Human spermbots for patient-representative 3D ovarian cancer cell treatment.

Cellular micromotors are attractive for locally delivering high concentrations of drug, and targeting hard-to-reach disease sites such as cervical cancer and early ovarian cancer lesions by non-invasive means. Spermatozoa are highly efficient micromotors perfectly adapted to traveling up the female reproductive system. Indeed, bovine sperm-based micromotors have shown potential to carry drugs toward gynecological cancers. However, due to major differences in the molecular make-up of bovine and human sperm, a key translational bottleneck for bringing this technology closer to the clinic is to transfer this concept to human material. Here, we successfully load human sperm with Doxorubicin (DOX) and perform treatment of 3D cervical cancer and patient-representative ovarian cancer cell cultures, resulting in strong anticancer cell effects. Additionally, we define the subcellular localization of the chemotherapeutic drug within human sperm, using high-resolution optical microscopy. We also assess drug effects on sperm motility and viability over time, employing sperm samples from healthy donors as well as assisted reproduction patients. Finally, we demonstrate guidance and release of human drug-loaded sperm onto cancer tissues using magnetic microcaps, and show the sperm microcap loaded with a second anticancer drug, camptothecin (CPT), which unlike DOX is not suitable for directly loading into sperm due to its hydrophobic nature. This co-drug delivery approach opens up novel targeted combinatorial drug therapies for future applications.

FOXD1 mutations are related to repeated implantation failure, intra-uterine growth restriction and preeclampsia.

BACKGROUND: Human reproductive disorders consist of frequently occurring dysfunctions including a broad range of phenotypes affecting fertility and women's health during pregnancy. Several female-related diseases have been associated with hypofertility/infertility phenotypes, such as recurrent pregnancy loss (RPL). Other occurring diseases may be life-threatening for the mother and foetus, such as preeclampsia (PE) and intra-uterine growth restriction (IUGR). FOXD1 was defined as a major molecule involved in embryo implantation in mice and humans by regulating endometrial/placental genes. FOXD1 mutations in human species have been functionally linked to RPL's origin. METHODS: FOXD1 gene mutation screening, in 158 patients affected by PE, IUGR, RPL and repeated implantation failure (RIF), by direct sequencing and bioinformatics analysis. Plasmid constructs including FOXD1 mutations were used to perform in vitro gene reporter assays. RESULTS: Nine non-synonymous sequence variants were identified. Functional experiments revealed that p.His267Tyr and p.Arg57del led to disturbances of promoter transcriptional activity (C3 and PlGF genes). The FOXD1 p.Ala356Gly and p.Ile364Met deleterious mutations (previously found in RPL patients) have been identified in the present work in women suffering PE and IUGR. CONCLUSIONS: Our results argue in favour of FOXD1 mutations' central role in RPL, RIF, IUGR and PE pathogenesis via C3 and PlGF regulation and they describe, for the first time, a functional link between FOXD1 and implantation/placental diseases. FOXD1 could therefore be used in clinical environments as a molecular biomarker for these diseases in the near future.

THBD sequence variants potentially related to recurrent pregnancy loss.

Recurrent pregnancy loss (RPL) is a frequently occurring disease, which is classified as idiopathic in more than 50% of cases. THBD, the endothelial cell receptor for thrombin, has been associated with distinct biological processes and considered a coherent RPL-related candidate gene. In the present study, we have sequenced the complete coding region of THBD in 262 patients affected by RPL. Bioinformatics analysis and screening of controls strongly suggested that the THBD-p.Trp153Gly mutation might be related to RPL aetiology. It could be used, after its validation by functional assays, as a molecular marker for diagnostic/prognostic purposes.

BMP15 c.-9C>G promoter sequence variant may contribute to the cause of non-syndromic premature ovarian failure.

BMP15 has drawn particular attention in the pathophysiology of reproduction, as its mutations in mammalian species have been related to different reproductive phenotypes. In humans, BMP15 coding regions have been sequenced in large panels of women with premature ovarian failure (POF), but only some mutations have been definitely validated as causing the phenotype. A functional association between the BMP15 c.-9C>G promoter polymorphism and cause of POF have been reported. The aim of this study was to determine the potential functional effect of this sequence variant on specific BMP15 promoter transactivation disturbances. Bioinformatics was used to identify transcription factor binding sites located on the promoter region of BMP15. Reverse transcription polymerase chain reaction was used to study specific gene expression in ovarian tissue. Luciferase reporter assays were used to establish transactivation disturbances caused by the BMP15 c.-9C>G variant. The c.-9C>G variant was found to modify the PITX1 transcription factor binding site. PITX1 and BMP15 co-expressed in human and mouse ovarian tissue, and PITX1 transactivated both BMP15 promoter versions (-9C and -9G). It was found that the BMP15 c.-9G allele was related to BMP15 increased transcription, supporting c.-9C>G as a causal agent of POF.

Granulocyte colony-stimulating factor (G-CSF): a mediator in endometrial receptivity for a patient with polycystic ovary (PCO) undergoing in vitro maturation (IVM).

Proliferative and secretory changes at the endometrial lining are the result of a complex intrauterine environment where sex steroid hormones and different local factors play an important role for endometrial thickening. Optimal endometrial thickness reflects an adequate maturation which is a key factor for embryo implantation. Here, we present a case of a woman with polycystic ovary who was treated using in vitro maturation (IVM) techniques. In addition, this patient showed a dyssynchrony between the endometrial phase characterised by endometrial thinning and the embryo development which had a negative impact for embryo implantation. A protocol using uterine perfusion of granulocyte colony-stimulating factor (G-CSF) was performed as an alternative treatment for the unresponsive endometrium. We found that uterine infusion of G-CSF quickly increased endometrial thickness resulting in a successful pregnancy and healthy born baby. These results suggest that G-CSF is a factor that participates during endometrial remodelling enhancing the synchronisation between uterine environment and embryo development.

INVO procedure: minimally invasive IVF as an alternative treatment option for infertile couples.

Intravaginal culture (IVC), also called INVO (intravaginal culture of oocytes), is an assisted reproduction procedure where oocyte fertilization and early embryo development are carried out within a gas permeable air-free plastic device, placed into the maternal vaginal cavity for incubation. In the present study we assessed the outcome of the INVO procedure, using the recently designed INVOcell device, in combination with a mild ovarian stimulation protocol. A total of 125 cycles were performed. On average 6.5 oocytes per cycle were retrieved, and a mean of 4.2 were placed per INVOcell device. The cleavage rate obtained after the INVO culture was 63%. The procedure yielded 40%, 31.2%, and 24% of clinical pregnancy, live birth, and single live birth rates per cycle, respectively. Our results suggest that the INVO procedure is an effective alternative treatment option in assisted reproduction that shows comparable results to those reported for existing IVF techniques.

La edad sobre el factor masculino y su efecto en la fertilidad de pareja / The effect of age on the male factor and its outcome regarding couples’ fertility

Objetivo: realizar un análisis de las anomalías en los parámetros seminales y su correlación con la edad por medio de espermogramas de pacientes queingresaron a la Unidad de Medicina Reproductiva GESTAMOS, por infertilidad.Métodos y materiales: se realizó un estudio descriptivo de corte transversal de 226 espermogramas obtenidos entre enero de 2005 y enero de 2008, loscuales se examinaron mediante el análisis estándar de acuerdo con la Organización Mundial de la Salud(OMS) y el protocolo del Laboratorio de Andrología del Centro de Fertilidad y Esterilidad CECOLFES, y se analizaron los índices de Correlación de Pearsonpor medio del programa BioStat® 2007 para el respectivo análisis estadístico.Resultados: se analizó un total de 226 espermogramas, los cuales presentaron 73,5 percent de alteraciones significativas en cualquiera de sus parámetros. La disminución en la movilidad progresiva rápida (astenozoospermia) se observó en 65 percent de los casos con tendencia a la baja, con relación a la edad en el 0,04 percent por año. Del mismo modo, se presentaron anomalías en la morfología espermática (teratozoospermia) en 52,2 percent de los espermogramas, con una tendencia al aumento en 0,02 percent de los casos por año. La disminución en la concentración espermática estuvo presente en 28 percent de las muestrascon un descenso en 0,01 x 106/mL por año.Conclusiones: se evidenció una disminución importante de la calidad espermática de la poblaciónestudiada superior a la reportada por la literatura mundial con más relevancia a medida que aumenta la edad de los pacientes, siendo la movilidad progresivarápida y la morfología las más afectadas, las cuales se encuentran estrechamente ligadas a la capacidad fecundante del paciente.

bjective: analysing abnormalities in seminal parameters and their correlation with age through spermograms of patients admitted tothe GESTAMOS Reproductive Medicine Unit for infertility.Methods and materials: this was a descriptive cross-sectional study of 226 spermograms collected between January 2005 and January 2008. Theywere examined by standard analysis according to WHO guidelines and CECOLFES Fertility and Sterility Centre’s Andrology Laboratory protocol.BioStat® 2007 statistical analysis software was used for analysing Pearson correlation Indices. Results: a total of 226 spermograms presented 73,5 percent significant changes in all their parameters. Decreased rapidly progressive mobility (astenozoospermy) was observed in 65 percent and such downward trend was related to age (0,04 percent per year). Abnormalitiesin sperm morphology (teratozoospermy) were presented in 52,2 percent, having an upward trend (0,02 percent per year). Decreased sperm concentrationwas observed in 28 percent (0,01 x 106/mL per year decrease).Conclusions: there was a significant decrease in the study population’s sperm quality which was greater than that reported in the literature. Thiswas more relevant in the sense that as patients’ age increased then the rapidly progressive mobility and morphology of their sperm became most affected,this being closely related to patients’ fertilising capacity.

Obstetric and perinatal outcome in 200 infants conceived from vitrified oocytes.

Cryopreservation of oocytes by vitrification is a promising new technique for assisted human reproduction. Any new technical development must be accompanied with data concerning obstetric and perinatal outcome. This study analysed the obstetric and perinatal outcomes in 165 pregnancies and 200 infants conceived following oocyte vitrification cycles in three assisted reproduction centres. The results indicate that the mean birth weight and the incidence of congenital anomalies are comparable to that of spontaneous conceptions in fertile women or infertile women undergoing in-vitro fertilization treatment. These preliminary findings may provide reassuring evidence that pregnancies and infants conceived following oocyte vitrification are not associated with increased risk of adverse obstetric and perinatal outcomes.

Nacimiento a partir de Fertilización in vitro en ciclo natural / Birth following in-vitro fertilization in a natural cycle

El ciclo natural combinado con fertilización in vitro (FIV) y transferencia embrionaria está mostrando ser una opción de gran beneficio especialmente para pacientes mayores de 35 años, ya que se disminuye el costo del tratamiento por la facilidad de no utilizar hormonas para estimulación ovárica y por tanto se elimina el riesgo de la utilización de hormonas exógenas, con el beneficio adicional de transferir los embriones obtenidos en un ambiente endometrial libre del efecto de la estimulación. Objetivo: describir el embarazo y nacimiento de un niño sano como resultado de fertilización in vitro en un ciclo menstrual normal sin utilización de estimulación exógena de gonadotropinas. Procedimiento: se realizó seguimiento folicular por ecografía a una paciente con factor tuboperitoneal, hasta que el folículo dominante alcanzó un tamaño de 16 mm y grosor endometrial mayor de 14 mm. Se aplicaron 10.000 UI de hCG y 36 horas después se realizó aspiración folicular. El folículo dominante con un oocito en metafase II (MII), se fertilizó por ICSI y a las 48 horas de cultivo, el embrión obtenido fue transferido al útero previa preparación endometrial. Resultados: se obtuvo un óvulo en MII, el cual fertilizó después de ICSI y se transfirió un embrión en 4 blastómeras grado I a las 48 h de la aspiración. Doce días después de la transferencia embrionaria se realiza prueba de embarazo cuantitativa con un resultado positivo y nacimiento de un bebé sano. Conclusiones: la obtención de un óvulo maduro sin estímulo hormonal y transferencia embrionaria en ciclo natural, ofrece una nueva alternativa al tratamiento de infertilidad independiente de la edad.

Successful ongoing pregnancies after vitrification of oocytes.

OBJECTIVE: To demonstrate the efficiency of vitrifying mature human oocytes for different clinical indications. DESIGN: Descriptive case series. SETTING: Cryobiology laboratory, Centro Colombiano de Fertilidad y Esterilidad-CECOLFES LTDA. (Bogotá, Colombia). PATIENT(S): Oocyte vitrification was offered as an alternative management for patients undergoing infertility treatment because of ovarian hyperstimulation syndrome, premature ovarian failure, natural ovarian failure, male factor, poor response, or oocyte donation. Mature oocytes were obtained from 33 donor women and 40 patients undergoing infertility treatment. INTERVENTION(S): Oocytes were retrieved by ultrasound-guided transvaginal aspiration and vitrified with the Cryotops method, with 30% ethylene glycol, 30% dimethyl sulfoxide, and 0.5 mol/L sucrose. Viability was assessed 3 hours after thawing. The surviving oocytes were inseminated by intracytoplasmic sperm injection. Fertilization was evaluated after 24 hours. The zygotes were further cultured in vitro for up to 72 hours until time of embryo transfer. MAIN OUTCOME MEASURE(S): Recovery, viability, fertilization, and pregnancy rates. RESULT(S): Oocyte vitrification with the Cryotop method resulted in high rates of recovery, viability, fertilization, cleavage, and ongoing pregnancy. CONCLUSION(S): Vitrification with the Cryotop method is an efficient, fast, and economical method for oocyte cryopreservation that offers high rates of survival, fertilization, embryo development, and ongoing normal pregnancies, providing a new alternative for the management of female infertility.

Determinación de deleciones en el cromosoma Y en hombres infértiles candidatos a técnicas de reproducción asistida / Determination of microdeletion in chromosome Y in infertile men, candidates to assisted

El compromiso espermático severo en el hombre infértil, puede ser el resultado de la presencia de deleciones en el cromosoma Y en la región AZFc (Factor de azoospermia región c). En este estudio se caracterizaron 97 hombres con infertilidad con una frecuencia del 6 porciento para la presencia de deleciones en la población estudiada. De estos hombres positivos para deleción en la región Yq (AZFc), nacieron tres niños mediante el uso de inyección intracitoplasmática (ICSI) que heredaron la mutación en el cromosoma Y, fundamentando la importancia de crear un programa de tamizaje para detectar la presencia de deleción en este cromosoma en la población de hombres infértiles candidatos para técnica de fertilización asistida (ART) mediante ICSI

Primera gestación lograda a partir de óvulos humanos vitrificados / First pregnancy achieved from human vitrified oocytes

La vitrificación de óvulos humanos tiene múltiples aplicaciones y demostró ser altamente efectiva en el manejo de una paciente con síndrome de hiperestimulación ovárica que se encontraba en tratamiento para infertilidad secundaria. Objetivo: mostrar la eficiencia de la vitrificación de oocitos aplicada a óvulos en metafase II (MII) de una mujer con síndrome de hiperestimulación ovárica. Procedimiento: se vitrificaron óvulos MII de una paciente con síndrome de hiperestimulación ovárica, para lo cual se utilizó la técnica cryotop y como agentes crioprotectores etilenglicol, dimetilsulfóxido (DMSO) y sacarosa. Dos meses después, una vez restablecida a la normalidad la condición clínica de la paciente, se desvitrificaron los óvulos, se utilizó la técnica de inyección intracitoplasmática de espermatozoides (ICSI) y 24 horas más tarde se evaluó fertilización , los cigotos obtenidos se cultivaron in vitro por 72 horas hasta el momento de la transferencia embrionaria intrauterina. Resultados: la técnica de vitrificación con cryotop demostró ser una herramienta efectiva para preservar óvulos en lugar de embriones, además de disminuir los problemas éticos, morales, legales y religiosos que ésta conlleva. Conclusiones: la vitrificación por cryotop proporciona altas tasas de supervivencia, fertilización, desarrollo embrionario y embarazo; así mismo, ofrece una nueva alternativa para el manejo del síndrome de hiperestimulación ovárica.

Methods in preimplantation genetic diagnosis.

Preimplantation genetic diagnosis (PGD) is a new strategy, orientated toward primary prevention of congenital anomalies in couples with reproductive risk, such as advanced maternal age, carriers of chromosomal abnormalities, and carriers of monogenic conditions. For these patients, PGD is an acceptable alternative to prenatal diagnosis, mainly in those countries where pregnancy interruption is forbidden by law. PGD effectively avoids the implications linked to traditional prenatal diagnosis. Centres that provide medical servicies on reproductive biomedicine are responsible for the development and improvement of this new prevention strategy. Thanks to advances in micromanipulation techniques, associated with recent progress in molecular genetics, PGD may be employed for any genetic condition in the future.