Sexual determination and differentiation during embryonic and fetal development of New Zealand rabbit females / Determinación y diferenciación sexual durante el desarrollo embrionario y fetal de conejos hembras de Nueva Zelanda

The aim of this study was to know the embryonic and fetal development of the female rabbit genital system (Oryctolagus cuniculus), describing its main phases and the moment of sexual differentiation. Eleven pregnant New Zealand female rabbits were used in different gestational phases. The day of coitus was determined as day 0. For each stage a minimum of two animals was considered. The samples were obtained every two days from the ninth day post-coitus (dpc) until the 28th dpc. The gestational period was divided in two: animals with undifferentiated sex (group 1) and animals with differentiated sex (group 2). The ages of embryos and fetuses were estimated through the crown-rump method. Subsequently, embryos and fetuses were dissected, fixed and processed to be embedded in paraffin (Histosec). The histological analysis was performed on sections stained with hematoxylin and eosin. Immunohistochemical analysis to determine sexual differentiation was performed on samples from the 16th, 18th and 28th dpc. Desert Hedgehog (Dhh) and Indian Hedgehog (Ihh) primary antibodies, respectively, were used to identify cells of the male and female germinal epithelium. The immunohistochemical results showed that at the 16th dpc, female sexual differentiation was evident, since positive expression of the Ihh protein was observed. Sexual differentiation was obtained through histological analysis on the 18th dpc and through anatomical observation of the external genitalia on the 24th dpc. Knowing the characteristics of the embryonic and fetal development of the female rabbit genital system as well as the moment of sexual differentiation make it possible to establish bases for future research that address the physiology and pathology of these organs. Thus, any alteration in the chain of events of sexual determination and differentiation must search for an explanation from the knowledge of the possible normal mechanisms affected.

El objetivo de esta investigación fue conocer el desarrollo embrionario y fetal del sistema genital femenino de conejo (Oryctolagus cuniculus), describiendo sus principales fases y el momento de la diferenciación sexual. Se utilizaron 11 conejos hembras gestantes neozelandesas, en diferentes fases gestacionales. El día del coito se determinó como día 0. Para cada etapa fue considerado un mínimos de dos animales. Las muestras fueron obtenidas cada dos días, a partir del noveno día post-coito (dpc) hasta el 28 dpc. El periodo gestacional fue dividido en dos: animales con sexo indiferenciado (grupo 1) y, animales con sexo diferenciado (grupo 2). Las edades de los embriones y los fetos fueron estimadas a través del método de crown-rump. Posteriormente, embriones y fetos fueron disecados, fijados y procesados para su inclusión en parafina (Histosec). El análisis histológico se realizó en secciones teñidas con Hematoxilina y Eosina. El análisis inmunohistoquímico para determinar la diferenciación sexual fue realizado en muestras de 16, 18 y 28 dpc. Para identificar células del epitelio germinativo masculino y feminino se utilizaron los anticuerpos primarios Desert Hedgehog (Dhh) e Indian Hedgehog (Ihh), respectivamente. Los resultados inmunohistoquímicos mostraron que a los 16 dpc se evidenció diferenciación sexual femenina, ya que se observó expresión positiva de la proteína Ihh. La diferenciación sexual, a través del análisis histológico fue obtenida a los 18 dpc y a través de la observación anatómica de los genitales externos a los 24 dpc. Conocer las características del desarrollo embrionario y fetal del sistema genital femenino de conejo, así como, el momento de la diferenciación sexual, permiten sentar bases para futuras investigaciones que aborden la fisiología y patología de estos órganos. Así, cualquier alteración en la cadena de eventos de la determinación y diferenciación sexual deberá buscar una explicación a partir del conocimiento de los posibles mecanismos normales afectados.

Transitando de embrión a feto: la metamorfosis de los cordados / Transiting from embryo to fetus: the metamorphosis of the chordata

Durante el período del desarrollo conocido como prefetal, el embrión cambia sus características ictiomórficas comunes a todos los vertebrados y adquiere gradualmente las formas propias de la especie que representa. Durante este período se forma la cara, involucionan los arcos faríngeos (branquiales) formándose el cuello, y aparecen los miembros. Se constituye, además, la hernia umbilical fisiológica, que consiste en la presencia de asas intestinales dentro del cordón umbilical. El sistema nervioso origina las vesículas telencefálicas, el diencéfalo, mesencéfalo, metencéfalo, y mielencéfalo. Este periodo corresponde a una etapa de máxima susceptibilidad ante los teratógenos que pueden generar malformaciones en todas las especies animales. El objetivo del presente trabajo es presentar los principales eventos acontecidos durante el periodo prefetal, además de una visión y opinión de los autores, proponiendo una nueva denominación a la etapa: periodo metamórfico.

During the period of development known as prefetal, the embryo changes its ictiomorphic characteristics common to all vertebrates and gradually acquires the proper forms of the species it represents. During this period the face is formed, the pharyngeal arches (branchial) involute forming the neck, and the limbs appear. In addition, the physiological umbilical hernia is constituted, which consists of the presence of intestinal loops inside the umbilical cord. The nervous system originates the telencephalic vesicles, the diencephalon, mesencephalon, metencephalon, and myelencephalon. This period corresponds to a stage of maximum susceptibility to teratogens that can generate malformations in all animal species. The objective of this paper is to present the main events that took place during the preferential period, as well as a vision and opinion of the authors, proposing a new name for the stage: metamorphic period.

Complicaciones obstétricas y neonatales del hipotiroidismo / Obstetrics and neonatal complications of hypothyroidism

Las patologías tiroideas son frecuentes y afectan alrededor del 10% de la población fértil femenina. El hipotiroidismo y la enfermedad tiroidea autoinmune traen aparejados trastornos de la fertilidad e implantación y desarrollo del embarazo. Durante el embarazo la glándula tiroides es puesta a prueba, poniéndose en marcha distintos mecanismos adaptativos. Se debe resaltar la importancia de un aporte suficiente de yodo antes y durante el embarazo. El adecuado funcionamiento tiroideo, tanto materno como fetal, juegan un rol fundamental para la normal evolución del embarazo y del desarrollo neuropsicointelectual del feto, ya que una disminución aún leve de la función tiroidea en la primera mitad del embarazo puede tener secuelas a largo plazo. Las hormonas tiroideas son fundamentales para el normal desarrollo del SNC y del cerebro fetal. Además de las complicaciones fetales, las pacientes hipotiroideas tienen mayores complicaciones en el embarazo tales como aborto, parto prematuro, retardo del crecimiento intrauterino y bajo peso al nacer. Sería ideal que la paciente hipotiroidea programe su embarazo, se halle adecuadamente tratada y con un control estricto durante el embarazo. Si bien no hay consenso para el screnning de rutina, hay grupos que postulan la utilidad de incluir un dosaje de TSH y ATPO en el primer trimestre del embarazo. El diagnóstico precoz y su adecuado tratamiento evitarán complicaciones muchas veces irreversibles en el embarazo y en el desarrollo neurológico fetal.

Study about the development of the temporomandibular joint in the human fetuses / Estudio del desarrollo de la articulacion temporomandibular en fetos humanos

The temporomandibular joint (TMJ) is a highly specialized articulation that differs from all the other synovial articulations for many reasons. In children, different from what we observe in adults, these articulations have rarely been studied under the morphofunctional aspect, mainly in the embryonary and fetal stages. In this study 10 fetuses with ages varying from 16 to 39 weeks of intrauterine life were used, and it could be observed that the fibers and thickness of the articular disc, as well as the articular capsule and the condylar process, suffer modifications according to age. It was also observed that the superior head of the lateral pterygoid muscle inserts itself in the articular disc and capsule in all the ages studied. Also, the maturation of the articular tissues, especially of the articular disc, as well as, the associated muscles, suggests that the TMJ was able to carry out mandibular movements since the 24th week of intrauterine life.

La articulación temporomandibular (ATM) es una articulación altamente especializada que se distingue de todas las otras articulaciones sinoviales por muchas razones. En los niños, a diferencia de lo que se observa en los adultos, estas articulaciones muy pocas veces han sido estudiadas en el aspecto morfofuncional, principalmente, en las etapas embrionaria y fetal. Se utilizaron 10 fetos con edades que variaban entre 16 a 39 semanas de vida intrauterina. Se pudo observar que las fibras y espesor del disco articular, así como la cápsula articular y el proceso condilar. Sufren modificaciones en función de la edad. También se observó que la parte superior del músculo pterigóideo lateral se inserta en el disco y en la cápsula articular en todas las edades estudiadas. Por otra parte, la maduración de los tejidos articulares, especialmente del disco articular, así como, de los músculos asociados, sugiere que la ATM es capaz de llevar a cabo los movimientos mandibulares, desde la 24 ª semana de vida intrauterina.

Curva de crecimiento intrauterino de recién nacidos peruanos / Intrauterine growth curves to live peruvian newborns

Objetivos: Obtener curvas de crecimiento intrauterino peruanas y analizar la influencia del sexo fetal, paridad, talla materna y región natural, en el peso fetal. Método: Enrolamiento prospectivo de recién nacidos vivos sucesivos en 29 Hospitales peruanos, nacidos en 2005 y registrados en el Sistema Informático Peri-natal (SIP2000). Se utilizó técnicas antropométricas recomendadas por el CLAP-OPS/OMS. Diagnóstico de edad gestacional por fecha de última menstruación. Se seleccionaron recién nacidos (RN) sin factores de riesgo para retardo de crecimiento intrauterino. Distribución de los pesos de los recién nacidos en percentiles. Resultados: 50.568 RN vivos fueron seleccionados de 99.439 nacimientos. Se consideró los percentiles 10, 50 y 90 y se calculó los percentiles 2,5 y 5 para clasificar al RN pequeño para la edad gestacional en leve, moderado o severo. El 55,7 por ciento de los recién nacidos presentaron 39 ó 40 semanas de gestación, con peso promedio de nacimiento de 3.295 y 3.400 gramos, con el percentil 10 de peso de 2750 y 2875 gramos, respectivamente. La multiparidad, talla materna alta, sexo fetal masculino y nacer en la costa produjeron peso de nacimiento significativamente mayores, entre las semanas 36 a 42 semanas. Conclusiones: Se recomienda usar estas curvas de crecimiento intrauterino para una mejor clasificación de los recién nacidos peruanos.

Objectives: To obtain peruvian intrauterine growth curves and analyze the influence of fetal sex, parity, maternal height and natural región in the fetal weight. Method: Prospective enrolment of successive live newborns of 29 Hospitals of Peru, born in 2005, and registered in the Perinatal Information System (SIP2000). Anthropometrics techniques recommended by the CLAP-OPS/OMS were used. Diagnosis of gestational age was made by date of the last menstrual period. Were selected newborns without risk factors for intrauterine growth retardation. Newborns weights distribution was made in percentile curves. Results: 50,568 newborns were selected from 99,439 births. Percentile 10, 50 and 90 and was calculated, and also percentile 2.5 and 5 to classify the newborn small for gestational age in mild, moderate or severe. The 55.7 percent of newborns had 39 or 40 weeks of gestation, with an average birth weight of 3,295 and 3,400 g, with a 10 percentile of 2,750 and 2,875 grams, respectively. The multiparity, high maternal height, male fetal sex and birth on the coast produced significantly higher birth weight, between weeks 36 to 42 weeks. Conclusions: It is recommended to use these intrauterine growth curves for better classification of the peruvians newborns.

Regulation of mouse embryo development by autocrine throphic factors

Embryo development depends on maternal and embryonic factors. When occurs in vitro, embryos secrete factors that stimulate their development. The purpose of this study was to investigate the possible effects of embryos at morula stage on mouse embryo development in vitro. To obtain conditioned media (CM), morulas were cultured in groups of 5 (CM5) or 10 (CM10) in microdrops of Ham-Fl0 culture medium during 24h and later they were removed. Subsequently, 365 morulas were cultured in CM5 and CM10 or in Ham-F10 media (as control group). No differences in blastocyst formation could be found between embryos cultured for 24h in Ham-F1O, CM5 or CM10 (49.66, 53.04, 60.00% respectively). However, CM5 significantly increased differentiation in embryos cultured for 48h as compared to Ham-FlO medium (80.00% and 64.14 respectively). The CM5 caused a significant increase in the hatching rate compared to Ham-F10 evaluated at 78 and 96h of culture (66.96 vs. 52.41% and 70.43 vs. 55.17%, respectively). After 72, 78 and 96h of culture the hatching rate for embryos cultured in CM10 was significantly higher than that in Ham-F10 (64.76 vs. 47.59%, 67.62 vs. 52.41% and 73.33 vs. 55.17%, respectively). At 48h of culture, differences between CM5, CMl0 and Ham-F10 were not observed. These results suggest that preimplantational mouse embryos produce trophic factor/factors that enhance the differentiation and hatching process

Embryonic development time of Penilia avirostris Dana, 1852 in a tropical bay in Brazil

O tempo de desenvolvimento na câmara incubadora dos embriões do cladócero Penilia avirostris Dana, 1852, foi estimado a partir do zooplâncton coletado na água superficial da Baía de Guanabara, Brasil, diariamente, durante 15 dias. A cada dia, foi anotado o estágio de maturidade dos embriões de 90 fêmeas partenogenéticas. O tempo de desenvolvimento total (do ovo até o nascimento) variou de 2 a 3 dias, sendo a fase imatura (estágios I e IV) geralmente mais longa (2 dias) que as fases intermediárias e maturas (1 dia, estágios V a XII). Resultados similares foram obtidos com a equação de Bottrell, que leva em conta a temperatura da água.

Dynamic and reversible changes of interstitial cell phenotype during remodeling of cardiac valves.

BACKGROUND AND AIM OF THE STUDY: The roles of cardiac valvular interstitial cells (VIC) in extracellular matrix remodeling in fetal development, adaptation and response to injury are largely unknown. METHODS: The phenotype of VIC was studied in health (normal adult human and sheep), development (fetal human and sheep), disease (human mitral valves with myxomatous degeneration), adaptation (clinical pulmonary to aortic valve autografts) and tissue-engineered heart valves matured in vitro and remodeled in vivo. Cell phenotype was assessed using expression of vimentin (V), alpha-smooth muscle actin (SMA, A), matrix metalloproteinase (MMP)-13/collagenase-3 (M), and SMemb (S). RESULTS: VIC in normal adult valves were predominantly quiescent fibroblasts immunoreactive to vimentin (89.7 +/- 2.5%), but not MMP-13 or SMemb, with only 2.5 +/- 0.4% of alpha-SMA-positive cells ('normal/quiescent' phenotype: V+/A-/M-/S-). In contrast, fetal VIC were mostly activated myofibroblasts ('developing/activated' phenotype: V+/A+/M+/S+), with 62.1 +/- 5.0% of cells staining positive for alpha-SMA. VIC in myxomatous valves, short-term autografts and engineered valves in vitro were also activated myofibroblasts with coexpression of vimentin, alpha-SMA (36.2 +/- 3.7%, 19.3 +/- 2.4%, and 60.3 +/- 9% positive cells, respectively), strong MMP-13 activity indicative of collagen remodeling, and SMemb ('remodeling/activated' phenotype: V+/A+/M+/S+). In contrast, VIC in long-term pulmonary autografts and engineered valve explants had a mostly fibroblast-like phenotype, with sparse alpha-SMA expression (6.0 +/- 1% and 5.4 +/- 1.0% positive cells) (V+/A-/M-/S-). CONCLUSION: Most VIC in normal valves were quiescent with a fibroblast-like phenotype. VIC in developing, diseased, adapting and engineered valves adjust to a dynamic environment through VIC activation and secretion of proteolytic enzymes mediating extracellular matrix remodeling ('developing/ remodeling/activated' phenotype), followed by a normalization of phenotype.

Effects of oxygen tension in the gas atmosphere during in vitro maturation, in vitro fertilization and in vitro culture on the efficiency of in vitro production of mouse embryos.

Effects of oxygen (O2) tension in the gas atmosphere during in vitro maturation (IVM), in vitro fertilization (IVF) and in vitro culture (IVC) on the efficiency of in vitro production of mouse embryos were examined. Mouse oocytes recovered from large antral follicles were subjected to IVM in Waymouth medium for 15, 16 and 17 hr under 5 or 20% O2 and then subjected to IVF and IVC under 5 or 20% O2 tension. Lowering the O2 tension in the gas atmosphere for IVM from 20 to 5% improved the cleavage rate after IVF when the oocytes were subjected to IVM for 15 hr; however, no improvement in the cleavage rate was observed when the culture period for IVM was extended to 16 and 17 hr. Lowering the O2 tension to 5% for IVM and IVC improved the development of the cleaved oocytes to the blastocyst stage, regardless of the culture period for IVM. However, the O2 tension for IVF had no remarkable effect on the subsequent embryonic development. These results demonstrate that 5% O2 is superior to 20% O2 for IVM and IVC, and suggest that 20% O2 for IVM may delay oocyte maturation and/or the acquisition of fertilizability and impair the developmental competence of oocytes.

Prenatal programming of postnatal obesity: fetal nutrition and the regulation of leptin synthesis and secretion before birth.

Exposure to either an increased or decreased level of intrauterine nutrition can result in an increase in adiposity and in circulating leptin concentrations in later life. In animals such as the sheep and pig in which fat is deposited before birth, leptin is synthesised in fetal adipose tissue and is present in the fetal circulation throughout late gestation. In the sheep a moderate increase or decrease in the level of maternal nutrition does not alter fetal plasma leptin concentrations, but there is evidence that chronic fetal hyperglycaemia and hyperinsulinaemia increase fetal fat mass and leptin synthesis within fetal fat depots. Importantly, there is a positive relationship between the relative mass of the 'unilocular' component of fetal perirenal and interscapular adipose tissue and circulating fetal leptin concentrations in the sheep. Thus, as in the neonate and adult, circulating leptin concentrations may be a signal of fat mass in fetal life. There is also evidence that leptin can act to regulate the lipid storage, leptin synthetic capacity and potential thermogenic functions of fat before birth. Thus, leptin may act as a signal of energy supply and have a 'lipostatic' role before birth. Future studies are clearly required to determine whether the intrauterine and early postnatal nutrient environment programme the endocrine feedback loop between adipose tissue and the central and peripheral neuroendocrine systems that regulate energy balance, resulting in an enhanced risk of obesity in adult life.

Differential growth of human embryos in vitro: role of reactive oxygen species.

OBJECTIVE: To examine the relationship of early human embryonic development with the level of reactive oxygen species (ROS) in the culture media on the first day (day 1 ROS) after insemination. DESIGN: A prospective study. SETTING: Patients undergoing assisted reproduction in a teaching hospital. PATIENT(S): Patients undergoing conventional IVF (n = 104; 115 cycles) and intracytoplasmic sperm injection (ICSI) (n = 91; 96 cycles) were included. Both fertilization and early cultures were performed in human tubal fluid with 5% serum substitute supplement. INTERVENTION(S): Day 1 ROS levels in the central well (sample) and the outer well (control) of each embryo culture dish were measured after overnight incubation by chemiluminescence assay using luminol as the probe. MAIN OUTCOME MEASURE(S): Fertilization rate and embryo quality at day 3 and 5 were recorded for each cycle. Age, parity, and demographic features were also compared. RESULT(S): High day 1 ROS levels in culture media were associated with low blastocyst rate, low fertilization rate, low cleavage rate, and high embryonic fragmentation with ICSI but not with conventional IVF. High day 1 ROS levels in culture media were associated with lower pregnancy rates in both IVF and ICSI cycles. CONCLUSION(S): Reactive oxygen species generated in culture media by day 1 may be an important biochemical marker for early embryonic growth. Increased embryonic fragmentation and slow cleavage rate may be partially attributed to early exposure of embryos to high ROS levels in ICSI cycles. Differential growth of ICSI embryos incubated under identical conditions may be in part due to differences in ROS levels of the culture medium surrounding these embryos.

Fgf18 is required for embryonic lung alveolar development.

Fgf18 is abundantly expressed in mouse embryonic lungs. To elucidate the roles of Fgf18 in mouse embryonic lung development, we examined the Fgf18-/- embryonic lungs. Although the sizes of the Fgf18-/- lungs were a little smaller in appearance than those of wild-type lungs, neither proximal nor distal airway branching in the Fgf18-/- lungs was impaired. However, the Fgf18-/- lungs at E18.5 had reduced alveolar space, thicker interstitial mesenchymal compartments, and many embedded capillaries. Cell proliferation in the Fgf18-/- lungs was also transiently reduced around E17.5, although the expression of marker genes for lung epithelial cells in the Fgf18-/- lungs was not impaired. The present findings indicate that the Fgf18 plays roles in lung alveolar development during late embryonic lung development stages. The cell proliferation during the terminal saccular stage stimulated by Fgf18 might play roles in the remodeling of the distal lung.

The developmental origins of well-being.

Low birthweight is now known to be associated with increased rates of coronary heart disease and the related disorders, stroke, hypertension and adult-onset diabetes. These associations have been extensively replicated in studies in different countries and are not the result of confounding variables. They extend across the normal range of birthweight and depend on lower birthweights in relation to the duration of gestation rather than the effects of premature birth. The associations are thought to be consequences of developmental plasticity, the phenomenon by which one genotype can give rise to a range of different physiological or morphological states in response to different environmental conditions during development. Recent observations have shown that impaired growth in infancy and rapid childhood weight gain exacerbate the effects of impaired prenatal growth. A new vision of optimal early human development is emerging, which takes account of health and well-being throughout life.

Regenerative medicine: stem cells and the science of monstrosity.

The nineteenth century science of teratology concerned itself with the study of malformations or "monstrosities", as they were then called. The first major contribution to the field was the work of Isidore Geoffrey Saint-Hilaire, Historie Generale et Particuliere des Anomalies de l'Organisation chez l'Homme et les Animaux, published in 1832, whose classifications formed the basis for the later experimental science of teratogeny, the art of reproducing monstrosities in animal embryos. In this article, I will argue that recent developments in the field of regenerative medicine can be situated in the tradition of teratological and teratogenic studies dating back to the nineteenth century. In particular, I will be interested in the historical link between studies in teratogenesis (the artificial production of teratomas) and stem cell research. Recent advances in stem cell research, I will suggest, return us to the questions that animated nineteenth century investigations into the nature of the monstrous or the anomalous. In the process, our most intuitive conceptions of "life itself" are undergoing a profound transformation.

PTENless means more.

Recent studies indicate that certain key molecules that are vital for various developmental processes, such as Wnt, Shh, and Notch, cause cancer when dysregulated. PTEN, a tumor suppressor that antagonizes the PI3 kinase pathway, is the newest one on the list. The biological function of PTEN is evolutionarily conserved from C. elegans to humans, and the PTEN-controlled signaling pathway regulates cellular processes crucial for normal development, including cell proliferation, soma growth, cell death, and cell migration. In this review, we will focus on the function of PTEN in murine development and its role in regulating stem cell self-renewal and proliferation. We will summarize the organomegaly phenotypes associated with Pten tissue-specific deletion and discuss how PTEN controls organ size, a fundamental aspect of development. Last, we will review the role of PTEN in hormone-dependent, adult-onset mammary and prostate gland development.

Developmental stage dependent regulation of DNA methylation and chromatin modification in a immature astrocyte specific gene promoter.

Astrocytes are generated from neuroepithelial cells after neurons during brain development. However, the mechanism of this sequential generation is not fully understood. Here, we show that a particular cytosine residue in the promoter of the gene encoding the immature astrocyte marker, S100beta, becomes demethylated, correlating with the time when the S100beta expression commences at embryonic day (E) 14. In addition, astrocyte-inducing cytokine, BMP2, increased histone acetylation around the CpG site in neuroepithelial cells at E14 but not E11 when S100beta expressing astrocytes are absent. Furthermore, binding of a methyl DNA binding protein, MeCP2, to the S100beta gene promoter in neuroepithelial cells was reduced at E14 compared to E11. Thus, demethylation of specific CpG site is suggested to be a critical determinant in regulating astrocyte differentiation in the developing brain.

Development of interlimb movement synchrony in the rat fetus.

In the fetal rat, interlimb synchrony is a prominent form of temporally organized spontaneous motor activity in which movement of different limbs occurs at nearly the same instant. In the present study, synchrony profiles were created for different pairwise combinations of limbs over the last 5 days of gestation. Observed rates of synchrony differentiated from randomized time series from Gestational Day 19 to Day 21 (E19-E21), with forelimb synchrony emerging earlier than that of other limb pairs. Synchrony profiles were elevated at the shortest intervals between successive limb movements, indicating that movements became more tightly coupled toward the end of gestation. Interlimb synchrony appears to be a robust method of quantifying fetal movement and may prove useful as a tool for assessing prenatal nervous system functioning.

Transfer of blastocysts and morulae on day 5.

OBJECTIVE: To limit the number of embryos transferred and reduce high-order multiple pregnancies without compromising a patient's opportunity to become pregnant. DESIGN: Retrospective, nonrandomized analysis of embryo development and patient outcome when embryos were transferred on day 5. SETTING: Private practice. PATIENT(S): Women undergoing in vitro fertilization (IVF) treatment. INTERVENTION(S): Extend embryo culture to day 5 before embryo transfer (ET) to reduce the number of embryos transferred and minimize high-order multiple pregnancies. MAIN OUTCOME MEASURE(S): Clinical pregnancy rate (CPR), implantation rate (IR), and live born rate (LBR) from expanding (expanded blastocysts and blastocysts) and nonexpanding (early blastocysts and morulae) embryos transferred on day 5. RESULT(S): Approximately 60% of patients had expanding embryos (EXE) on day 5. Forty percent of patients having an ET had transferable non-expanding embryos (NEE). Patients with EXE had higher CPR and LBR compared to patients with NEE. Implantation rate and multiple pregnancy rate (MPR) were also higher for patients with EXE. The miscarriage rate (MCR) for patients with EXE and NEE was not different. Approximately 5.5% of patients did not have an ET, with most (>98%) of the ET failures from patients with <==3 two pronuclei (2PN) embryos. The number of 2PN embryos had an effect on CPR, LBR, MPR, and the number of patients having cryopreservation. CONCLUSION(S): Day 5 ET allows for the selection of embryos with the highest implantation potential as evidenced by acceptable pregnancy rates for patients with either EXE or NEE, without the need to transfer more than two embryos.

Impact of retained embryos on the outcome of assisted reproductive technologies.

OBJECTIVE: To assess the impact of embryo retention in the embryo transfer catheter followed by "immediate" retransfer on pregnancy outcome in women undergoing assisted reproduction. DESIGN: Retrospective analysis of embryo transfer following in vitro fertilization. SETTING: Assisted reproductive technology practice in a university in vitro fertilization program. PATIENT(S): In vitro fertilization charts for 1,812 embryo transfer cycles representing 1,139 patients between January 1997 and March 2002 were reviewed. INTERVENTION(S): None. MAIN OUTCOME MEASURE(S): Pregnancy rate, implantation rate, delivery rate. RESULT(S): Three embryo transfer cycles were excluded from analysis because of missing data, leaving 1,364 embryo transfers during oocyte recovery cycles and 445 embryo transfer cycles using thawed embryos. Seventy-one embryo transfers (3.9% of all transfers) were complicated by finding retained embryos after the initial embryo transfer-all retained embryos were immediately retransferred. There was no difference in the frequency of retained embryos during oocyte retrieval versus thawed embryo cycles. The pregnancy, implantation, and delivery rates per embryo transfer were not negatively affected by embryo(s) retained in the transfer catheter. Age, fresh versus frozen embryo, use of ultrasound during the procedure, or transferring physician did not influence pregnancy outcome. CONCLUSION(S): Immediate retransfer of embryos retained in the catheter following the initial transfer attempt did not have an adverse effect on pregnancy outcome.

Extent of nuclear DNA damage in ejaculated spermatozoa impacts on blastocyst development after in vitro fertilization.

OBJECTIVE: To determine whether the extent of ongoing apoptotic cell death measured as the presence of DNA strand breaks in spermatozoa affects embryo development to the blastocyst stage in IVF. DESIGN: A prospective comparative study. SETTING: A university IVF clinic and a private IVF clinic. PATIENT(S): Men (n = 49) undergoing infertility treatment with IVF. INTERVENTION(S): After density gradient centrifugation preparation, part of the sperm sample was used for infertility treatment, and the rest was fixed in paraformaldehyde. Strand breaks in DNA that are indicative of apoptosis were detected by the in situ DNA nick end labeling (TUNEL) technique. A total of 15,000 spermatozoa from each sample were evaluated for TUNEL reactivity by flow cytometry. MAIN OUTCOME MEASURE(S): Percentage of ejaculated spermatozoa with DNA strand breaks indicative of apoptosis, blastocyst development rate, and pregnancy rate. RESULT(S): Blastocyst development showed a significant negative correlation with percentage TUNEL positivity in spermatozoa. When 20% was used as a cutoff for TUNEL positivity in sperm samples, the percentage of blastocyst development was 50% higher in the <20% TUNEL-positivity group (n = 27) compared with those with >/=20% TUNEL positivity (n = 22; 44.7% blastocyst development vs. 29.8%). Clinical pregnancy rates in these two groups were 52% vs. 44%, respectively. CONCLUSION(S): The extent of nuclear DNA fragmentation in prepared ejaculated spermatozoa used in IVF negatively correlates with blastocyst development. A larger series of patients needs to be assessed to determine whether this paternal effect on blastocyst development may also affect pregnancy outcome.