although there were no histologic differences. Reduced stromal cell estrogen receptor and increased glandular cell progesterone receptor staining was found in all tamoxifen-treated endometria regardless of the diagnosis. CONCLUSION: The tamoxifen-associated changes in endometrial steroid receptors support an estrogenic effect that is independent of histologic diagnosis and duration of use. This may contribute to the pathogenesis of tamoxifen-associated polyps and carcinomas. (Am J Obstet Gynecol 1997;176:129-37.)

Objective To identify the incidence, risk factors, and obstetric/perinatal outcomes associated with monozygotic twins (MZTs) after IVF. Design Nested case-control. Setting University-based center. Patient(s) The IVF cycles eventuating in pregnancy from 2000–2009. Intervention(s) None. Main Outcome Measure(s) The MZT incidence, chorionicity/zygosity, pregnancy/neonatal outcome. Result(s) Of 6,223 gestations, 131 MZTs were diagnosed (2.1% incidence; 2.0% in autologous and 2.7% in donor IVF cycles), 10 were dichorionic, and 121 were monochorionic. Controlling for all risk factors, young oocyte age, extended culture (noncleavage embryos transferred on/after day 4), and year of IVF treatment cycle were significantly associated with MZT. When assessing factors associated with specific MZT placentation, day 3 assisted hatching correlated more with dichorionic MZT, whereas extended culture and advanced day 5 embryonic stage correlated with monochorionic MZT. Comparing monozygotic to dizygotic multigestation outcomes, MZT fared worse; however, once controlling for triplet gestation, only gestational age at delivery remained significantly compromised in the monozygotic group. Conclusion(s) After IVF the incidence of MZT is high, with young oocyte age, year of treatment, and extended culture (or embryo stage at transfer) conferring greatest risk. Regarding MZT type, assisted reproductive technology (ART) procedures may influence the timing of embryonic splitting (i.e., division in early embryonic development may be influenced by zona pellucida [ZP] manipulation whereas later splitting may occur during delayed implantation). Poor obstetric/perinatal outcome is significantly impacted by the presence of an "extra" fetus, as high-order multiple gestation concurrent with an MZT conveyed the worst prognosis.

Midodrine, a peripheral α-adrenergic agonist, was evaluated in 7 female and 2 male patients with familial dysautonomia (FD), a disorder characterized by decreased sympathetic innervation. Prior to and after three months of midodrine treatment, each patient's response to postural change was assessed by arteriosonde readings of blood pressure and heart rate, corrected QT-interval measurements, Doppler evaluation of renal blood flow and circulating atrial natriuretic peptide (ANP) levels. The initial midodrine dose (2.5 mg three times daily) was raised until subjective symptoms improved. Doses were reduced if patients felt jittery or developed erect hypertension (systolic > 180 mmHg or diastolic > 110 mmHg). Midodrine, at an average dose of 0.25 mg/kg per day, improved subjective symptoms in all patients. With treatment, magnitude of blood pressure responses was variable. Although mean erect blood pressure did not increase significantly for the aggregate, it did increase in six of nine patients. In addition, the QTc interval normalized and erect renal perfusion improved. Changes in supine mean blood pressure and supine circulating ANP correlated directly. We judge midodrine to be useful in management of orthostatic hypotension in patients with familial dysautonomia.

To explore patient goals and quality of life (QOL) via a prospective registry and compare fertility preservation (FP) outcomes before, during, and after cancer therapy.Of 35 patients entering the registry from 3/2008 to 3/2010, 29/35 completed the study survey and agreed to follow-up, and 31/35 completed treatment. Survey results and FP outcomes were analyzed.Most patients rated the impact of cancer treatment on fertility of highest importance at baseline and 1-year follow-up. QOL scores were overall positive at both intervals. Patients naïve to any cancer treatment (n = 12) had more gametes frozen than patients with prior cancer treatment (n = 19) with no difference in age or gonadotropin dosage. For patients awaiting cancer treatment, the median time from consultation to oocyte retrieval was 25 days. Cancer treatment sequalae posed challenges to optimal FP outcomes.Fertility preservation remains a significant issue for cancer patients. With early reproductive endocrinologist referral, cancer treatment delay is minimized and FP outcomes are optimized.

BACKGROUND: To minimize the potential for harmful inheritable conditions, donors are rigorously screened according to standard guidelines, yet such guidelines may not be sufficient to exclude egg donors with certain known inheritable conditions. We compared universal screening of oocyte donors with Tay-Sachs, Fragile X, karyotype and Minnesota Multiphasic Personality Inventory-2 (MMPI-2) versus standard American Society of Reproductive Medicine (ASRM) guidelines that do not include such testing. METHODS: In this 12 year retrospective cohort study, results of enhanced universal screening of all anonymous oocyte donor candidates from 1997 to 2008 at a university hospital oocyte donation program were reviewed. Primary outcomes were the frequency of oocyte donor candidates excluded as a result of enhanced universal screening (Tay-Sachs, Fragile X, karyotypic analysis and MMPI-2) versus basic screening according to ASRM guidelines. RESULTS: Of 1303 candidates who underwent on-site evaluation, 47% passed the screening process, 23% were lost to follow-up and 31% were excluded. Genetic and psychological factors accounted for the most common reasons for candidate exclusion. Enhanced genetic screening excluded an additional 25 candidates (19% of all genetic exclusions) and enhanced psychological screening excluded an additional 15 candidates (12% of all psychological exclusions). Altogether enhanced screening accounted for 40 candidates (10%) of the total pool of excluded candidates. CONCLUSIONS: Although our study is limited by its retrospective nature and center-specific conclusions, we show that enhanced comprehensive screening can exclude a significant number of candidates from an oocyte donor program and should be encouraged to assure optimal short-term and long-term outcomes for pregnancies achieved through oocyte donation.

In an 8-year review of ectopic pregnancy (EP) rates in donor egg recipients and standard patients undergoing in vitro fertilization–embryo transfer (IVF-ET) at a large university-based program, we report an EP rate of 0.6% in donor egg recipients and 0.9% in standard IVF patients, a difference that is not statistically significant. Donor egg recipients were found to have a significantly lower incidence of tubal disease compared with standard IVF patients; however, tubal disease was not found to be an independent risk factor for EP in our practice, perhaps owing to aggressive management of tubal disease.

To characterize incidence, chorionicity, amnionicity, and pregnancy outcome for monozygotic twin pregnancy (MZT) after IVF.Retrospective review.University-based fertility center.Autologous and oocyte donation IVF cycles eventuating in 4,976 clinical gestations from 2000 to 2007.None.MZT incidence, chorionicity, zygosity, pregnancy outcome.Ninety-eight MZTs were diagnosed after first-trimester ultrasound evaluation (2% incidence). The incidence in cycles transfering autologous oocytes was 1.7% but was 3.3% with donor oocytes; however, women35 years old using their own oocytes displayed a similar rate (3.1%) to women using donor oocytes. Eighty MZTs occurred after fresh day-5 transfer; only 14 followed fresh day-3 transfer (2.6% vs. 1.2%). The MZT incidence in day-3 transfers without hatching was not different from those with hatching (1.3% vs. 1.1%). In addition, MZT incidence did not differ significantly whether or not ICSI was performed (2.4% vs. 2.0%). Four MZTs occurred after frozen-thawed embryo transfer (0.8% incidence). Ninety-five percent of all placental arrangements were confirmed as monochorionic-diamniotic on obstetric ultrasounds.These findings confirm a higher incidence of MZT after IVF. Monochorionic-diamniotic implantations were increased, whereas monochorionic-monoamniotic were not. The MZT risk factors included young age and extended culture, but not zona penetration or cryopreservation.

This study assessed 1908 embryos, including those with abnormal numbers of pronuclei, in IVF cycles from July 2001 to December 2006 in which preimplantation genetic screening (PGS) was performed on day 3 post-retrieval and ‘euploid’ embryos transferred the following day. PGS-intracytoplasmic sperm injection and PGS-translocation cycles were excluded. At 18 h post-insemination, the zygote distribution was 19% 0PN, 4% 1PN, 69% 2PN and 8% 3PN. No pregnancy occurred following 0PN or 1PN embryo transfers. A retrospective, blinded morphological ranking of all embryos on day 3 was performed and the results compared with PGS; no 0PN or 1PN embryo would have been chosen for transfer based on morphological superiority alone. Blastocyst formation occurred in 1PN embryos (29%) but not in 0PN embryos when evaluated on day 5. Euploid karyotypes were reported for biopsies of 0PN (3%), 1PN (5%) and 2PN (19%) embryos (P = 0.015, 1PN versus 2PN). A Y chromosome was observed in 0PN (17%) and 1PN (32%) embryos; surprisingly, 91% of these Y chromosome-bearing embryos were aneuploid. Many different meiotic and fertilization errors can result in 0PN or 1PN zygotes; these results indicate that the resultant embryos should not be transferred, especially when normally fertilized embryos are available.

Objective To compare rates of implantation, pregnancy, miscarriage, multiple gestation, and selective reduction between patients undergoing day 5 (d5) and day 3 (d3) ETs. Design Retrospective cohort study. Setting University-based IVF center. Patient(s) The first d5 ET cycle of patients 42 years of age from 2003 to 2006 was compared with a historical control of first cycle d3 ET patients 42 years of age from 1996 to 1999 who would have met current d5 ET criteria. Intervention(s) None. Main Outcome Measure(s) Rates of implantation, clinical pregnancy, miscarriage, live birth, high order multiple pregnancy (HOMP), and selective reduction. Result(s) D5 ET patients had higher implantation rates (39% vs. 30%), with no difference in the no-transfer rate. D5 ET patients had lower rates of HOMP (2.5% vs. 11%) and HOMP delivery (0.7% vs. 3.5%), multiple pregnancy (27% vs. 33%), multiple delivery (19% vs. 26%), and twin delivery (18% vs. 23%). There were fewer selective reductions of HOMP with d5 ET (1.7% vs. 3.8%). Conclusion(s) Extended culture improves embryo selection through increased implantation, facilitating fewer embryos per transfer, which lowers multiple gestation rates and the need for HOMP reduction.

Objective To determine whether early measurement of the serum cytokines interleukin-2 receptor (IL-2R), IL-6, and IL-8 along with human chorionic gonadotropin (hCG) and progesterone (P 4 ) can differentiate an ectopic from an intrauterine gestation. Design Retrospective analysis. Setting University-based fertility center. Patient(s) 75 women who underwent treatment with in vitro fertilization (IVF) and subsequently had an ectopic gestation (n = 15), spontaneous abortion (SAB) (n = 30), or term delivery (TD) (n = 30). Intervention(s) Serum samples were obtained 14 (day 28) and 21 (day 35) days after oocyte retrieval. Main Outcome Measure(s) Serum concentrations of IL-2R, IL-6, IL-8, P 4 , and hCG. Result(s) Median hCG readings on day 28 and day 35 were statistically significantly lower in the ectopic gestation group than in those with spontaneous abortion or term delivery. On day 28, median IL-8 levels were lower in the ectopic gestation group when compared with all intrauterine gestations combined. No statistically significant differences in IL-2R or IL-6 levels were noted between groups. Despite P 4 supplementation, median day-35 P 4 levels were lower in ectopic gestation than in the spontaneous abortion and term delivery cycles. Conclusion(s) In the setting of a rise or plateau in hCG levels, low day-28 IL-8 and day-35 P 4 levels suggested an extrauterine implantation. This assay combination may facilitate earlier diagnosis of an ectopic gestation when pregnancy location is unclear.

Objective To assess the clinical value of transabdominal ultrasound (TAS)–assisted embryo transfer on outcomes of in vitro fertilization–embryo transfer (IVF-ET) in comparison to the "clinical touch" method of transcervical embryo transfer by one physician and to determine if transabdominal ultrasound should be applied to all cases of embryo transfer in this practice. Design A retrospective comparison study. Setting A university-based IVF practice. Patient(s) Two hundred forty-nine patients who underwent transcervical transfer of fresh embryos created using autologous oocytes performed by the same physician from July 1, 2003, to June 30, 2004. Intervention(s) On selected days, at time of embryo transfer, transabdominal ultrasound was performed to guide catheter placement depth approximately 1 cm from the uterine fundus. Main Outcome Measure(s) The presence of at least one gestational sac on ultrasound was compared in the two study groups; additionally, the clinical pregnancy rate (presence of fetal cardiac activity observed on ultrasound), the ectopic pregnancy rate, the biochemical pregnancy rate, and the implantation rate (number of gestational sacs identified on ultrasound per number of embryos transfered) between groups was evaluated. Characteristics of the two cohorts were analyzed to verify similarities between the treatment and control groups, including age of recipient, prior IVF history, day of transfer (day 3 or day 5 after retrieval), difficulty of transfer, the use of a tenaculum, and the quality and number of embryos transfered. Result(s) No statistical difference was seen in the presence or number of gestational sacs following embryo transfer with or without transabdominal ultrasound guidance. Conclusion(s) No additional advantage is conferred when using transabdominal ultrasound to perform embryo transfer. In experienced hands, the "clinical touch" method of embryo transfer yields equivalent results to transabdominal ultrasound–guided embryo placement. However, in patients with a prior history of difficult uterine sounding or embryo transfer, transabdominal ultrasound guidance may still play a role.

In humans, the live birth rate drops precipitously with increasing maternal age, and this decline is associated with increases in the incidence of oocyte and embryo aneuploidy. Preimplantation aneuploidy screening has improved pregnancy outcome by significantly lowering the miscarriage rate. Nevertheless, aneuploidy screening only identifies the affected embryos; it does not attempt to correct the underlying biologic problem. Anomalies in chromosome segregation can result from a dysfunctional first or second meiotic division in the egg or develop after fertilization during the first few mitoses of early embryonic development. In both instances, ooplasmic anomalies may account for the nuclear problem. Low cell levels of cytoplasmic proteins (e.g., cytoskeletal elements, enzymes, energy stores, cell cycle regulatory proteins) may lead to a dysfunctional division of chromosomes during egg maturation or following fertilization. Ooplasmic injection is a micromanipulation technique that has produced pregnancies in patients with a history of poor-quality, fragmented embryos. Germinal vesicle transfer is a research procedure used to investigate the ooplasmic-nuclear interplay regulating cell cycle, maturation, and fertilization. Both these techniques may prove to be effective in improving the quality of eggs from patients of advanced maternal age.

Objective: To establish prognostic relevance of parameters assessed in oocyte donation cycles. Design: Retrospective analysis. Setting: Large university-based donor oocyte program. Patient(s): All oocyte recipient cycles achieving embryo transfer from September 1995 to October 1998. Intervention(s): None. Main Outcome Measure(s): Pregnancy. Result(s): Recipient age and reproductive status, day 9 and 12 serum estradiol (E2) levels and a progesterone (P) level obtained 2 days after initiation of hormonal therapy did not correlate with pregnancy. Endometrial thickness, but not endometrial pattern, was useful in predicting pregnancy outcome. The clinical pregnancy and live-birth rate in cycles where the endometrial thickness was less than 8 mm was significantly lower when compared to cycles with an endometrial thickness ≥9 mm. Cycles where optimal quality embryos were transferred had the highest implantation (36%), clinical pregnancy (63%) and live birth (54%) rates and these rates were significantly higher than those of cycles where only poor quality embryos were available for transfer (10% implantation, 17% clinical pregnancy, and 8% live birth rates, respectively; P

Objective: To compare implantation and pregnancy rates in oocyte recipients undergoing a two-embryo versus three-embryo transfer, 3 days after retrieval. Design: Retrospective comparative analysis. Setting: University-based in vitro fertilization center. Patient(s): All oocyte recipients undergoing embryo transfer from January 1, 1997 through August 31, 1999. Intervention(s): Recipients received two or three embryos. Main Outcome Measure(s): Implantation, and clinical and multiple pregnancy rates. Result(s): Seventy-three recipients underwent a two-embryo transfer, and 376 had three embryos replaced. The numbers of oocytes retrieved (12.7 ± 0.89 vs. 13.1 ± 0.36) and embryos obtained (8.05 ± 0.65 vs. 8.77 ± 0.27) did not differ between the two-embryo and three-embryo transfer groups, nor did the proportion of patients with embryo cryopreservation (54.3% vs. 42.6%, respectively). There was no significant difference in pregnancy or implantation rates when comparing those patients with a two-embryo transfer to those with a three-embryo transfer. Significantly, 13.8% of the pregnancies in the three-embryo transfer group were triplet. Conclusion(s): Reducing the number of embryos transferred in an oocyte donation cycle can lower the incidence of triplet pregnancies without significantly lowering the overall pregnancy rate.

We evaluated whether mouse oocytes reconstructed by germinal vesicle (GV) transfer can develop to blastocyst stage. The oocytes were artificially activated with sequential treatment of A23187 and anisomycin; fertilization was then established by transfer or exchange of pronuclei with those of zygotes fertilized in vivo. Type 1 zygotes were constructed by placing the male haploid pronucleus from a zygote into the cytoplasm of an oocyte that underwent GV transfer, in-vitro maturation and activation; for type 2 zygotes, the female pronucleus was removed from a zygote and replaced with the female pronucleus of an oocyte subjected to GV transfer, in-vitro maturation and activation. Karyotypes of activated oocytes and type 2 zygotes were also subjected to analysis. When cultured in human tubal fluid (HTF) medium, reconstructed oocytes matured and, following artificial activation, consistently developed a pronucleus with a haploid karyotype; the activation rate for this medium was two- to three-fold higher than that of oocytes cultured in M199 (87% versus 30% respectively). Following transfer of a male pronucleus, only 47% of the type 1 zygotes developed to morula or blastocyst stage and embryo morphology was poor. In contrast, 73% of the type 2 zygotes developed to morula or blastocyst stage, many even hatching, with few morphological anomalies. Normal karyotypes were observed in 88% of the type 2 zygotes analysed. These observations demonstrate that the nucleus of a mouse oocyte subjected to sequential nuclear transfer at GV and pronucleus stages is, nonetheless, capable of maturing meiotically, activating normally and supporting embryonic development to hatching blastocyst stage. In contrast, the developmental potential of the cytoplasm of such oocytes appears to be compromised by these procedures.

Objective: To determine whether electrically stimulated Ca 2+ influx can "rescue" fertilization and early embryogenesis in human oocytes that fail to fertilize after intracytoplasmic sperm injection (ICSI). Design: Prospective, randomized trial of a laboratory procedure. Setting: A research laboratory at a university medical center. Patient(s): Discarded oocytes from ICSI-IVF cycles. Intervention(s): Oocytes (n = 104) that showed no evidence of fertilization 16–24 hours after ICSI were assigned to three treatment groups: group 1 (one direct current electrical pulse at 1.36–1.50 kV/cm for 40–60 μs), group 2 (three pulses every 15–20 minutes), or group 3 (treated the same as group 2 but with no electrical stimulation). Main Outcome Measure(s): After stimulation, the oocytes were cultured in vitro for 3–5 days. Oocytes that displayed two pronuclei and a second polar body within 16 hours were considered to have fertilized normally. Fertilization and embryo cleavage rates were compared between groups. Result(s): Fertilization occurred in 26 (70%) of 37 and 38 (78%) of 49 group 1 and 2 oocytes, respectively, but in only 5 (27%) of 18 group 3 oocytes. Within 3 days, group 2 embryos routinely developed beyond the two-cell to four-cell stage (61% versus 13% in group 1); 11% of these oocytes developed to the morula or early blastocyst stage. Sex chromosome analyses indicated 10 male and 8 female embryos. Conclusion(s): Oocytes that fail to fertilize by 24 hours after ICSI can resume apparently normal fertilization and early embryonic development in response to electrical stimulation. Moreover, the degree of cytoplasmic activation as determined by the number of pulses applied affects fertilization efficiency and early embryonic development.

Preimplantation genetic diagnosis was performed in 61 day 3 embryos obtained by in-vitro fertilization from seven patient carriers of haemophilia, Marfan's syndrome, Bloch-Sulzemberg syndrome (incontinentia pigmentosa) or X chromosome-linked immune deficiency, retinitis pigmentosa, and FG syndrome, which is characterized by mental retardation and hypotonia. After multiplex polymerase chain reaction, 16 embryos were diagnosed as being unaffected, and these were transferred to the uterus on the following day (day 4). Of these embryos, six (37.5%) implanted, resulting in the delivery of a singleton and a twin pregnancy, a late second trimester miscarriage (twins at week 20) and a first trimester miscarriage at week 8. All the diagnoses were confirmed by amniocentesis. We report for the first time a late day 4 transfer of biopsied human embryos undergoing preimplantation genetic diagnosis. This transfer schedule allows an extra day to perform genetic analyses on single blastomeres and to monitor any adverse effect of the biopsy procedure.